Guidelines in dermoscopy
Transcript
Guidelines in dermoscopy
EDITORIALS G ITAL DERMATOL VENEREOL 2005;140:301 Guidelines in dermoscopy A. W. KOPF D r. Chimenti and his colleagues are to be congratulated in the recommendation to develop guidelines for this important addition to the clinical diagnosis of pigmented lesions of the skin with emphasis on malignant melanoma. The authors summarize that dermoscopy, in the hands of those experienced with the technique, has proven to enhance the in vivo diagnostic accuracy of malignant melanoma. Furthermore, it has been shown that dermoscopy raises the confidence of the physician in differentiating malignant melanoma from other pigmented lesions of the skin. The innovative algorithm for the management of pigmented skin lesions (Figure 2) is a useful road map for the application of dermoscopy which allows the physician to decide whether to biopsy or to follow the lesion. Importantly, using dermoscopy, the clinician can decide that biopsy is not needed which leads to cost containment of medical care. IN THIS ISSUE SEE PAGE 329 Address reprint request to: A.W. Kopf, MD, Clinical Professor of Dermatology, New York University School of Medicine, 350 Fifth Avenue, Suite 7805, New York, NY 10118-0189, USA. E-mail [email protected] Vol. 140 - N. 4 Department of Dermatology New York University School of Medicine New York, NY, USA Table I and Table II present succinct and meaningful definitions for melanoma-specific dermoscopic criteria and the main dermoscopic features of the most difficult pigmented lesions that must be differentiated from melanoma. In toto, this comprehensive guideline and its accompanying rich bibliography contain the essential aspects of dermoscopy not only for the beginner but also for the expert. It is becoming abundantly clear that any physician who accepts the responsibility for the differential diagnosis of pigmented lesions of the skin needs to learn dermoscopy. The Guidelines by Chimenti et al. is an excellent source for the current understanding of this most useful diagnostic tool, the primary conclusion of which is whether a lesion should be biopsied or not! GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 301 G ITAL DERMATOL VENEREOL 2005;140:303-8 What’s new in hereditary epidermolysis bullosa A. N. LIN E pidermolysis bullosa (EB) is a group of genetically determined disorders characterized by various degrees of cutaneous and mucosal fragility. It is found worldwide. In the last few decades, much has been learnt about their genetic basis and clinical features. As recently as the early 1980’s, the classification of EB was based entirely on clinical features. Many subtypes were recognized, often identified with confusing eponyms. In an attempt to better understand these disorders, investigators have established national registries of patients in various countries around the world. These registries allowed investigators to identify and study large numbers of patients with this rare disease. The results have been impressive. Investigators have identified at least 10 mutations underlying all major forms of EB. They have refined and simplified the classification of major forms of EB, and increased our understanding of their clinical features. Based on our knowledge of the newly identified mutations, prenatal diagnosis is no longer based on histological study of fetal skin biopsy specimens, but has been transformed into a molecular based technique. Perhaps most excitingly, researchers have begun to study gene therapy as a potential cure for these devastating diseases. In this issue of Giornale Italiano di Dermatologia e Venereologia, Tadini et IN THIS ISSUE SEE PAGE 358 Address reprint requests to: A. N. Lin, MD, Associate Professor, Division of Dermatology and Cutaneous Sciences, University of Alberta, Edmonton, AB, Canada Vol. 140 - N. 4 Division of Dermatology and Cutaneous Sciences University of Alberta, Edmonton, AB, Canada al. present the data gathered by the Italian EB Registry, further enhancing our understanding of these diseases. Identification of mutations in EB EB simplex Classically, EB has been classified into simplex, junctional, and dystrophic forms. EB simplex (EBS) is caused by mutations in the genes encoding for keratin 5 (KRT5) and Keratin 14 (KRT14). In most patients, it is transmitted as an autosomal dominant form. In the 2000 revised classificatioin system, based primarily on data gathered by the American EB Registry, 4 subtypes are recognized.1 Weber-Cockayne EBS is generally considerd to be the most common type of EBS.2 It primarily affects the hands and feet, making it one of the most readily recognizable types. Patients may not even realize they have EB until they begin vigorous physical activity, such as joining the army. Extracutaneous involvement tends to be mild or absent. Tadini et al. present the interesting finding that they found 67 cases of Weber-Cockayne EBS, slightly fewer than their 75 cases of Koebner EBS. In GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 303 LIN WHAT’S NEW IN HEREDITARY EPIDERMOLYSIS BULLOSA Koebner EBS, blistering is generalized with relative sparing of palms and soles. The most severe form is EBS-Dowling Meara, with generalized blistering present at or shortly after birth, progressive palmoplantar keratoderma, and mucous membrane involvement. Blisters often are grouped, resulting in “herpetiform” arrangement. A distinctive feature on electron microscopy is clumping of tonofilaments. The mottled pigmentation subtype features hypopigmented reticulated macules, and corresponds with an increased number of melanosomes within basal keratinocytes, dermal macrohages, and Scwann cells.3 It is a very rare type, and was not observed in the data presented by Tadini et al. This raises the possibility that there may be regional variations in its prevalence, although it may simply reflect the extremely uncommon prevalence of this subtype. Tadini et al. reported 1 case of EBS with muscular dystrophy, another rare subtype. These patients present with blisters at birth, followed by late onset muscular dystrophy, and abnormalities of the nail and teeth, sometimes associated with laryngeal webs and urethral strictures. It is caused by mutations in the gene encoding plectin (PLEC1), an intermediate filament inter-acting protein. Plectin is present not only in the hemidesmosome, but also in sarcolemma of the muscle, findings that may explain the association with muscular dystrophy. Because of the close association of plectin with hemidesmosome, it has been proposed that EBS with muscular dystrophy should be reclassified into a new type, called hemidesdmosomal type.3 In contrast to most forms of EBS, EBS with muscular dystrophy is inherited as an autosomal recessive disorder. EBS-Ogna is another rare form of EB, previously classified as EBS, which has now been shown to be due to a missense mutation in PLEC1,3 and which is now classified as another form of hemidesmosomal EB.3 The plectin mutation has been demonstrated in the original kindred from the Norwegian village of Ogna, and also in a German family with similar phenotype.3 These patients present with hemorrhagic blisters of the skin, but do not have muscular dystrophy.3 The vast majority of EBS is transmitted as an autosomal dominant trait, but autosomal recessive mutations have been identified in KRT14 in 7 families.3 Junctional EB Junctional EB is transmitted invariably as an autosomal recessive trait, and is caused by mutations in 304 gene encoding the subunits of laminin 5 (LAMA3, LAMB3, and LAMC2). The Herlitz type is one of the most serious types of EB. Patients present with widespread blistering at birth. A characteristic feature is exuberant granulation tissue on the face, especially around the mouth. Enamel hypoplasisa is common, leading to caries and loss of teeth. Laryngeal involvement is a rare but serious complication, and patients who develop stridor will require immediate ENT consultation to evaluate the airway, and tracheotomy is sometimes required.2 Because of the widespread blistering, death in the first few years of life is common, often due to combination of factors such as sepsis, severe anemia, and malnutrition. In the non-Herlitz type, patients exhibit a milder phenotype, but generalized blistering is also present at birth, and enamel hypoplasia is also common.1 It is interesting to note that Shabbir syndrome, also known as layrngo-onycho-cutaneous syndrome, is a recessively inherited disorder that features skin fragility and exuberant granulation tissue around the eyes and larynx. Investigators have recently uncovered mutations in LAMA3,3 raising the possibility that this disorder may in fact be related to Herlitz-JEB. Hemidesmosomal EB Generalized atrophic benign EB (GABEB) is a form of EB originally classified as JEB. In addition to cutaneous fragility, patients have nail dystrophy, scarring alopecia of the scalp, dental abnormalities, loss of eyelashes, and patchy hyperpigmentation of the skin. It is caused by mutations in the gene encoding the 180kDa BPAG, a transmembrane hemidesmosomal protein that is also known as type XVII collagen. Because of the close association of this protein with the hemidesmosome, this type of EB has been reclassified as a new type, the hemidesmosomal type,3 along with the types formerly known as EBS-muscular dystrophy and the Ogna variant of EB, discussed previously in this review. EB with pyloric atresia (EB-PA) is another form of EB that was formerly classified as a form of junctional EB. It features blistering at birth, in association with pyloric atresia. Often, there is a history of polyhydramnios during the pregnancy, a clue that the newborn may have some form of gastric outlet obstruction. If this is suspected, then an upright abdominal film should be done. If pyloric atresia is present, the X-ray will show the single bubble sign, reflecting a large bubble of GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 WHAT’S NEW IN HEREDITARY EPIDERMOLYSIS BULLOSA swallowed air that is trapped in the stomach. Immediate surgical intervention is then necessary for survival. With early surgical correction of the pyloric stenosis, favourable outcome is possible. EB-PA is caused by one of the genes encoding the subunit polypeptides of the alpha6-beta4 integrin (ITGA6 and ITGB4). Because of the close association of this integrin with hemidesmosomes, EB-PA has also been reclassified as a hemidesmosomal type of EB.3 Dystrophic EB Dystrophic EB (DEB) is caused by mutations in the gene encoding type VII collagen (COL7A1). Over 100 mutations in this gene have been described in various forms of dystrophic EB. Type VII collagen is the principal constituent of anchoring fibrils, which normally ensure cohesion between the epidermis and dermis. As a result, separation occurs below the lamina densa, and electron microscopy shows absence or reduced numbers of anchoring fibrils. DEB can be inherited in both autosomal recessive and dominant forms. The recessively inherited Hallopeau-Siemens form (RDEB-HS) is one of the most devastating types of EB. Patients present with widespread blistering at birth, and soon develop progressive fusion of the digits in childhood, resulting in considerable functional disability. Because the esophagus is lined with stratified squamous epithelium like the skin, patients can also develop esophageal blisters that can lead to scarring and stenosis. Dysphagia is then a major clinical problem, leading to anemia and malnutrition. Some patients may require esophageal dilation, but this may damage the already fragile mucosa and may cause perforation. Bypassing the stenosed portion of the esophagus with an isoperistaltic segment of the colon has been performed, but this is a major operation. Favourable results have been observed with gastrostomy, allowing introduction of nutrients directly into the stomach.2 Patients with the non-Hallopeau Siemens form of RDEB (RDEB-nonHS) have a milder phenotype. They still present with generalized blistering at birth, and may also develop partial fusion of the digits. An unusual variant is the inverse type, in which blisters develop mainly at inverse sites such as the axilla and groin, and severe oral and esophageal involvement are common.2 The dominant type of dystrophic EB is also caused by mutations in the gene encoding type VII collagen. Patients develop relatively minor blistering, but nail Vol. 140 - N. 4 LIN dystrophy, scarring, and mucosal involvement can also occur. Some patients present with distinctive white papules, called albopapuloid lesions. Clinical observations By studying large numbers of EB patients identified through a national registry, investigators have made important observations about clinical aspects of EB. These include extracutaneous involvements that have remained poorly understood until recently, including involvement of the kidney, eyes, and prevention of skin cancer in the dystrophic types. Ocular involvement With its stratified squamous epithelium like the skin, the cornea is subject to erosions and ulcers in EB. In their study of 3 280 patients enrolled in the American EB Registry, Fine et al.4 reported an association between ocular involvement and disease severity. They found that at least one episode of corneal erosions and blisters occurred in 74.1% of patients with RDEB-HS, and in 47.5% of patients with JEB-H. Symblepharons and ectropions were most often seen in the inverse type of RDEB and JEB-H. Also, blindness resulting from cumulative corneal scarring was seen in 6.46% of patients with RDEB-HS. All patients with EB who present with painful tearing eyes should be assessed by an ophthalmologist to prevent cumulative corneal scarring. Renal disease Individual case reports have suggested that renal failure may occur in some patients with EB. The causes of the renal failure have included poststreptococcal glomerulonephritis (presumably secondary to frequent cutaneous infection), amyloidosis, and chronic mechanical obstruction. Fine et al.5 analyzed data concerning 3 280 EB patients enrolled with the American EB Registry, and found 9 patients who were reported to have died of renal failure. Among these 9 patients, clinical and laboratory data permitted classification in 7 patients. Five of these patients had RDEB-HS, one had RDEB-non HS, and one had JEB-non Herlitz. They found that the cumulative risk for death from renal failure among patients with RDEB-HS was 12.3% by age 35 years, and recommended surveillance for early kidney involvement should be part of the routine evaluation of adults with RDEB and JEB. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 305 LIN WHAT’S NEW IN HEREDITARY EPIDERMOLYSIS BULLOSA Squamous cell carcinoma Squamous cell carcinoma remains a leading cause of death in patients with recessive dystrophic EB. They tend to arise in chronically eroded skin, and may present as heaped up granulation tissue, or thick keratotic plaques. In contrast to squamous cell carcinomas that arise from actinically damaged skin in patients without EB, these cancers are biologically aggressive, and metastasize widely. Approximately 85% of all patients with RDEB-HS will have developed at least one cutaneous squamous cell carcinoma by age 45 years.6 Furthermore, most patients die of metastatic squamous cell carcinoma within 5 years of diagnosis of the first tumor.7 In patients with xeroderma pigmentosum and renal transplants, studies have shown that chronic therapy with isotretinoin may prevent development of squamous cell carcinoma. In 2004, Fine et al.7 performed an open study of 20 patients with RDEB (5 had RDEB-HS, 15 had RDEB-nonHS) who were aged 15 years or older. Each patient was given isotretinoin daily, starting at 0.1 mg/kg/day, and the dose was increased monthly by 0.1 mg/kg/day until either maintenance dose was achieved (0.5 mg/kg/day), or the patient became intolerant of the next higher dose. Treatment was continued for 8 months in 19 patients. One patient terminated treatment after 3 months because of hypertriglyceridemia and transient abdominal pain, consistent with drug-induced acute pancreatitis, but all symptoms resolved within 3 days. Other patients experienced side effects that were mild, including skin dryness and fragility, epistaxis, and pruritus. Interestingly, over half the patients reported reduced blister formation while taking low-dosage isotretinoin, but this effect was lost as each patient approached the target maintenance dosage of 0.5 mg/kg/day. This finding may reflect in vitro data suggesting that isotretinoin may modulate collagenase synthesis by RDEB fibroblasts.7 These data suggest that isotretinoin is tolerated at the dosage studied in RDEB patients, and set the stage for this agent to be studied as a possible chemopreventive agent against squamous cell carcinoma in RDEB. Prenatal diagnosis Because EB can be such a devastating disease, families with pregnancies at risk often request prenatal diagnosis. In the past, this depended on examination of 306 fetal skin biopsy with electron microscopy and/or immunohistochemistry. However, fetal skin biopsy can only be obtained rather late in the pregnancy, often after 17 weeks, and is associated with a relatively high rate of miscarriage. Also, this method requires examination of the biopsy specimen by an expert in ultrastructure of fetal skin. With identification of the mutations underlying all major forms of EB, investigators have made impressive progress towards DNA-based prenatal diagnosis. Pfendner et al.8 recently reviewed the experience of DNA-based prenatal diagnosis performed at the DeBRA Molecular Diagnostics Laboratory at Jefferson Medical College. Since 1993, investigators at that center performed 144 DNA-based prenatal diagnoses in 121 families at risk for RDEB (63 pregnancies), JEB (69 pregnancies), EB-PA (6 pregnancies), and EBS (6 pregnancies). In most cases with DEB, and in all cases with JEB and EBS, the diagnosis was confirmed by demonstration of specific mutations. Prenatal testing was done on DNA isolated from chorionic villi or amniocytes obtained in the first or second trimester. Their overall accuracy was greater than 98% (2 unexplained discordant results out of 144 samples submitted), showing that DNA-based prenatal diagnosis is a very accurate and reliable method to assess pregnancies at risk. Gene therapy One of the most exciting advances in EB research is the prospect of gene therapy. Research on this has focused on junctional and recessive dystrophic EB, 2 of the most serious forms. Junctional EB Important advances in gene therapy for EB have described in a recent review.9 In early work concerning junctional EB, investigators have isolated keratinocytes from a patient with severe Herlitz JEB, and showed a homozygous mutation of the LAMB3 gene. These cells were unable to synthesize lamin-5, and hence were not able to assemble hemidesmosomes. Investigators used a retroviral construct expressing human beta3 cDNA to successfully transduce the JEB keratinocytes, which were then able to synthesize and secrete mature heteterotrimeric lamin-5. The use of viral vectors, however, is associated with GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 WHAT’S NEW IN HEREDITARY EPIDERMOLYSIS BULLOSA numerous logistical and biosafety concerns. Investigators have therefore developed non-viral approaches to JEB gene therapy. Using PhiC31 integrase, investigators have successfully integrated a laminin-5 beta3 expression plasmid into the genome of primary keratinocytes from four unrelated patients with JEB.9 Recently, investigators have used another nonviral vector (“Sleeping Beauty” transposable element) to successfully integrate the LAMB3 cDNA into the genomes of epidermal holoclones from six unrelated JEB patients.10 These cells also regenerated human JEB skin on SCID mice that was normalized at the level of laminin 5 protein expression, hemidesmosome formation, and blistering. Recessive dystrophic EB Woodley et al.11 have recently reviewed advances in gene therapy for recessive dystrophic EB. Keratinocytes from patients with inherited dystrophic EB cannot make type VII collagen, and show various abnormalities when compared with normal keratinocytes, For example, they are enlarged, elongated, and attach poorly to extracellular matrix. However, when these cells are transduced with a lentiviral vector encoding human gene for type VII collagen, the cells begin to permanently synthesize and secrete type VII collagen, and all the abnormal morphologic features became normal. They showed normal morphology, attached to extracellular matrix, and migrated in a normal fashion. Investigators have created two animal models in which 3 kinds of skin equivalents were transplanted onto mice. The first kind consisted of cells obtained from patients with severe recessive dystrophic EB, the second consisted of the same cells that had been “gene corrected” by stable integration of human type VII collagen and are able to synthesize and secrete type VII collagen, and the third consisted of normal human keratinocytes and fibroblasts. The skin equivalents that were not gene corrected showed features of RDEB, with fragile epidermal-dermal attachment, and virtually absence of anchoring fibrils. However, the skin equivalents made with gene-corrected cells showed presence of anchoring fibrils and type VII collagen at the epidermal dermal interface.11 These results suggest that it may be possible to perform ex vivo gene therapy, a process in which one takes skin biopsies from patients with RDEB, and then stably transfect the cells with the human COL7A1 Vol. 140 - N. 4 LIN gene, giving them the ability to synthesize and secrete type VII collagen.11 One can theoretically expand these cells into large sheets, and transplant them back onto the patient with RDEB. However, this procedure would be technically difficult, and the fragile graft would easily be lost. In a recent report, Woodley et al.12 injected recombinant human type VII collagen into immunocmpetent SKH mice. The injected protein was incorporated onto the basement membrane zone and remained stable for at least 6 weeks. Sera from 10 mice were evaluated for antibodies to type VII collagen, and these antibodies were found in 6 of the 10 mice. However, none of the mice lost weight or showed any untoward effects. Also, the antibodies did not prevent further incorporation of human type VII collagen in the basement membrane zone when later injected into new areas of the mouse’s skin. In the same report, Woodley et al.12 studied RDEB skin tissues regenerated on immunodeficient mice. These tissues retained the RDEB phenotype, with histologic evidence of dermal-epidermal separation and absence of human type VII collagen staining. However, intradermal injection of recombinant type VII collagen into the RDEB skin corrected the blistering and restored type VII collagen expression at the basement membrane zone. These studies yielded important insights to the prospect of protein-based gene therapy. References 1. Fine J-D, Eady RAJ, Bauer EA, Briggaman RA, Bruckner-Tuderman L, Christiano A et al. Revised classification system for inherited epidermolysis bullosa: Report of the second International Concensus Meeting on diagnosis and classification of epidermolysis bullosa. J Am Acad Dermatol 2000;42:1051-66. 2. Lin AN, Carter DM. Epidermolysis Bullosa: Basic and Clinical Aspects. New York:Springer-Verlag;1993. 3. Uitto J, Richard G. Progress in epidermolysis bullosa: genetic classification and clinical implications. Am J Med Genet C Semin Med Genet 2004;131C:61-74. 4. Fine J-D, Johnson LB, Weiner M, Stein A, Cash S, Deleoz J et al. Eye involvement in inherited epidermolysis bullosa: experience of the National Epidermolysis Bullosa Registry. Am J Ophthalmol 2004;138:254-62. 5. Fine J-D, Johnson LB, Weiner M, Stein A, Cash S, Deleoz J et al. Inherited epidermolysis bullosa and the risk of death from renal disease: experience of the National Epidermolysis Bullosa Registry. Am J Kidney Dis 2004;44:651-60. 6. Fine J-D, Bauer EA, McGuire J, Moshell A Epidermolysis Bullosa: clinical epidemiologic, and laboratory advances and the findings of the National Epidermolysis Bullosa Registry. Baltimore: Johns Hopkins University Press;1999. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 307 LIN WHAT’S NEW IN HEREDITARY EPIDERMOLYSIS BULLOSA 7. Fine J-D, Johnson LB, Weiner M, Stein A, Suchindran C. Chemoprevention of ssquamous cell carcinoma in recessive dystrophic epidermolysis bullosa: results of a phase 1 trial of systemic isotretinoin. J Am Acad Dermatol 2004;50:563-71. 8. Pfendner EG, Nakano A, Pulkinen L, Christiano A, Uitto J. Prenatal diagnosis for epidermolysis bullosa: a study of 144 consecutive pregnancies at risk. Prenat Diagn 2003;23:447-56. 9. Bauer JW, Laimer M. Gene therapy of epidermolysis bullosa. Expert Opin Bio Ther 2004;4:1435-43. 308 10. Ortiz-Urda S, Lin Q, Yant SR, Keene D, Kay MA, Khavari PA. Sdustainable correction of junctional epidermolysis bullosa via transposonmediated nonviral gene transfer. Gene Ther 2003;10:1099-104. 11. Woodley DT, Chen M. Epidermolysis bullosa: then and now. J Am Acad Dermatol 2004;51:S55-7. 12. Woodley DT, Keene DR, Atha T, Huang Y, Lipman K, Li W et al. Injection of recombinant human type VII collagen restores collagen function in dystrophic epidermolysis bullosa. Nat Med 2004;10: 693-5. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 G ITAL DERMATOL VENEREOL 2005;140:309-13 Inherited Epidermolysis Bullosa Registries. Whither Hence? J. D. FINE 1, 2 I n the current issue of the Journal Tadini et al. reports on the Italian experience with a nationally based registry of epidermolysis bullosa (EB) patients. This is an important contribution to our literature, since it provides an opportunity to compare data generated in Europe with those produced since 1986 within the United States by the National EB Registry.1 These registries differ not only in their geographic origin but also in the source of data collection, the number of enrollees, and the duration of follow-up. The Italian registry is a hospital-based one, involving hospitalbased participating dermatologists throughout Italy. Data generated in this manner are similar to those obtained via a hospital-based survey performed in Japan many years ago.2 The American Registry, based on data collection by investigators at four regionally distinct medical schools, has instead used rigorous epidemiological case finding techniques, in conjunction with the recruitment advantage of a free, centralized, diagnostic laboratory, referrals from outside physicians, and self-referrals from other patients and their affected family members, to identify and recruit its study subjects.3 Given these differences in study design and methodology, one might expect underreporting of milder cases in the Italian registry, since IN THIS ISSUE SEE PAGE 358 Address reprint reqeusts to: Dr. J. D. Fine, Divisions of Dermatology and Pediatrics, Vanderbilt University School of Medicine, c/o VMG Dermatology, 1900 Patterson Street, Suite 100 Nashville, TN 37203, USA. E-mail: [email protected] Vol. 140 - N. 4 1Divisions of Dermatology and Pediatrics Vanderbilt University School of Medicine Nashville, TN, USA 2National Epidermolysis Bullosa Registry Nashville, TN, USA few mild cases of EB would likely present for evaluation within a hospital setting. Despite such differences, however, the overall prevalence (10.1 cases per million in Italy vs 8.2 in the U.S.) and incidence rates (20.1 cases per one million live births in Italy vs 19.6 in the U.S.) for inherited EB are nearly identical, supporting the validity of the data already published by the American EB registry,2 as well as validating the effectiveness of the ongoing registry in Italy in its case finding efforts. Based on the close consistency of these demographic parameters within both otherwise distinct study populations, it seems appropriate to summarize the experience of the American EB Registry and then to suggest how the Italian registry can further add to our overall understanding of this disease. What have we already learned from the American EB registry, based on nearly 19 years of collection of cross-sectional data on nearly 3 300 patients and longitudinal data on approximately 425 subjects randomly selected from the projects’ overall study population? First, we now have accurate estimates of the prevalence and incidence of each of the major EB types and subtypes, as well as overall rates for EB as a whole.2 GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 309 FINE INHERITED EPIDERMOLYSIS BULLOSA REGISTRIES Given the rarity of some minor EB subtypes even within a population as large as the United States, however, it must be stressed that these latter calculated rates, similar to those reported in this issue of the Journal by Tadini et al., can be used only as rough estimates, since statistically these numbers are not as firm as those derived from the more common EB subtypes. Second, our data have documented that most of the cutaneous features in EB, most notably scarring, milia, and nail dystrophy, can occur not only in dystrophic EB but also in junctional and simplex patients. For example, approximately 30%, 11%, and 33% of all EB simplex patients enrolled in the American registry had scarring, milia, and nail dystrophy, respectively.4 As a practical correlate, this means that the clinician cannot reliably use cutaneous morphological features, either singly or in combinations, to accurately assist in the diagnosis and classification of EB patients, in the absence of concurrent laboratory testing. Indeed, we were unable to achieve sensitivities and specificities of greater than 90% with any of these findings, singly or in combinations of up to three findings, even for those findings which have previously been believed to be good surrogate markers of different EB subtypes. Third, we were able to use our extensive database to develop representative diagrams depicting the relative frequency of cutaneous disease activity in each of the major EB subtypes.5 These diagrams, for the first time, clearly demonstrated that considerable overlap exists among all forms of EB, as relates to both sites and relative frequency of skin involvement, further raising concerns over the use of cutaneous findings as surrogate diagnostic markers for the purpose of subclassification. These newer observations will undoubtedly affect any further revisions of the classification system for EB which we proposed in 1999 as a result of early data generated on behalf of the National EB Registry.6 Fourth, we have been able to determine the frequency with which specific cutaneous findings arise over time.4 As a result, we now know that some features commonly used to recognize junctional and dystrophic EB, to include scarring, nail dystrophy, and acral webbing, may not be present during early infancy, the time during which confirmation of diagnosis is the most emotionally charged. In contrast, other features, such as exuberant granulation tissue, may disappear with increasing age, making them useful hints for diagnosis only when they are present. Application of lifetable analysis technique to our extensive dataset has also 310 allowed us to be able to predict, at any given age of the patient, where he or she will likely have each of these cutaneous findings. Fifth, we have been able to determine the frequency with which different extracutaneous complications occur, as stratified by individual EB subtype.7 This includes quantitation of numerous complications within each of the major organ systems or anatomic sites, to include the external eye, oral cavity, gastrointestinal tract, genitourinary tract, lungs, heart, musculoskeletal system, and bloodstream. A recently published example is a summary of the frequency with which different complications arise within the genitourinary tract in each of the major EB subtypes.8 It should be emphasized that the ability to perform detailed analyses, especially lifetable ones, is a reflection of the power that can be derived from a rare disease registry which has large numbers of well characterized patients, since accurate estimates of such frequencies are dependent on the robustness of the size of the study population, its generalizability to an entire population of affected patients, and the length of longitudinal followup which was performed. As one derivative of such work, we now have quantitative data to demonstrate that most subtypes of EB are prone to develop at least several of the extracutaneous complications which were previously believed to occur in only the most severe forms of inherited EB.7 Analogous to what we did with cutaneous findings, we have been able to use lifetable analyses to predict the cumulative and conditional risk of an EB patient developing any of these extracutaneous complications over time, as stratified by EB type and subtype. This is important, since it provides useful clinical information to the physician as to when these patients need to be screened most carefully for early signs of extracutaneous complications (i.e., early surveillance should have a beneficial impact on the success of medical or surgical intervention). These types of data also provide information on the natural history of EB, as pertains to extracutaneous disease activity. Several years ago, for example, we reported on the cumulative risk of EB subtypes developing esophageal strictures and upper airway obstruction.9 In the case of esophageal strictures, about 12% and 80% of all HallopeauSiemens recessive dystrophic EB (RDEB-HS) patients could be predicted to develop this by ages 2 and 20, respectively, as would 25% of all Herlitz JEB (JEB-H) patients develop tracheolaryngeal stenosis or obstruction by age 3. Such data argue for meticulous surveil- GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 INHERITED EPIDERMOLYSIS BULLOSA REGISTRIES lance during infancy for both of these outcomes, since esophageal strictures will impair nutritional intake and growth if not corrected by dilatation, and upper airway obstruction in the setting of JEB may be lifethreatening. More recently published examples of the types of analyses possible from a rare disease registry include estimates for the cumulative risk of EB patients developing hand and foot deformities,10 and corneal blisters and scars.11 Not surprisingly, mitten deformities were primarily seen in RDEB-HS, occurring as early as the first year of life, and were present in virtually all patients by age 25. In regard to corneal injury, blisters occurred more frequently and earlier than scarring, and arose primarily in patients with RDEB-HS and JEB-H. Sixth, and possibly the most important consequence of our database, has been our ability to rigorously address the issue of skin derived cancers in inherited EB. We made this a major goal of this project from the onset, since many older case reports and small case series had suggested that tumors might be prevalent in at least some of the EB subtypes. As a result of 16 years of systematic data collection by the American registry, we now know the following about squamous cell carcinomas (SCCs) and EB.12 First, SCCs are most commonly seen in RDEB-HS, but also occur in other forms of RDEB, as well as in junctional EB. In contrast, there is no increased cumulative risk over lifetime of SCCs developing in EB simplex or DDEB, when compared to the non-EB population, despite suggestions to the contrary in some older publications which were based on case reports. Second, the onset of SCCs is usually first seen within the latter half of the 2nd decade of life, and by age 40 nearly 80% of all RDEB-HS patients will have developed at least one SCC (JD Fine, unpublished data , 2005). Third, we now know that multiple primary SCCs are the rule, and that they usually arise on extremities within areas of chronic scarring or nonhealing erosions. Fourth, using lifetable analyses we have demonstrated that about 80% of all of our RDEB-HS patients die of metastatic SCC within five years of the diagnosis of their first tumors, despite having undergone aggressive and presumably successful surgical excision of the primary tumor. Fifth, there is no increased risk of EB patients developing internal cancers. Sixth, we have found that there is a small but significant increased cumulative risk (about 2%) of malignant melanoma arising by as early as age 12 in patients with RDEB-HS, and a higher than expected cumulative risk of basal cell carcino- Vol. 140 - N. 4 FINE mas arising in patients with Dowling-Meara EB simplex by mid-adulthood, when compared to observations within the overall American population at the same ages (JD Fine, unpublished data, 2005). These collective findings suggest that tumor surveillance for malignant melanoma in EB patients is of importance only in RDEB-HS, and should be done during early childhood. In contrast, surveillance for SCCs and basal cell carcinomas is not necessary until at least mid young adulthood, but must become an integral part of patient care thereafter, given the risk of mortality from SCCs in the setting of RDEB. Data collected on behalf of the American EB Registry has also permitted determination within each major EB subtype of the risk of death from a variety of causes other than cancer, to include failure-to-thrive and sepsis (each a risk primarily in infants with junctional EB), other infections (pneumonia; other), and renal failure.13 For example, we now know that the cumulative risk of death from renal failure in RDEBHS patients is approximately 12%,14 and that such renal disease usually occurs by young to mid adulthood. The collection of such a large patient cohort has also allowed us the opportunity to study several other issues pertinent to inherited EB. For example, we have used a randomly selected subset of our patients to try to address issues such as: a) the annual cost of care, stratified by age and EB subtype; b) the relative availability of insurance reimbursement for hospitalized and outpatient services, medications, wound healing products, and nutritional supplements; c) the psychological impact of this disease on affected children, adults, parents and their family units;15 and d) the impact of this disease on basic function (activities of daily living) and pain.16 Registries also provide the opportunity for the harvesting and banking of DNA, cells, and blood and tissue specimens from a large number of well characterized patients. These samples can be then used by investigators worldwide as new opportunities arise for basic research. Over the past 10 years, for example, our patient population has served as a major source for tissues from which many of the molecular defects in EB have subsequently been detected. Finally, rare disease registries can serve as a major resource from which patients can be recruited for clinical trials. This is invaluable, since it will be otherwise impossible for any investigator working in one GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 311 FINE INHERITED EPIDERMOLYSIS BULLOSA REGISTRIES geographical area to successfully recruit a sufficient number of well characterized patients to be able to rigorously test the efficacy of a clinical intervention and have any hope of achieving statistically interpretable conclusions. Recently, for example, we used our cohort to ascertain the clinical efficacy of tetracycline in EB simplex 17 and to determine whether systemic isotretinoin might be safe enough to be used as a possible chemopreventive agent against SCCs in patients with RDEB.17, 18 How, then, can the Italian EB Registry be used most effectively, and what questions can be answered as a result of its well defined and growing patient cohort? First, it will certainly be very important for the Registry to attempt to collect data on selected extracutaneous complications and outcomes of interest, so that their findings can be compared with the published experience of its American registry counterpart. This will allow us to determine whether there are ethnic or immunogenetic differences within these 2 geographically distinct populations that might result in significant differences being observed in the risk for development of one or more complications across these 2 different populations. Were such differences noted, then this would suggest the need to employ different surveillance strategies in Italy, compared with those proposed by us for implementation within the United States. Second, the Italian registry can rigorously test whether differences in methods of treatment in Europe and the United States significantly impact differentially on patient morbidity or mortality. As a correlate, it is possible that differences in surveillance or approach to evaluation and care by physicians in different countries may result in differences in the relative severity of some extracutaneous complications seen within these geographically distinct EB populations. Third, comparisons may also suggest that inherent differences in the overall nature of the health care systems (sources of funding of medical care; relative access to specialists; other) in these 2 countries might similarly contribute to differences in clinical outcomes. Although the latter is less likely to be the case in 2 highly industrialized countries, this is clearly a major issue elsewhere in the world. For example, when we attempted to recruit young adult RDEB-HS patients in South America to participate in an internationally based clinical trial, we were surprised to learn that 312 very few of these patients survived early infancy, undoubtedly a reflection of gross differences in the availability and quality of care afforded to EB patients in less industrialized parts of the world. Fourth, the Italian EB Registry will provide a substantial patient population from which patients can be recruited to participate in basic research or in clinical trials. Given the many regulatory constraints on clinical trials currently imposed by the Food and Drug Administration within the United States, the availability of patients within the Italian EB Registry might provide the opportunity to test some hypotheses more rapidly in Europe than can be presently done in North America. The development of a rigorous EB registry in Europe is long overdue. It is therefore wonderful to see that a vibrant one is now in place in Italy under the direction of Professor Tadini. This registry will serve as a major reference source for future clinical trials in Europe that require participation of more than only a few patients with EB. From my perspective as head of the American EB Registry, I look forward to many fruitful collaborations in the future with my dermatology counterparts in Italy. References 1. Fine JD, Johnson LB and Suchindran CM. The National Epidermolysis Bullosa Registry. J Invest Dermatol 1994;102:54S-56S. 2. Fine JD, Johnson LB, Suchindran C, Moshell A, Gedde-Dahl T. The epidemiology of inherited EB: findings within American, Canadian, and European study populations. In: Fine JD, Bauer EA, McGuire J, Moshell A, editors. Epidermolysis bullosa: clinical, epidemiologic, and laboratory advances, and the findings of the National Epidermolysis Bullosa Registry. Baltimore: Johns Hopkins University Press; 1999. p.101-13. 3. Fine JD, Johnson LB, Suchindran C, Carter DM, Moshell A. The National Epidermolysis Bullosa Registry: organization, goals, methodologic approaches, basic demography, and accomplishments. In: Fine JD, Bauer EA, McGuire J, Moshell A, editors. Epidermolysis bullosa: clinical, epidemiologic, and laboratory advances, and the findings of the National Epidermolysis Bullosa Registry. Baltimore: Johns Hopkins University Press; 1999.p.79-100. 4. Fine JD, Johnson LB, Suchindran C, Bauer EA, Carter DM, McGuire J et al. Cutaneous and skin-associated musculoskeletal manifestations of inherited EB: the National Epidermolysis Bullosa Registry experience. In: Fine JD, Bauer EA, McGuire J, Moshell A, editors. Epidermolysis bullosa: clinical, epidemiologic, and laboratory advances, and the findings of the National Epidermolysis Bullosa Registry. Baltimore: Johns Hopkins University Press; 1999.p.114-46. 5. Devries DT, Johnson LB, Weiner M, Fine J-D. Relative extent of skin involvement in inherited epidermolysis bullosa (EB): composite regional anatomic diagrams based on the findings of the National EB Registry, 1986-2002. J Am Acad Dermatol 2004;50:572-81. 6. Fine J-D, Eady RAJ, Bauer EA, Briggaman RA, Bruckner-Tuderman L, Christiano A et al. Revised classification system for inherited epidermolysis bullosa: report of the Second International Con- GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 INHERITED EPIDERMOLYSIS BULLOSA REGISTRIES 7. 8. 9. 10. 11. 12. sensus Meeting on diagnosis and classification of epidermolysis bullosa. J Am Acad Dermatol 2000;42:1051-66. Fine JD, Johnson LB, Suchindran C, Bauer EA, Carter DM, McGuire J et al. Extracutaneous features of inherited EB: the National Epidermolysis Bullosa Registry experience. In: Fine JD, Bauer EA, McGuire J, Moshell A, editors. Epidermolysis bullosa: clinical, epidemiologic, and laboratory advances, and the findings of the National Epidermolysis Bullosa Registry. Baltimore: Johns Hopkins University Press; 1999.p.147-74. Fine J-D, Johnson LB, Weiner M, Stein A, Cash S, DeLeoz J, et al. Genitourinary complications of inherited epidermolysis bullosa (EB): experience of the National EB Registry and review of the literature. J Urol 2004;172:2040-44. Fine JD, Johnson LB, Moshell A, Suchindran C. The risk of selected major extracutaneous outcomes in inherited epidermolysis bullosa: lifetable analyses of the National Epidermolysis Bullosa Registry study population. In: Fine JD, Bauer EA, McGuire J, Moshell A, editors. Epidermolysis bullosa: clinical, epidemiologic, and laboratory advances, and the findings of the National Epidermolysis Bullosa Registry. Baltimore: Johns Hopkins University Press; 1999.p.193205. Fine J-D, Johnson LB, Weiner M, Stein A, Cash S, DeLeoz J, et al. Pseudosyndactyly and musculoskeletal deformities in inherited epidermolysis bullosa (EB): experience of the National EB Registry, 1986-2002. J Hand Surg (British and European Volume) 2005;30B: 14-22. Fine J-D, Johnson LB, Weiner M, Stein A, Cash S, DeLeoz J, et al. Eye involvement in inherited epidermolysis bullosa (EB): experience of the National EB Registry. Am J Ophthalmol 2004;138:254-62. Fine JD, Johnson LB, Suchindran C, Bauer EA, Carter DM, McGuire J et al. Cancer and inherited epidermolysis bullosa: lifetable analyses Vol. 140 - N. 4 FINE of the National Epidermolysis Bullosa Registry study population. In: Fine JD, Bauer EA, McGuire J, Moshell A, editors. Epidermolysis bullosa: clinical, epidemiologic, and laboratory advances, and the findings of the National Epidermolysis Bullosa Registry. Baltimore: Johns Hopkins University Press; 1999.p.175-92. 13. Fine JD, Johnson LB, Suchindran C, Bauer EA, Carter DM, McGuire J et al. Premature death and inherited epidermolysis bullosa: contingency table and lifetable analyses of the National Epidermolysis Bullosa Registry study population. In: Fine JD, Bauer EA, McGuire J, Moshell A, editors. Epidermolysis bullosa: clinical, epidemiologic, and laboratory advances, and the findings of the National Epidermolysis Bullosa Registry. Baltimore: Johns Hopkins University Press; 1999. p.206-24. 14. Fine J-D, Johnson LB, Weiner M, Stein A, Cash S, DeLeoz J et al. Inherited epidermolysis bullosa (EB) and the risk of death from renal disease: experience of the National EB Registry. Am J Kidney Dis 2004;44:651-60. 15. Fine J-D, Johnson LB, Weiner M, Suchindran C. Impact of inherited epidermolysis bullosa on parental interpersonal relationships, marital status, and family size. Br J Dermatol 2005;152:1009-14. 16. Fine J-D, Johnson LB, Weiner M, Suchindran C. Assessment of mobility, activities and pain in different subtypes of epidermolysis bullosa. Clin Exp Dermatol 2004;29:122-27. 17. Weiner M, Stein A, Cash S, DeLeoz J, Fine J-D. Tetracycline and epidermolysis bullosa simplex: a double-blind, placebo-controlled, crossover randomized clinical trial. Br J Dermatol 2004;150:613-14. 18. Fine JD, Weiner M, Stein A, Suchindran C, Johnson LB. Systemic isotretinoin and recessive dystrophic epiderolysis bullosa (RDEB): results of a Phase 1 clinical trial. J Invest Dermatol 2001;117:543. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 313 G ITAL DERMATOL VENEREOL 2005;140:315-6 Cutaneous malignant melanoma risk assessment A. R. SCHWARTZ H ow does one evaluate risk factors for cutaneous malignant melanoma? This can be a challenging question. After all, a goal in life is to minimize risk, an especially appealing concept for one of the deadliest of all cancers, melanoma, for which the incidence has been rising in America and Europe. In this issue Rubegni et al.1 of the University of Siena have done an impressive evaluation of melanoma risk in 140 Italian natives of Tuscany in describing their results of a casecontrol study of melanoma risk factors conducted in Tuscany during one winter extending from October 2002 to May 2003. They demonstrated a highly significant difference between controls and melanoma patients in nevi number and the presence of atypical nevi, constitutional skin color and eye color. This study was unique in that it employed 6 quantitative variables representing objective skin color, measured with a Minolta CR-300 colorimeter consisting of a detector and a microcomputer. They concluded that objective skin color measurements need to be combined with phenotypic parameters and sun exposure history for precise ascertainment of individual melanoma risk. Their work is consistent with other studies, including one by Dabkowski et al.2 of a Polish population in which an increased number of nevi (especially atypical ones), fair skin, and blue/green eyes, as well as IN THIS ISSUE SEE PAGE 373 Address reprint requests to: R. A. Schwartz MD, Professor & Head -Dermatology, New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103-214, USA. E-mail: [email protected] Vol. 140 - N. 4 Department of Dermatology New Jersey Medical School, Newark, NJ, USA intense UV exposure and sunburns, were important risk factors for melanoma development. Others have shown additional risk factors including freckling, family history of melanoma, and certain chromosomal mutations or polymorphisms.3-10 Identifying these factors can facilitate recognition of precursor lesions and the early diagnosis of melanoma, which can save lives. A critical element of reducing melanoma risk is prevention, particularly in childhood.3 Childhood ultraviolet exposure may be a critical factor, particularly in predisposed individuals. Physicians and health educations need to play an active role in the education and motivation of children to reduce solar exposure. Clearly, there are healthful benefits in some ultraviolet light reaching human skin in order to maintain adequate vitamin D metabolism. However, those at high risk for skin cancer need to be identified and then advised on protective measures to lessen the impact of ultraviolet light on their skin. Efforts such as those of Rubegni et al.1 and Dabkowski et al.2 are particularly valuable in this regard. In addition, there is a need to investigate the role of specific regulatory proteins, adhesion molecules, and other factors in the promotion of human melanocytic neoplasia, in order to facilitate improved understanding of this epidemic and reduce the risk of developing GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 315 SCHWARTZ CUTANEOUS MALIGNANT MELANOMA RISK ASSESSMENT melanoma. Disorders such as oculocutaneous albinism, xeroderma pigmentosum and congenital neurocutaneous melanosis can serve as important model diseases.11-17 Perhaps someday there will be effective gene therapy, or at least viable options for mending human genes.18 10. References 11. 1. Rubegni P, Sbano P, Cevenini G, Risulo M, Stanghellini E, Barbini P et al. Methological procedure for evaluation of risk factors for cutaneous malignant melanoma in a representative sample of the Tuscan population. G Ital Dermatol Venereol (in press). 2. Dabkowski J, Omulecki A, Zalewska A. Identification of melanoma risk factors in the Polish population. Dermatol Surg 1997;23:1039-42. 3. Azfar RS, Schwartz RA, Berwick M. Primary melanoma prevention in children. G Ital Dermatol Venereol 2004;139:267-72. 4. Desmond RA, Soong SJ. Epidemiology of malignant melanoma. Surg Clin North Am 2003;83:1-29. 5. Rokuhara S, Saida T, Oguchi M, Matsumoto K, Murase S, Oguchi S. Number of acquired melanocytic nevi in patients with melanoma and control subjects in Japan: nevus count is a significant risk factor for nonacral melanoma but not for acral melanoma. J Am Acad Dermatol 2004;50:695-700. 6. Youl P, Aitken J, Hayward N, Hogg D, Liu L, Lassam N et al. Melanoma in adolescents: a case-control study of risk factors in Queensland, Australia. Int J Cancer 2002;98:92-8. 7. Fargnoli MC, Piccolo D, Altobelli E, Formicone F, Chimenti S, Peris K. Constitutional and environmental risk factors for cutaneous 316 8. 9. 12. 13. 14. 15. 16. 17. 18. melanoma in an Italian population. A case-control study. Melanoma Res 2004;14:151-7. Lefkowitz A, Schwartz RA, Janniger CK. Melanoma precursors in children. Cutis 1999;63:321-4. Kaszuba A, Schwartz RA, Trznadel-Budzko E, Dobrska-Drobnik G, Seneczko M. Malignant melanoma. Part I - Epidemiology and etiopathogenesis. Nowa Klinika 2001;8:769-73. Kaszuba A, Seneczko F, Schwartz RA, Trznadel-Budzko E, Kaszuba A. Malignant melanoma. Part II - Clinical types, diagnostics and contemporary methods of treatment. Nowa Klinika 2001;8:773-9. Spicer MS, Stampien TM, Lambert WC, Schwartz RA, Harmon C, Fitzgerald-Bocarsly P. Severe xeroderma pigmentosum associated with numerous melanomas, no other skin tumors, high natural killer cell activity, normal interferon production, and a benign course. J Cutan Pathol 1997;24:126. Leibowitz E, Janniger CK, Schwartz RA, Lambert WC. Xeroderma pigmentosum. Cutis 1997;60:79-84. Papadopoulos AJ, Schwartz RA, Sarasin A, Lambert WC. Xeroderma pigmentosum variant in a Greek patient. Int J Dermatol 2001;40:442-5. Cruz MA, Cho ES, Schwartz RA, Janniger CK. Congenital neurocutaneous melanosis. Cutis 1997;60:178-81. Okulicz JF, Shah RS, Schwartz RA, Janniger CK. Oculocutaneous albinism. J Eur Acad Dermatol Venereol 2003;17:251-6. Centurión SA, Schwartz RA. Oculocutaneous albinism type 2. Acta Dermatovenerol Alp Panonica Adriat 2003;12:32-6. Kraemer KH, Lee MM, Andrews AD, Lambert WC. The role of sunlight and DNA repair in melanoma and nonmelanoma skin cancer. The xeroderma pigmentosum paradigm. Arch Dermatol 1994;130: 1018-21. Cleaver JE. Mending human genes: a job for a lifetime. DNA Repair (Amst) 2005; 4:635-8. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 G ITAL DERMATOL VENEREOL 2005;140:317-20 Prediction of photodynamic efficacy A. D. TOSCA, M. P. STEFANIDOU P hotodynamic therapy (PDT) refers to light activation of a tumor-localizing photosensitizer to generate highly reactive oxygen intermediates, causing selective tissue injury and necrosis by oxidizing essential cellular components, vascular damage and/or inflammatory reaction and immune host response. Recently, it has been shown that apoptosis is also involved in tumour cell death after topical photodynamic therapy with 5-aminolevulinic acid (ALA-PDT) within 1 day in patients with actinic keratoses (AK). The easy accessibility of the skin to the light exposure has led to an increasing interest for PDT application in dermatology. Photodynamic therapy is mainly associated with the treatment of cancer, but is also being applied to premalignant and benign diseases. Photodynamic therapy is an alternative treatment modality for superficial non-melanoma skin tumours and various inflammatory, viral and other diseases, with potentially high effectiveness and low morbidity. Topical ALA-PDT has become a therapeutic option of growing interest. Its main advantage is the absence of generalized cutaneous photosensitivity. Topical ALA-PDT involves photosensitization with endogenous porphyrins and activation with visible light. Almost all types of cells in the human body are able to synthesise heme. The principle of ALA-PDT is that 5IN THIS ISSUE SEE PAGE 381 Address reprint requests to: Dr. A. D. Tosca, Department of Dermatology, University Hospital of Heraklion, 71110 Heraklion, Crete, Greece. E-mail:[email protected] Vol. 140 - N. 4 Department of Dermatology University Hospital, Heraklion, Crete, Greece aminolevulinic acid (ALA) in excess results in a builtup of intracellular porphyrins and especially protoporphyrin IX (PpIX), an extremely potent photosensitizer and fluorescence emitter. In situ conversion of ALA to PpIX is accomplished in normal and neoplastic keratinocytes to a different degree, by enzymes in the heme pathway resulting to selective accumulation in target-tissue and tissue-specific phototoxic effects. The relative accumulation of PpIX in diseased tissue is not specific for neoplastic disease and has been shown after the application of ALA to benign proliferative skin conditions, such as viral warts and condylomata 1 and psoriasis. The number of possible clinical indications expanded besides oncology and now encompasses several inflammatory and viral skin diseases as a consequence of experimental findings demonstrating that PDT can promote apoptotic cell death and modulate immune activities of the skin. Additionally, the application of ALA leads to accumulation of PpIX in hair follicles and sebaceous glands, suggesting the potential use for disorders of skin appendages such as acne, alopecia areata, hypertrichosis. New applications for ALA-PDT in dermatology are presently being investigated such as psoriasis, plaque-stage cutaneous T-cell lymphoma, Darier’s dis- GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 317 TOSCA PREDICTION OF PHOTODYNAMIC EFFICACY ease, Kaposi sarcoma, port-wine stains, lichen sclerosus, scleroderma, Hailey-Hailey disease. The literature related to the use of topical ALAPDT is extensive for the treatment of actinic keratoses, basal cell carcinoma and Bowen’s disease.2 Aminolevulinic acid, a metabolite of the heme biosynthesis was the first agent to receive regulatory approval for the treatment of AK in conjunction with blue light in dermatology, in 1999 (Levulan, DUSA Pharmaceuticals, Inc, Valhalla, NY). Several clinical studies report high response rates in superficial basal cell carcinoma (BCC) ranging from 79% to 100% and AK from 81% to 100%.2-4 For nodular BCC the results are more disappointing, with reported response rates ranging from 10% to 42%. The challenge to the dermatologist remains to optimize outcomes following PDT therapy. The efficacy of the treatment is dependant on many variables. Small modifications of the treatment procedure may have quite a significant impact on the outcome. Controlled comparative studies or data that sufficiently allow to compare the different modalities are still missing. The success of treatment requires an optimal interplay among different parameters, such as type and drug dose, selection of light source, depth of light penetration into tissue and light dose, treatment schedules, criteria of tumour and patient selection. An efficient photodynamic agent should have several properties: selective retention or uptake by the target tissue, high absorbance in the useful wavelength range for optimal tissue penetration, high quantum yield of singlet oxygen, fast clearance from serum and healthy tissue, short time interval between drug application and its accumulation into lesion, high chemical purity, low systemic toxicity and side effects, lack of mutagenic potential. Aminolevulinic acid is the natural precursor of PpIX which is formed endogenously via the biosynthetic pathway of heme. Advantages of ALA-PDT are: localized and short-term photosensitivity and rapid photodegradation by light illumination. The induction of a more lipophilic ester-group (ALA- methylester, methyl-aminolevulinate) seems to enhance the selectivity and deeper penetration of the photosensitizer. Discomfort and the intensity of pain may be lower during PDT with methyl-aminolevulinate than with ALA. Rossi et al.5 in this issue describe a study of PDT using topical methyl-aminolevulinate (Metvix, PhotoCure ASA, Oslo, Norway) to treat 170 AK of the 318 face and scalp. A non-coherent red light source emitting at 630 nm was employed with light intensity 70100 mW/cm2 and light dose of 37 J/cm2, with complete response rate of 90% in 6 months. Photoexitation of the photosensitizer by light corresponding to its absorption spectrum is a basic requirement in PDT. The light used for PDT can be provided by incoherent light sources or laser systems. Basic considerations for the choice of a light source are the following: the emitted wavelength should match an absorption peak of the photosensitizer used and the fact that longer wavelengths penetrate deeper into tissues. The depth of tissue penetration depends on the wavelength, the absorption by endogenous chromophores and the absorption by the sensitizing drug (self-shielding), and is limited by optical scattering within the tissue. It might even be of advantage to use a broad band light source, since photosensitizer’s photoproducts with other absorption peaks are formed during PDT. Protoporphyrin IX has its maximum absorption in the Soret band and additional smaller peaks in the green and red regions. However, with red light the interactions with chromophors of the skin (melanin, hemoglobin) are minimized, leading to a sufficient penetration of light into skin. Regarding the light dose in PDT the fluence and the intensity used are different for oncologic and nononcologic indications. For PDT the light source has to provide sufficient intensity of light, up to 40 mW/cm2 for non-oncologic indications and up to 150 mW/cm2 for oncologic indications. Basically, the fluence of the light should be less than 150 J/cm2 and the intensity of the light source less than 200 mW/cm2 in order to avoid photothermal effects. The time interval between drug administration and sufficient photosensitizer concentration determines the optimal time point of light exposure and has been estimated for various photosensitizers and route of administration. Irradiation should be performed when an optimal ratio of photosensitizer levels in tumor versus normal tissue is reached. Some authors emphasized tumour particularities that might lead to decreased responsiveness. Thick nodular BCC and hypertrophic AK are rather resistant, although surgical debulking of nodules and curettage of scales, or repetition of the treatment may improve clearance rates. Morpheiform and pigmented BCC are almost always resistant to the treatment. However, different photosensitizers and PDT regimens may result in different cellular response patterns GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 PREDICTION OF PHOTODYNAMIC EFFICACY even in the same type of tumour, while many specific responses may be effected only in a narrow window of time, PDT dose, or both. It becomes clear that there is a need to monitor parameters other than delivered light dose and irradiance and to correlate them with ALA-PDT. Prediction of PDT efficacy could be made on the basis of indirect data from fluorescence time course kinetics and the direct biologic tissue response such as clinical erythema development after PDT. The determination of the time course of photosensitizer’s fluorescence in skin lesion is crucial for effective PDT in order to maximize PDT effectiveness. Chromophores such as porphyrins have the ability to absorb energy when excited by light of certain wavelength. The emission of light following absorption of incident photons is termed “fluorescence”. Blue light is mainly applied for fluorescence detection, due to its reparability from the red fluorescence of PpIX generated. The penetration of blue light is only a few tenth of millimeter and the respective fluorescence is generated only in the superficial part of the skin. This fluorescence yields hardly any information from deeper structures inside the diseased tissue. The light intensity used for fluorescence excitation is at least twenty-fold lower as compared to PDT. Since the PpIX accumulation after ALA-PDT is a gradual process, fluorescence kinetics could be used to determine the ideal time point application to initiate the irradiation. Imaging spectroscopy using digital cameras (CCD) can be used to study the time course kinetics and spatial distribution of fluorescence.6 The equipment needed for fluorescence imaging is a light source for excitation, a digital camera for detection, a system of optical filters and the software for data processing. The fluorescence images are recorded in a dark room. In our set-up the power density of the light source is very low (approximately 0.5 mW/cm2) and the exposition time very short, in order to avoid possible photobleaching of the photosensitizer. Excitation is performed by light of 425±10 nm. For the study of fluorescence kinetics, serial in vivo fluorescence images are captured. For any image the medians of fluorescence intensity are calculated after correcting for background autofluorescence and the sequential integrated fluorescence signals are then plotted versus time. Two parameters are evaluated: the maximal fluorescence intensity, as compared with the zero time baseline intensity (photosensitizer accumulation effi- Vol. 140 - N. 4 TOSCA ciency) and the correlation of fluorescence spatial distribution with the area of the lesion assessed with visual light (photosensitizer localization efficiency). Serial, in vivo and real-time measurements of fluorescence intensity are of particular advantage when an endogenous photosensitizer is used, which might be synthesized in various amounts over time in different tumors of the same type, depending of the metabolic activity of the target cell. Optimal destruction of skin lesion is therefore achieved while the surrounding skin is left intact. In vivo fluorescence kinetics evaluation over time showed that AK and BCC developed maximum fluorescence emission intensity between 4 to 4.5 h after application. Despite fluorescence intensity values until 14 h the irradiation starting is chosen to be early 3.5 to 5 h when high localization efficiency was also noted, ensuring the selectivity of the procedure.6 Some authors, based on the principle that the intensity of the emitted fluorescence is a function of the amount of the sensitizer present, claimed that the fluorescence of skin lesions after ALA application using a Wood’s lamp or laser-induced fluorescence provided an estimate for prediction of PDT.7 However, no direct correlation was found between fluorescence intensity and clinical response by other authors.8 On the other hand, the optimal time for PDT could be deduced from the time-dependent concentration of PpIX, since fluorescence kinetics is a temporal process. In order to maximize PDT effectiveness irradiation of the treatment area should occur at the time of sufficiently increased concentration of the endogenous photosensitizer within the lesion and of optimal ratio of photosensitizer levels in tumour: normal tissue. Tope et al.9 studied fluorescence kinetics in BCC after oral administration of ALA and showed a full thickness PpIX accumulation in all BCC histological subtypes and maximal tumour: normal skin fluorescence ratios 1 to 3 h after ALA ingestion. Based on the cumulative evidence of selective erythema during ALA-PDT which might be closely correlated with the tissue photo-induced actions, we studied the in vivo skin color changes, as represented by erythema development by means of a remote machine vision system in correlation with the clinical and histological responses of AK and BCC subjected to treatment.3 A remarkable correlation of erythema development and effective tissue response to PDT was found. The skin erythema imaging is a reliable notable GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 319 TOSCA PREDICTION OF PHOTODYNAMIC EFFICACY marker of the phototoxic effect and, thereafter, of the PDT effectiveness. It is interesting that contrary to the UVB-induced erythema, where higher doses provoke even stronger reaction, during ALA-PDT the progressive increase of energy leads the skin erythema to a saturation level. This is probably owed to PpIX photobleaching due to self reaction with the released singlet oxygen and free radicals, and this may explain why further increase of the light dose has no effect on both the tumour response and the erythema development. Laser Doppler measurements for assessing blood flow were used. By means of the Laser Doppler Perfusion Imager it is possible to record blood flow in the superficial and reticular dermis over a large surface and estimate a representative mean perfusion. Wang et al. have investigated the perfusion in superficial BCC and found an increased blood perfusion after topical PDT. A full picture of the therapeutic effectiveness of ALA-PDT remains the histological evaluation. Alternative methods allow for control of PDT efficacy. Techniques to measure photosensitizer concentration such as microdialysis and to evaluate light fluence within tissue, tumour tissue oxygen consumption and radical generation are being developed to assist PDT treatment. Microdialysis is a technique for investigation of drug-penetration. A semipermeable dialysis catheter is placed in the dermis and perfused by a solution. Depending on the concentration gradient, molecules in the surrounding extracellular space which are at a higher concentration diffuse into the dialysis fibre and the opposite takes place in the case of molecules of lower concentration. Microdialysis samples before and after ALA application are analysed with an ion exchange high performance chromatograph.11 The penetration of ALA in tumour area is rapid and after 15 minutes the concentration is high and stable. On the other hand, virtually no ALA penetrates healthy skin. The availability of oxygen within the tissue undergoing PDT treatment is an important parameter that can limit direct tumour kill. Since singlet oxygen arises from ground state oxygen, rapid and substantial reduc- 320 tion of tissue oxygen tension upon illumination during PDT were reported through damage of the vascular system and through O2 consumption in the oxidative reactions taking place. The rates of singlet oxygen generation and therefore tissue oxygen consumption/depletion are high when both tissue photosensitizer levels and the fluence rate of light are high. The fluence rate must be adjusted downward to slow oxygen consumption sufficiently to facilitate the maintenance of tissue pO2 levels during treatment. The potential for PDT in the treatment of several skin conditions is promising, but rigorous trials must be performed. Further studies are required to confirm the optimal treatment parameters and reliable predictors of the therapeutic outcome. References 1. Stefanaki IM, Georgiou S, Themelis GC, Vazgiouraki EM, Tosca AD. In vivo fluorescence kinetics and photodynamic therapy in condylomata acuminata. Br J Dermatol 2003;149:972-6. 2. Kalka K, Merk H, Mukhtar H. Photodymamic therapy in deramtology. J Am Acad Dermatol 2000;42:389-413. 3. Tosca AD, Balas CJ, Stefanidou MP, Katsantonis JC, Georgiou SK, Tzardi MN. Photodynamic therapy of skin malignancies with aminolevulinic acid. Emphasis on anatomical observations and in vivo erythema visual assessment. Dermatol Surg 1996;22:929-34. 4. Morton CA, MacKie RM, Whitehurst C, Moore JV, McColl JH. Photodynamic therapy for basal cell carcinoma – effect of tumour thickness and duration of photosensitizer application and response. Arch Dermatol 1998;134:248-9. 5. Rossi R, Mavilia L, Ghersetich I, Lotti TM. Photodynamic therapy of actinic keratoses with methyl-aminolevulinate (Metvix). G Ital Dermatol Venereol (in press) 6. Stefanidou M, Tosca A, Themelis G, Vazgiouraki E, Balas K. in vivo fluorescence kinetics and photodynamic therapy efficacy of δ-aminolevulinic acid-induced porphyrins in basal cell carcinomas and actinic keratoses; implications for optimization of photodynamic therapy. Eur J Dermatol 2000;10;351-6. 7. Svanberg K, Andersson T, Killander D, Wang I, Stenram U, AndersonEngels S et al. Photodynamic theory of non-melanoma malignant tumors of the skin using topical δ-aminolevulinic acid sensitization and light irradiation. Br J Dermatol 1994;130:743-51. 8. Fijan S, Hönigsmann H, Ortel B. Photodynamic therapy of epithelial skin tumours using delta-aminolevulinic acid and desferrioxamine. Br J Dermatol 1995;133:282-8. 9. Tope WD, Ross EV, Kollias N, Martin A, Gillies R, Anderson RR. Protoporphyrin IX fluorescence induced in basal cell carcinoma by oral δ-aminolevulinic acid. Photochem Photobiol 1998;67:249-55. 10. Wennberg A, Larko O, Lonnroth P, Larson G, Krogstad A. Deltaaminolevulinic acid in superficial basal cell carcinomas and normal skin –a microdialysis and perfusion study. Clin Exp Dermatol 2000;25:317-22. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 G ITAL DERMATOL VENEREOL 2005;140:321-3 Lyme Borreliosis up-to-date J. HERCOGOVÁ L yme borreliosis (LB) is a systemic infectious disease caused by Borrelia burgdorferi sensu lato transmitted by Ixodes ricinus ticks. The incidence of LB is estimated up to 70-100 cases per 100 000 inhabitants in Europe. Skin manifestations of the disease were described in 1883 by Buchwald, nevertheless the causal organism was discovered by Burgdorfer only in 1982. Clinical manifestations of the disease include mainly cutaneous, nervous, cardiovascular, locomotor signs and symptoms. LB might present in three stages: 1st early localized disease (erythema migrans [EM], borrelial lymphocytoma [BL] or lymphocytoma borreliensis, and lymphadenitis), 2nd early disseminated disease (flu-like symptoms, secondary lesions of EM, acute neuroborreliosis, dysrhytmias, atrio-ventricular block, myocarditis, acute arthritis, etc.), and 3rd late disseminated disease (chronic neuroborreliosis, chronic arthritis, acrodermatitis chronica atrophicans [ACA]).1 Every tick-bite does not cause skin manifestations, EM develops only in 50% of infected patients and 30-50 % of patients with EM does not report any tick or insect bites. Each stage of LB might have a typical skin manifestation, some of them are pathognomic for LB, namely, annular EM in all patients and BL localized on the ear lobe in children. IN THIS ISSUE SEE PAGE 417 Address reprint requests to: Dr. J. Hercogová, Department of Dermatology, 2nd Medical School, Charles University Prague, Czech Republic, University Hospital Bulovka, Budinova 2, 180 81 Prague 8, Czech Republic. E-mail: [email protected] Vol. 140 - N. 4 Department of Dermatology, 2nd Medical School Charles University Prague, Czech Republic EM is the most frequent manifestation of Borrelia infection, it represents also 85% of all skin manistations of LB. Three types of EM are recognized based on the colour of the lesion: annular patch with central clearing (EM anulare), homogenous patch (EM maculare) and target-like lesions (EM concentricum). EM is mostly solitary. The histopathological picture is non-specific: superficial dermatitis, perivascular and periadnexal infiltrate predominantly consists from lymphocytes with some few plasma cells. Another skin manifestation of the 1st stage is BL. It is a rare manifestation of LB (only 5% of skin involvement), it is a red-violet papule (BL papulare) or plaque (BL infiltratum), few mm to 5 cm in diameter. The lesion is usually solitary, localized predominantly on the ear lobe, nose tip, areola mammae, scrotum, and above the bone prominences. Histopathological picture of BL is characterized by superficial and deep dermatitis composed of lymphocytic infiltrates localized periadnexally and perivascullary in the upper and middle dermis, with the presence of plasma cells in the inflammatory infiltrate. Both, EM and BL could be accompanied by regional lymphadenopathy. Second stage of LB, includes not only skin involvement, but also extracutaneous manifestations. The spirochete can disseminate soon after the tick bite and neu- GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 321 HERCOGOVÁ LYME BORRELIOSIS UP-TO-DATE rologic symptoms or arthritis can occur in early or late in the course.2 Those could be neurologic (lymphocytic meningitis, cranial neuritis also with peripheral facial nerve palsy, radiculoneuropathy, rarely encephalomyelitis), cardiovascular (carditis), locomotor (arthritis, typically in one or both knees, myositis) and others (hepatitis, conjunctivitis, incl. general signs and symptoms – malaise, headache, fatique etc.). Secondary EM follows the primary EM after one week, EM lesions are annular and smaller than the primary one. Third LB stage of late infection includes involvement of the skin, joints, nerves, fatique could be a very important symptom. Characteristic skin manifestation is ACA which appears late in the course of LB (months or years after infection). ACA is much less frequent compared to EM, but its diagnostics could be very difficult. It represents 10% of all cutaneous manifestations of LB. Initial oedema and diffuse dark erythema localized predominantly on the protrudent parts of the limbs changes into an atrophy of the skin and the adnexa. Distinct clinical types could be recognized in ACA patients – inflammatory ACA stage is mainly macular (ACA maculare) in 75% of ACA patients, or oedematous (ACA oedematosa) in 13% of patients, atrophic stage of ACA is present in 12 % of our ACA patients and it could be of various types: ACA teleangiectatica – teleangiectasias predominate, ACA fibromatosa – fibrous nodules above the bone prominences, typically localized above elbow joints, ulnar aspects of the forarms and above the interdigital and carpophalangeal joints, and ACA atrophicans sensu stricto – atrophy of the skin and underlying tissue. Histopahology of ACA lesions varies on the duration of the skin lesions. At the beginning, band-like infiltrate of lymphocytes with plasma cells, histiocytes, oesinophils is seen in the upper dermis, later inflammation resolves and is prominent around vessels and adnexa, compact orthokeratosis of the epidermis and epidermis atrophy follows, sometimes dilatation of vessels in the upper dermis (histopathological background of teleangiectasiae). After some years, atrophy of the dermis, incl. elastic fibres and adnexa is present. In fibromatous ACA type, collagen bundles are concentrically composed. Half of ACA patients suffer also from the joint and peripheral nerve involvement.3 The infection heals spontaneously in some patients, in the others the disease continues even after the proper treatment. Besides those characteristic manifestations some authors hesitate, if other signs or symptoms also belong to the clinical picture of LB. Diffuse reversible alope- 322 cia, pseudopelade Brocq, morphoea and lichen sclerosus et atrophicus, cutaneous marginal zone B-cell lymphoma, anetoderma, idiopatic atrophoderma Pasini-Pierini, progressive facial hemiatrophy, are discussed in the literature.4-7 The diagnosis of LB should be based on a presence of three factors together: opportunity for a tick exposure, a characteristic clinical manifestation (both local and systemic) and a confirmation of B. burgdorferi infection, with exception of pathognomic skin manifestations, i.e. anular erythema migrans (but illness of longer than 30 days duration is required for IgG immunoblot positivity) and papular BL on the ear lobe in a child.1 Direct proof of borrelial infection include: 1) histopathological detection of the microorganisms in the tissue by the light or the electron microscope, 2) isolation of B. burgdorferi (sensitivity of isolation of the skin samples in non-treated patients is 50 %), 3) conventional, mainly nested polymerase chain reaction (PCR) or LighCycler real-time PCR or template DNA hybridization using TaqMan fluorogenic probe. Those systems are capable to identify 1-10 cultivated spirochaete. Isolation of borrelial DNA from clinical samples is difficult as they contain abundance of host DNA. Specificity of PCR is evaluated in the Southern blots using specific probes, restriction enzymes or sequencing of PCR products.8, 9 Indirect laboratory tests include: 1) indirect immunofluorescence, 2) ELISA assay, using the whole-cell sonificated antigen or recombinant borrelial antigens. Serum specimen with a positive test result is further tested with immunoblotting. However, immunoblotting still has many problems. True standardisation of an immunoblotting method for diagnosis of LB would require agreement on the strains used for antigen preparation. This approach would not be possible in Europe due to different local prevalence of species and strains of Borrelia burgdorferi sensu lato and also heterogenecity within those strains.10 LB is both underdiagnosed and overdiagnosed, and we believe variability of symptoms is in close relationship to different Borrelia serotypes. B. burgdorferi sensu lato which has been subdivided into three genospecies: B. burgdorferi sensu stricto, B. afzelii and B. garinii. Some studies show that B. afzelii represents a dominant human skin isolate in Europe, B. garinii is mainly connected to neuroborreliosis and B. burgdorefri sensu stricto appears to be the major pathogen in Lyme arthritis. The other Borreliae (e.g. B. valaisiana) were not proved to cause the human disease. Further- GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 LYME BORRELIOSIS UP-TO-DATE HERCOGOVÁ more, ACA is connected with B. afzelii OspA serotype 2, infection with B. garinii OspA serotype 4 correlates with neuroboreliosis, and B. burgdorferi sensu stricto appears to play the major role in arthritis.11-13 There are still some unanswered questions concerning LB. We do not know, if co-infections with the other tick-born pathogens, especially Anaplasma phagocytophilum are important, and if B. burgdorferi could be terratogenic while the infection is present during pregnancy, how to treat and prevent the disease with its serious consequences. The agent of human granulocytic ehrlichiosis (HGE), A. phagocytophilum, was identified in the USA in 1994 and later in Europe. The vectors of HGE are nymphs and adults of I. ricinus tick.14, 15 Coinfection may alter the clinical manifestation and response to treatment of LB and therefore they should be considered in differential diagnosis when evaluating persons who are at the risk for tick-borne diseases. Certain clinical features (e.g. trombocytopenia or leukopenia) which are not typical for LB should suggest these coinfections. Transmission of the agents of LB and HGE by individual ticks is equally efficient and independent. Most dually infected ticks are able to transmit both pathogens to a susceptible host.16 Borrelia infections during pregnancy were considered dangerous, more recent studies have refused some of these fears. A causal association with Borrelia infection was not proven in any infant born to 105 mothers with EM during pregnancy, however, two abortions and six preterm babies, including one who had cardiac abnormalities and two who died shortly after delivery were observed.17 Problematic effect of LB treatment is based on possible chronic character of infection, non-sufficient knowledge on pharmacodynamic interactions of antimicrobial agents with Borreliae, documented failure of therapy. Currently, beta-lactams, macrolides and tetracyclines are used. The duration of treatment (minimum 14 days) and the daily dose depends on the LB stage and the clinical manifestations. New antimicrobial agents (fluoroquinolones, ketolids) are under evaluation.18 Some studies show that a one dose prophylaxis with doxycycline (200 mg) could decrease the risk of LB transmission after a tick-bite.19 Recommendations to prevent LB include avoiding exposure to tick bites by limiting outdoor activities in tick-infested locations, using tick repellents, tucking in clothing and frequent skin inspection for early detection and correct removal of ticks. Antibiotic prophylaxis has Vol. 140 - N. 4 not been shown to be effective and no vaccination available in Europe until nowadays. References 1. McGinley-Smith DE, Tsao SS. Dermatoses from ticks. J Am Acad Dermatol 2003;49:363-92. 2. Sood SK. Lyme disease. Pediatr Inf Dis J 1999;18:913-25. 3. Hercogová J, Brzonǒvá I. Lyme disease in central Europe. Curr Opin Infect Dis 2001;14:133-7. 4. Hercogová J. Borrelia burgdorferi: a protagonist in Lyme disease, a bystander in morphoea? J Eur Acad Dermat Ven 2002;16:98-9. 5. Roggero E, Zucca E, Mainetti C, Bertoni F, Valsangiacomo C, Pedrinis E et al. Eradication of Borrelia burgdorferi infection in primary marginal zone B-cell lymphoma of the skin. Hum Pathol 2000;31:263-8. 6. Trevisan G, Rees DHE, Stinco G. Borrelia burgdorferi and localized scleroderma. Clin Dermatol 1994; 12: 475-9. 7. Weide B, Walz T, Garbe C. Is morphea caused by Borrelia burgdorferi? A review. Br J Dermatol 2000;142:636-44. 8. Xu Y, Bruno JF, Luft BJ. Detection of genetic diversity in linear plasmids 28-3 and 36 in Borrelia burgdorferi sensu stricto by subtractive hybridization. Mikrob Pathol 2003;25:269-78. 9. Zore A, Ruzic-Sabljic E, Maraspin V, Cimperman J, Lotric-Furlan S, Pikelj A et al. Sensitivity of culture and polymerase chain reaction for the etiologic diagnosis of erythema migrans. Wien Klin Wochenschr 2002;114:606-9. 10. Robertson J, Guy E, Andrews N, Wilske B, Anda P, Granstrom M et al. A European multicenter study of immunoblotting in serodiagnosis of Lyme borreliosis. J Clin Microbiol 2000;38:2097-102. 11. Manconi RT, Hohenberger S, Jauris-Heipke S. Genetic analysis of Borrelia garinii OspA serotype 4 strains associated with neuroborreliosis: evidence for extensive genetic homogeneity. J Clin Microbiol 1999;37:3965-70. 12. Ornstein K, Berglund J, Nilsson I, Norrby R, Bergstrom S. Characterization of Lyme borreliosis isolates from patients with erythema migrans and neuroborreliosis in southern Sweden. J Clin Microbiol 2001;39:1294-8. 13. Luneman JD, Krause A. Heterogenita von Borrelia burgdorferi: Atiopathogenetische Relevanz und Klinische Implikationen. Z Rheumatol 2003;62:148-54. 14. Hulínská D, Votýpka J, Plch J, Vlcek E, Valesova M, Bojar M et al. Molecular and microscopical evidence of Ehrlichia spp. and Borrelia burgdorferi sensu lato in patients, animals and ticks in the Czech Republic. Microbiologica 2002;25:437-48. 15. Santino I, Del Piano M, Sessa R, Favia G, Iori A. Detection of four Borrelia burgdorferi genospecies and first report of human granulocytic ehrlichiosis agent in Ixodes ricicnus ticks collected in central Italy. Epidemiol Infect 2002;129:893-97. 16. Levin ML, Fish D. Acquisition of coinfection and simultaneous transmission of Borrelia burgdorferi and Ehrlichia phagocytophila by Ixodes scapularis ticks. Infect Immun 2000;68:2183-6. 17. Maraspin V, Cimperman J, Lotric-Furlan S, Pleterski-Rigler D, Strle F. Erythema migrans in pregnancy. Wien Klin Wochenschr 1999;111:933-40. 18. Hunfeld KP, Kraiczy P, Kekoukh E. Standardised in vitro susceptibililty testing of Borrelia burgdorferi aganist well-known and newly developed antimicrobial agents. Possible implications for new therapeutic approaches to Lyme disease. Int J Med Microbiol 2002;291 Suppl 33:125-37. 19. Nadelman RB, Nowakowski J, Fish D, Falco RC, Freeman K, McKenna D et al. Prophylaxis with single-dose doxycycline for the prevention of Lyme disease after an Ixodes scapularis tick bite. N Engl J Med 2001;345:79-84. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 323 G ITAL DERMATOL VENEREOL 2005;140:325-7 Photodynamic therapy. When and how? L. R. BRAATHEN Photodynamic therapy Dermatological University Clinic, Inselspital Bern, Switzerland T he term photodynamic therapy (PDT) includes the presence of a photosensitizer in the tissue which is then activated by light to produce reactive oxygen species in particular singlet oxygen which then damage and kill the cells.1 In Europe the only registered drug for topical PDT is the methyl aminolevulinate (MAL) which is present in a 16% concentration in the cream Metvix®. MAL is taken-up by the highly active cancer cells which then produce large amounts of photosensitive porphyrins, above all protoporphyrin IX, which then makes the cell photosensitive to red light. By illuminating the tissue singlet oxygen is produced which then damages and kills the cells. Because MAL has a high cancer tissue selectivity it can also be used for diagnostic purposes, by illuminating the MAL-treated area 3 h after application with blue light the cancer tissue demonstrates red/pink fluorescence and thus clearly delineating the tumor tissue. This procedure, called fluorescence detection, enables dermatologists to perform guided biopsies or guided tumor resections. Using a newly developed technical system using a camera and digital images one can document the findings before treatment and also when applied after the successful treatment to demonstrate the efficacy of the treatment.1 IN THIS ISSUE SEE PAGE 381 Address reprint requests to: L. R. Braathen, MD, PhD, MHA, Dermatological University Clinic, Inselspital, 3010 Bern, Switzerland. E-mail: [email protected] Vol. 140 - N. 4 Topical photodynamic therapy procedure Before application of the MAL cream (Metvix®) one should remove hyperkeratosis or crusts with gentle abrasion. Nodular basal cell carcinomas (BCCs) more than 3 mm thick should be debulked. Bleeding can be stopped by compression with a physiological saline-soaked cloth or gaze. After the bleeding has stopped the cream is applied to the lesion under occlusion for 3 h. It is also possible to leave it on for several hours more. Thereafter, the occlusive dressing is removed, the cream is wiped off and the lesion is illuminated with high intensity red light, which using lamps with light emission diodes (LED) technology takes 8-10 min. Most patients have light or moderate pain mainly during the illumination, a few have stronger pain which need to be treated. Spraying water to the treated area during the illumination helps as does also an experienced nurse talking calmly to the patient during the illumination. Treatment of basal cell carcinoma MAL-PDT for BCCs has been extensively studied over the last years. In one study Solèr et al. studied the long-term effects on MAL-PDT in 59 patients with 350 BCCs. The patients were followed for 2-4 years (mean GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 325 BRAATHEN PHOTODYNAMIC THERAPY. WHEN AND HOW? 35 months) and had an overall cure rate of 79% with a recurrence rate of 11% at 35 months and cosmetic outcome excellent or good in 98% of the completely responding lesions.2 Another multicenter study investigated MAL-PDT in patients with superficial and/or nodular BCCs. Clinical remission rate 3 months after treatment was 92% for superficial and 87% for nodular BCCs.3 Compiled data from several trials demonstrated complete clearance rate of 87% for superficial BCCs and 71% for nodular BCCs.4 Overall one can conclude that MAL-PDT is an efficient therapy which can also be repeated if recurrence occurs. In all studies a good to excellent cosmetic result was reported. Actinic keratosis MAL-PDT of actinic keratosis (AK) has been investigated in several prospective studies and compared with cryotherapy. Randomized multicenter prospective studies of MAL-PDT compared to cryotherapy have been performed in Europe and Australia. The complete response rates with PDT in these studies were 69% and 91% as compared to 68% and 75% for cryotherapy. All studies demonstrated an excellent or good cosmetic outcome of PDT in close to a 100% of the patients.5, 6 PDT is especially well suited for larger areas with many AKs, i.e. field cancerization areas. Bowen’s disease and incipient squamous cell carcinoma In the largest existing study of treatment of Bowen’s disease MAL-PDT was compared with cryotherapy and 5-fluorouracil (5-FU) in a controlled European multicenter study (40 centers). A total of 225 patients with 275 lesions were included in the study and MALPDT induced a complete response in 93% of lesion compared to 86% with cryotherapy and 83% with 5FU. After 12 months the overall lesion cure rate was 74% with MAL-PDT, 65% with cryotherapy and 62% with 5-FU.7 Methyl aminolevulinate-photodynamic therapy in field cancerization areas Field cancerization areas are usually sun exposed areas, e.g. scalp, face, ears, dorsal aspects of hands, and 326 décolleté which have clinical signs of seriously sun damaged skin and recurrent AKs, BCCs, and spinocellular carcinomas (SCC). These patients have, when being treated with cryotherapy or surgery, often multiple scars and white spots from previous treatments. MAL-PDT is very well suited for treating such larger areas. Organ transplanted and immunosuppressed patients These patients demonstrates increased skin cancer frequency. They should be regularly seen by dermatologists and their AKs, BCCs, and SCCs should be treated at an early stage. This is especially important for the SCCs to prevent metastases. We include all transplanted patients with immunosuppressive treatment in a skin care program including surveillance of their skin and treatment of their non-melanoma skin cancer lesions. Conclusions PDT is rapidly evolving to become a routine therapy in dermatology. It is well documented through controlled studies and thus evidence based with efficacy comparable to other commonly used standard treatments. The British Photodermatology Group has already published guidelines for topical PDT.8 The advantages of PDT includes simultaneous treatment of larger areas with multiple lesions; relatively short healing period and high patient preference because of the excellent cosmetic outcome. PDT can also be repeated in the same area if needed. References 1. Szeimies RM, Karrer S, Abels C, Landthaler M, Elmets CA. Photodynamic therapy in dermatology. In: Krutmann J, Hönigsmann H, Elmets CA, Bergstresser PR editors. Dermatological phototherapy and photodiagnostic methods. Berlin: Springer, 2001. p. 209-47. 2. Solèr AM, Warloe T, Berner A, Giercksky KE. A follow-up study of recurrence and cosmesis in completely responding superficial and nodular basal cell carcinomas treated with methyl 5-aminolaevulinate-based photodynamic therapy alone and with prior curettage. Br J Dermatol 2001;145:467-71. 3. Horn M, Wolf P, Wulf HC, Warloe T, Fritsch C, Rhodes LE et al. Topical methyl aminolevulinate photodynamic therapy in patients with basal cell carcinoma prone to complications and poor cosmetic outcome with conventional treatment. Br J Dermatol 2003;149: 1242-9. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 PHOTODYNAMIC THERAPY. WHEN AND HOW? 4. Zeitouni NC, Oseroff AR, Shieh S. Photodynamic therapy for nonmelanoma skin cancers. Mol Immunol 2003;39:1133-6. 5. Szeimies RM, Karrer S, Radakovic-Fijan S, Tanew A, Calzavara-Pinton PG, Zane C et al. Photodynamic therapy using topical methyl 5aminolevulinate compared with cryotherapy for actinic keratosis: a prospective randomized study. J Am Acad Dermatol 2002;47:25862. 6. Freeman M, Vinciullo C, Francis D, Spelman L, Nguyen R, Fergin P et al. A comparison of photodynamic therapy using topical methyl aminolevulinate with single cycle cryotherapy in patients with actinic Vol. 140 - N. 4 BRAATHEN keratosis: a prospective, randomized study. J Dermatol Treat 2003;14:99-106. 7. Morton C, Horn M, Leman J, Tack B, Bédane C, Tjioe M et al. A placebo controlled European study comparing MAL-PDT with cryotherapy and 5-fluorouracil in patients with Bowen’s disease. J Eur Acad Dermatol Venereol 2004;18 Suppl 2:415. 8. Morton CA, Brown SB, Collins S, Ibbotson S, Jenkinson H, Kurwa H et al. Guidelines for topical photodynamic therapy: report of a workshop of the British Photodermatology Group. Br J Dermatol 2002;146:552-67. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 327 GUIDELINES G ITAL DERMATOL VENEREOL 2005;140:329-47 Guidelines in dermoscopy S. CHIMENTI 1, G. ARGENZIANO 2, A. DI STEFANI 1, L. ANDREASSI 3, P. CARLI 4, V. DE GIORGI 4, G. FERRARA 5, A. FERRARI 6, S. GASPARINI 7, G. L. GIOVENE 8, M. LOMUTO 9, G. MAZZOCCHETTI 10, G. PELLACANI 11, R. PELLICANO 9, K. PERIS 6, D. PICCOLO 6, M. A. PIZZICHETTA 12, P. RUBEGNI 3, M. SCALVENZI 13, S. SEIDENARI 11, S. SERRESI 14, I. STANGANELLI 15, B. GIANNOTTI 4 T he guidelines that we propose reflect the state of the art in dermoscopy at the time the report was prepared. Results of in-progress study might require some changes to the conclusions or recommendations reported in the following. Adherence to these guidelines should always take into consideration care and conscientiousness in the interpretation of dermoscopic criteria. The final outcome is to establish the most accurate preoperative diagnosis and the most proper management, in light of all the circumstances presented by the individual patient. Guidelines can never replace individual medical responsibility. The significance of dermoscopy Dermoscopy is a non-invasive technique widely used in daily practice for the early diagnosis of melanoma.1 It has been reported that clinical examination alone is 65-80% sensitive in the diagnosis of melanoma.2 A recent systematic review of the literature demonstrated that dermoscopy improves the diagnostic accuracy of melanoma up to 35% compared to the naked eye.3 This diagnostic improvement can be achieved only if the observer has a good degree of Address reprint requests to: Prof. S. Chimenti, Clinica di Dermatologia, Università degli Studi di Roma Tor Vergata, PTV-Policlinico di Tor Vergata, Viale Oxford 81, 00133 Roma (Italy). E-mail: [email protected] Vol. 140 - N. 4 1Department of Dermatology Università degli Studi di Roma Tor Vergata, Rome 2Department of Dermatology 2nd University of Naples, Naples 3Department of Dermatology, University of Siena, Siena 4Department of Dermatology, University of Florence, Florence 5Department of Pathology, Ospedale G. Rummo, Benevento 6Department of Dermatology, University of L’Aquila, L’Aquila 7Private practice, Terni 8Private practice, Perugia 9Department of Dematology, S. Giovanni Rotondo 10Department of Dermatology, P.O. di Lanciano 11Department of Dermatology, University of Modena e Reggio Emilia Modena e Reggio Emilia 12Department of Oncology Centro di Riferimento Oncologico, Aviano 13Divion of Dermatology, University Federico II, Naples 14Divion of Dermatology, I.N.R.C.A. Hospital, Ancona 15Department of Oncology and Dermatology CPO, Ravenna and Niguarda Hospital, Milan experience in dermoscopy, otherwise for the untrained or less experienced examiners diagnostic accuracy can decrease as compared to the naked eye.4, 5 Therefore, adequate training is necessary for the effective application of this technique.6-8 In addition, the value of formal dermoscopy teaching courses conducted by a qualified and expert staff should be highlighted. Furthermore, the inclusion of dermoscopy training with- GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 329 CHIMENTI GUIDELINES IN DERMOSCOPY dermoscopic criteria otherwise not visible with the naked eye.11 100 90 80 70 Impact of dermoscopy in the clinical management of pigmented skin lesions % 60 50 40 30 20 10 0 Specificity Clinical examination ABCD Sensitivity Pattern analysis Combined approach 7-point checklist Figure 1.—Specificity and sensitivity for melanoma diagnosis of clinical examination and of the different diagnostic algorithms. in teaching programs for residents in dermatology could be recommended. Integration of clinical and dermoscopic examination Integration of dermoscopy in the context of physical examination has been shown to improve the preoperative diagnosis of melanoma (Figure 1). In 1996 Menzies et al. 9 demonstrated that 9 out of 107 (8%) melanomas did not have any melanoma-specific dermoscopic criteria and were reported as featureless melanomas. Such lesions were excised only on the basis of recent changes to the lesions as observed by the patients. Because the great importance of the clinical evolution of the lesion, the letter E (= evolution of the lesion) has been added to the ABCD rule of dermoscopy.10 Moreover, the percentage of correctly diagnosed melanomas is higher for in vivo dermoscopy (face to face with the patient) compared with dermoscopy performed on slide images of the same cases.11 This also implies that some clinical parameters such as the age and skin phototype of the patient, number and characteristics of the other nevi, location and history of the lesion, can be crucial for increasing diagnostic accuracy. In the presence of any suspicious clinical data the dermatologist may focus attention on slight or less noticeable 330 The role of dermoscopy in the clinical management of pigmented skin lesions (PSL), has been recently evaluated. In order to verify whether this technique may decrease the number of surgical excisions of benign PSL, a series of lesions consecutively excised and histologically diagnosed were retrospectively evaluated both clinically and dermoscopically.12 The results showed that, although the sensitivity for melanoma was 90%, all malignant skin neoplasms (melanomas and basal cell carcinomas, BCC) were correctly classified as equivocal lesions to be excised. In addition, 40% of clinically suspicious (false positives) PSL, were correctly diagnosed as benign lesions by dermoscopic examination thus avoiding unnecessary surgery. Therefore, the use of dermoscopy to establish whether a PSL should be biopsied or not allows us to significantly improve the clinical diagnosis and management of PSL.12 An algorithm for the management of PSL showing a combined clinical and dermoscopic approach is reported in Figure 2. This algorithm was designed on the basis of the mean activity of a reference center for PSL screening and early diagnosis of melanoma. Two-step procedure for dermoscopic diagnosis Recently a diagnostic method for the dermoscopic diagnosis of PSL was validated and proposed as standard at the Consensus Net Meeting on Dermoscopy (CNMD). The CNMD, organized in 2000 via the Internet between 40 experts from 14 countries, had the objective to investigate some important issues in dermoscopy such as the better definition and standardization of dermoscopic terminology, and the reproducibility and validity of the different criteria and diagnostic algorithms.13 This diagnostic method in dermoscopy is based on a two-step procedure. The first step aims to differentiate melanocytic and non melanocytic PSL. The criteria to define a specific lesion of a melanocytic nature are: pigmented network, brown globules, streaks, homogeneous blue GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 GUIDELINES IN DERMOSCOPY CHIMENTI ~65% PSL = CLINICAL EXAMINATION PSL (approximately 1 melanoma out of 100 patients observed) Diagnosis of benign lesion ~35% clinically EQUIVOCAL lesions Follow-up in selected cases DERMOSCOPY (Pattern analysis, ABCD, 7-Point) Suspicious for Melanoma COMBINED APPROACH: at least 1 diagnosis (clinical or dermoscopic) of melanoma 40% of clinically equivocal lesions: diagnosis of benign lesion EXCISION Follow-up in selected lesions Figure 2.—Algorithm for the management of pigmented skin lesions (PLS). Data related to the mean activity of a reference center for melanoma screening. pigmentation and parallel pattern.13 If none of those criteria can be identified, one should recognize the presence of criteria for the diagnosis of seborrheic keratosis (milia-like cysts, comedo-like openings, fingerprint-like structures, cerebriforme pattern with fissures and ridges), BCC (arborizing vessels, leaf-like structures, large blue-gray ovoid nests, multiple bluegray globules, spoke-wheel areas and ulceration), and vascular lesions (red-blue lacunas, red-bluish to reddish-black homogeneous areas).14 In the absence of any of the above mentioned criteria, the pattern is defined as non-specific and should be considered suspicious for the diagnosis of melanocytic lesion.13 The second step is useful for differentiating benign melanocytic lesions from melanoma, and includes different diagnostic algorithms: modified pattern analysis,14, 15 ABCD rule,16 Menzies method,9 and sevenpoint checklist.17 According to the results of the CNMD, all diagnostic methods exhibited high sensitivity in the diagnosis of melanoma, although pattern analysis has shown a significantly higher specificity as compared to the other algorithms. However, it should be emphasized that pattern analysis requires a higher degree of experience in dermoscopy.13 Recently the Vol. 140 - N. 4 three-point checklist has been proposed to allow nonexpert dermoscopists to increase their sensitivity to melanoma diagnosis (albeit with a decrease in specificity).18 The three-point checklist is a simplified method based on the evaluation of only 3 dermoscopic criteria: asymmetry of the lesion, the presence of an atypical pigmented network and of blue-white structures (defined as the presence of any type of blue and/or white color). The three-point checklist could represent a dermoscopic method for melanoma screening also in the hands of non-experts.18 Melanoma-specific dermoscopic criteria In recent years, several studies have demonstrated the validity and reproducibility of certain dermoscopic criteria, significantly associated to melanoma diagnosis, and therefore defined as melanoma-specific criteria 13-15, 19-28 (Table I). Of the various global features, the multicomponent pattern, defined as the combination of 3 or more dermoscopic patterns in a given PSL, was the most predictive for the diagnosis of melanoma.13 The globular, cobblestone, homoge- GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 331 CHIMENTI GUIDELINES IN DERMOSCOPY TABLE I.—Melanoma-specific dermoscopic criteria showing a statistically predictive value for melanoma diagnosis, and corresponding histopathologic substrates.13-15, 19-28 Melanoma-specific dermoscopic criteria Multicomponent pattern Atypical pigment network Irregular streaks Regression structures Irregular dots/globules Irregular blotches Blue-whitish veil Asymmetry Dermoscopic description Combination of 3 or more dermoscopic patterns in a given PSL Black, brown or gray network, with irregular holes and thick lines, sharply interrupted at the periphery of the lesion Irregular bulbous or linear structures, irregularly distributed at the edge of the lesion, not clearly associated with pigment network lines White scar-like areas and/or blue pepperinglike granules. Usually corresponding to a clinically flat part of the lesion Black, brown, round to oval variously sized structures, irregularly distributed within the lesion Association with melanoma diagnosis (Odds ratio) 13 4.3 Thickened and irregularly broadened rete ridges; a loss of rete ridges may occur with the progression of the melanoma Confluent junctional nests of melanocytes 9,3 Thickened papillary dermis with fibrosis and/or variable amounts of melanophages Pigment aggregates within the stratum corneum (or even a sign of pagetoid invasion of the epidermis) / nests of melanocytes at the dermo-epidermal junction or papillary dermis Black, brown and/or gray structureless areas Hyperpigmentation throughout the epidermis with irregular shape and asymmetrical and/or upper dermis distribution within the lesion Irregular structureless area of a confluent blue Acanthotic epidermis with focal hypergranupigmentation with an overlying whitish losis above sheets of heavily pigmented “ground-glass” film, usually corresponmelanocytes in the dermis ding to a clinically elevated part of the lesion Asymmetry in shape of the lesion and in distribution of colors and structures (as calculated by both ABCD rule and Menzies method) 5.4 neous, and starburst pattern were highly predictive of a diagnosis of benign melanocytic lesions.13 Atypical pigment network, irregular streaks and regression structures were the local features that showed the highest association with melanoma, followed by irregular dots/globules, irregular blotches, and a bluewhitish veil.13 In contrast, the typical pigment network, regular dots/globules, regular streaks and regular blotches were associated with benign melanocytic lesions.13 Structural asymmetry of the lesion, as assessed by the ABCD rule or Menzies method, was also significantly associated with melanoma.13 All melanoma-specific criteria have a well defined histopathological substrate (Table I) and their presence should always be investigated in a PSL. The observation of melanoma-specific criteria is almost always sufficient to decide to surgically excise and histopathologically examine a given lesion. 332 Histopathologic substrates 5.8 4.8 4.1 2.9 13.7 (asymmetry on both axes to ABCD rule) 43.8 (asymmetry ac-cording to Menzies method) The problem of false negatives and equivocal lesions False negatives in dermoscopy mainly include melanomas which are misdiagnosed and, therefore, not referred for surgical excision. False negative melanomas may essentially simulate a benign melanocytic lesion such as Clark nevus and Spitz/Reed nevus, or may lack any distinctive dermoscopic criteria (featureless melanoma), or shows hypo- or no pigmentation.9, 14, 29 From a practical point of view, when a lesion is clinically suspicious and, at first glance, is dermoscopically benign, it is crucial to perform an accurate dermoscopic examination in order to identify any subtle atypical features. However, the diagnosis of melanoma should always be suspected when a lesion shows a non-specific pattern. In addition, clinical history is an essential integration to dermoscopy GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 GUIDELINES IN DERMOSCOPY CHIMENTI when featureless lesions are diagnosed.10 In hypopigmented lesions the detection of an atypical vascular pattern (milky-red areas/globules, linear-irregular vessels or a combination of dotted and linear-irregular vessels) 29, 30 can be highly suggestive of melanoma, especially if associated with other criteria such as irregular blotches, dots/globules, regression structures or a blue-whitish veil.30 In cases of pink or amelanotic lesions, vascular patterns alone may not be sufficient for the diagnosis of melanoma, and should be integrated with clinical information such as age, sex, personal or family history of melanoma, number and sites of lesions, time of onset and description of any changes over time. A combined approach (dermoscopic examination and clinical data) may help in the early detection of amelanotic melanoma.30 Moreover, a percentage of lesions that are equivocal by clinical and dermoscopic examination, may still be equivocal after histopathologic examination.31 The limit between benign and malignant lesions is not clear for lesions such as junctional Clark nevi (and melanoma in situ) or atypical Spitz/Reed nevi (and spitzoid melanomas). Skin lesions within this “gray zone” should be surgically excised or followed up closely (1-3 months) by dermoscopy in order to detect a potential asymmetric enlargement or changes in dermoscopic features.32, 33 Recently a new dermoscopic classification of Clark nevi has been proposed to select specifically those lesions which should be surgically excised.34 An eccentric peripheral hyperpigmentation or the presence of 3 different structures in a given lesion (reticular, globular and homogeneous pattern) were significantly more frequently found in malignant than in benign melanocytic lesions and this implies the surgical removal of the lesion.35 A model of possible natural evolution over time has been described with sequential dermoscopic examinations for spitzoid lesions: from a globular pattern to a starburst pattern,36 and subsequently the disappearance of streaks at the periphery of the lesion and the presence of a central homogeneous pigmentation.37, 38 In a percentage of spitzoid lesions the detection of a superficial black network (that histopathologically corresponds to focal areas of pigmented parakeratosis, producing a black reticulated appearance on the horizontal plane) can be useful for the diagnosis of benignancy.39 The age of the patient is an important clinical parameter for the management of Vol. 140 - N. 4 these lesions: surgical excision should be performed for any spitzoid lesion occurring in adult patients, while in a child, a spitzoid lesion showing typical dermoscopic features could be dermoscopically monitored over time.40 Melanocytic skin lesions showing features of regression (blue-white structures such as white scar-like depigmentation and blue pepper-like granules) may be difficult to classify clinically and dermoscopically. A recent study of dermoscopic-pathologic correlation on clinically equivocal melanocytic lesions with blue-white structures demonstrated that the majority of nevi with regression exhibited blue areas with a central distribution and involving <50% of the lesion surface, while histopathologic equivocal lesions revealed a combination of white and blue areas with an irregular distribution and involving >50% of the lesion surface.41 Based on those results, an algorithm was proposed that can be applied to the management of lesions exhibiting dermoscopic features of regression: lesions showing a low degree (<10%) of blue-white structures can be dermoscopically monitored over time; in contrast, lesions with a high degree of regression (>50%) or with a moderate degree (between 10% and 50%) of regression along with the presence of a combination of blue and white areas should be surgically excised and histopathologically examined.41 Dermoscopic follow-up and modification during time of pigmented skin lesions There are 2 main reasons why a patient must be periodically examined: first, some patients have a higher risk of developing a melanoma (i.e. personal or family history of melanoma, total nevus count, skin phototype I-II); second, to monitor the possible evolution of single only moderately atypical lesions (not suspicious for melanoma) over time. In addition, dermoscopic follow-up is useful in patients with multiple nevi, often clinically atypical, which would be practically impossible to remove simultaneously.1, 42, 43 In a recent study the characteristics of growing melanocytic nevi were described: in a series of 1 612 common nevi, 5% showed an enlargement in a mean follow-up period of 12 months.44 Dermoscopy revealed a peripheral symmetric rim of brown globules in 50% of enlarging nevi, due to the junctional activity of melanocytes. Although this phenomenon was more common in the under-20s, symmetrical enlargement alone (without any other atypical feature) did not indi- GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 333 CHIMENTI GUIDELINES IN DERMOSCOPY cate malignancy, as demonstrated by histopathological examination of these lesions.44 However, enlarging lesions in adults, especially if showing a peripheral rim of brown globules, should be carefully monitored over time (3 months dermoscopic follow-up) or surgically excised.45 Other studies demonstrated the efficacy of dermoscopic follow-up in detecting patterns of modification of PSL over time. 46 Atypical nevi showed focal enlargement without substantial structural dermoscopic changes. In contrast, melanomas showed focal enlargement associated with a change in shape as well as appearance of dermoscopic structures such as irregular black dots, atypical network, regression structures, irregular streaks or a blue-whitish veil.47 The detection of changes in dermoscopic criteria (i.e. extension or loss of pigment network, distribution or number of black dots and/or hypopigmented areas or regression structures), in a dermoscopic follow-up examination, should suggest the surgical excision of the lesion. Similarly, the appearance of atypical dermoscopic features such as irregular black dots, atypical network, regression structures, irregular streaks, a blue-whitish veil and atypical vascular pattern, are indicative of a suspicious lesion to be removed. Moreover, Menzies et al.32 demonstrated that a short term follow-up (median 3 months) of 318 only moderately atypical lesions revealed 7 early dermoscopically featureless melanomas. Such melanomas did not show any atypical dermoscopic criteria but could be identified only by morphologic changes over a short period of time.32 Remarkably there are some risks in performing a dermoscopic follow-up of atypical lesions. In a recent study, some authors 48 claimed that the uncritical use of sequential imaging cannot be recommended, since the usefulness of this technique depends on the experience in the interpretation of follow-up images and on the patient’s compliance with a scheduled followup program over time. The selection of patients and lesions submitted for follow-up examination must be carefully performed in order to avoid the risk of leaving a melanoma unexcised.33 Recently Carli et al.49 demonstrated in a randomized study on 938 subjects, that dermoscopic follow-up of equivocal lesions is associated with a reduction in the number of PSL excised for diagnostic verification but also with a non-negligible occurrence of initial melanomas left unexcised. 334 Furthermore, concerning the dermoscopic modification of melanocytic lesions, the effects of ultraviolet irradiation, including increased pigmentation and irregular distribution of the pigment, increased dimension of brown globules, decrease of hypopigmented areas and less visibility of the pigment network are well known.50-53 Sun-induced morphological changes are transient and presumably related to activating melanocytes.50-53 Several studies emphasize the need to re-examine the lesions 4-6 weeks after sun exposure, since the differentiation from melanoma can be difficult in the period following ultraviolet irradiation.50-53 In general, dermoscopic follow-up examination over time should be performed only for moderately atypical lesions, which are not elevated on the skin surface, with no melanoma-specific features or history of changes. Nodular lesions showing atypical features should never be submitted to a dermoscopic follow-up, since it is not possible to rule out the diagnosis of nodular melanoma. In such cases surgical excision is mandatory. Main dermoscopic features of the most difficult pigmented skin lesions for clinical management and the differentiation with melanoma The dermoscopic detection of melanoma-specific criteria, as discussed in a previous section, should always lead to surgical excision of a given lesion. In Table II 13, 14, 30, 34-36, 38, 40, 41, 54-70 the main dermoscopic features of the most difficult PSL that commonly represent false negatives or false positives, and some clues for an accurate differential diagnosis and a better clinical management are reported. In Clark nevi, as already mentioned, the detection of an eccentric peripheral hyperpigmentation or the presence of reticular, globular and homogeneous structures in the same lesion is important to recommend surgical excision, because the same characteristics can frequently be found in melanoma.34, 35 If a patient shows multiple lesions with those dermoscopic features, it is reasonable to excise the most atypical ones and to perform a short term digital follow-up in the others.32, 34 For nevi showing dermoscopic features of regression a model for the management is reported in Table II.41 Spitz/Reed nevi (spindle and/or epithelioid cell nevi, including the pigmented variant, previously consid- GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 GUIDELINES IN DERMOSCOPY CHIMENTI TABLE II.—Main dermoscopic features of the most difficult PSL (equivocal lesions and false negatives) with clues for differential diagnosis and clinical management. Skin lesions Equivocal lesions 1) Clark nevus with eccentric hyperpigmentation 2) Clark nevus with regression 3) Spitz/Reed nevus False negatives 1) Melanoma simulating the following PSL: — Clark nevus — Spitz/Reed nevus — Dermal nevus — Pigmented lesions of the face — Acral nevus — Ungueal lesions — Labial and genital melanosis — Blue nevus — Congenital nevus — Recurrent nevus — Irritated nevi — Reticulated lentigo — Basal cell carcinoma — Seborrheic keratosis — Vascular lesions 2) Melanoma with non-specific pattern 3) Amelanotic melanoma 4) Melanoma metastasis Vol. 140 - N. 4 Main dermoscopic features and management recommendations Excision if solitary lesion; short term follow-up (3 months) if multiple lesions with this dermoscopic feature in the same patient 32, 34, 35 Excision if regression >50% of the lesion surface; follow-up if regression <10%; excision if regression 10-50% together with the a combination of blue and white areas 41 Excision in adult patients,40 short term digital follow-up if typical dermoscopic features in children 36, 38 Short term follow-up (3 months) if multiple atypical lesions in the same patient;32, 34, 35 excision if solitary lesion even if slightly atypical. Excision if simultaneous presence of reticular, globular and homogeneous structures 35 Excision in adult patients,40 short term digital follow-up if typical dermoscopic features in children 36, 38 Differential criteria to evaluate: — cobblestone pattern, comma-like vessels and hair favor a dermal nevus 14 — asymmetry, blue-whitish veil, irregular dots/globules and atypical vascular pattern favor a melanoma, also with the presence of a cobblestone pattern 13 Dermoscopic follow-up in nodular lesion should be avoided. Diagnostic criteria to evaluate: — annular-granular structures — asymmetric pigmented follicular openings — rhomboidal structures 54 surgical excision or incisional biopsy in suspicious (blue-gray) areas in broad lesions Surgical excision of the lesions showing:70 — parallel-ridge pattern — atypical or multicomponent pattern Incisional biopsy if:55-57 — brown background and longitudinal brown to black lines, irregular in shape, coloration, thickness and parallelism. — micro-Hutchinson sign Dermoscopic follow-up in doubtful lesions 56 Incisional biopsy in lesions with variegated coloration and irregular distribution of the pigment 58 Excision of nodular lesions Excision when dermoscopically atypical or clinical history unclear 59 Rule out a melanoma metastasis 60 Dermoscopic follow-up by dermoscopic images of:61, 62 — entire lesion if possible — representative areas of the architectural pattern — borders — special interest areas Excision or incisional biopsy of suspicious lesions Atypical dermoscopic features (irregular streaks and dots/globules),63 definite clinical history is essential if re-excise or not the lesion Close follow-up (1-2 weeks); excision in unsolved cases Thickened, dark brown to black pigmented network, with irregular and asymmetric mashes.64 Excision in equivocal lesions Arborizing vessels, leaf-like areas, large blue-gray ovoid nests, multiple blue-gray globules, spoke wheel areas and ulceration, with the absence of pigment network 65, 66. Surgical excision is the elective treatment — evaluate the number of milia-like cysts and comedo-like openings: often numerous in seborrheic keratosis, few in melanoma 67 — excision in suspicious lesions, showing false pigment network and/or pseudo-globular structures 67-69 — Haemangioma: well circumscribed red lacunas (must be differentiated from milky red areas (less defined) in melanoma 14 — Pyogenic granuloma: excision and histopathologic exam in adults Excision of the lesion in the absence of specific dermoscopic criteria 14 Suspicious dermoscopic criteria in pink lesions requiring excision 29, 30 — nodular ulcerated lesion — pigment remnants, especially if blue-gray in color — milky red areas — atypical vascular pattern (dotted and/or linear-irregular vessels) — homogeneous bluish pigmentation — diffuse non-homogeneous brown-bluish pigmentation — red-bluish globular pattern 60 GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 335 CHIMENTI GUIDELINES IN DERMOSCOPY ered as a distinctive entity named Reed nevus) can dermoscopically show different dermoscopic patterns: starburst, globular, reticular, homogeneous, hypopigmented and atypical.37 Often these lesions can hardly be differentiated from a spitzoid melanoma, either dermoscopically or histopathologically. Consequently, any spitzoid lesion in adult patients should be surgically excised 40 while in childhood, if the spitzoid lesion shows typical dermoscopic features, could be closely followed-up over time, avoiding unnecessary excisions of benign lesions.36, 38 Dermal nevi (Unna and Miescher nevi) are usually characterized by a homogeneous pigmentation (that on the face appears as a pigment pseudonetwork), by a cobblestone pattern and comma-like vessels.14 In addition, dermal nevi may show exophytic papillary structures and irregular crypts.14 The detection of asymmetry, a blue-whitish veil, irregular dots/globules and atypical vascular pattern is suspicious of melanoma.13 When a nodular lesion is observed, a follow-up is never recommended in the presence of even a minimal diagnostic suspicion. Pigmented lesions of the face show a characteristic pigment pseudonetwork, due to the distribution of the pigment around follicular ostia.14 Criteria to diagnose a lentigo maligna include: annular-granular structures (of blue-gray color) and asymmetric pigmented follicular openings.54 Rhomboidal structures and homogeneous pigmented areas of follicular invasion are indeed associated with progression to an invasive melanoma (lentigo maligna melanoma).54 Surgical excision should always be performed when these features are observed, while an incisional (punch) biopsy can be performed in large lesions, preferentially within the bluish black areas. Acral nevi (nevi of the palms and soles) show a characteristic parallel pattern due to the disposition of the pigment along the sulci. The parallel-furrows pattern, the lattice-like pattern and the fibrillar pattern are typical of benign acral melanocytic lesions while the parallel-ridge pattern is associated with acral lentiginous melanoma.70 Lesions showing atypical or multicomponent patterns should be surgically excised and histopathologically examined. Ungueal lesions should be distinguished from subungueal hemorrhage, melanocytic nevi and druginduced longitudinal melanonychia. Subungueal hemorrage is characterized by a clinical history of trauma and dermoscopically by roundish sharply 336 demarked black-reddish areas and by blood spots or purple-to-black dots. Melanocytic nevi show a brown coloration of the background and the presence of regular lines in shape and parallelism, and drug-induced longitudinal melanonychia reveals a grayish coloration of the background and the presence of regular thin lines.55 When a diagnosis can not be made with certainty, a close dermoscopic follow-up can be useful to establish the final diagnosis.56 Subungueal melanoma dermoscopically shows a brown background and longitudinal brown to black lines, irregular in shape, coloration, thickness and parallelism. Important is the detection of micro-Hutchinson sign (pigmentation of the periungueal skin and cuticle, visible only at dermoscopic examination) that, although rarely detected, is suspicious of melanoma.55, 57 In equivocal lesions, an incisional biopsy, including the matrix, is required. Labial and genital melanosis are benign pigmented lesions dermoscopically characterized by a diffuse background pigmentation with a granular or globular (often with aligned globules) or linear-curvilinear, frequently parallel, intensification of the pigment, light brown to brown or grayish in coloration.71 Dermoscopic follow-up of lesions showing even a slightly irregular pigmentation is recommended. In equivocal lesions showing variegated coloration and irregular distribution of the pigment, dermoscopy can help to identify the most atypical zone in order to perform an incisional biopsy at the diagnostic site.58 Blue nevi are easily identified for their typical homogeneous blue pigmentation, common locations on the extremities and without history of changes. In some cases, characterized by yellow-whitish areas, related to the presence of fibrosis, the main differential diagnosis is melanoma. Because of the important significance of blue structures in dermoscopy, a careful examination of these lesions is always recommended and a biopsy is suggested when lesions are dermoscopically atypical or the clinical history is unclear.59 Furthermore, melanoma metastasis may simulate a blue nevus.60 Dermoscopy can also be useful to further characterize and follow up congenital nevi, although their dermoscopic features are quite variable. The cobblestone pattern is the most common, followed by a multicomponent pattern consisting of multiple colors, dots/globules, pigmented network, hypopigmented areas and homogeneous blue areas.61 Recently, other GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 GUIDELINES IN DERMOSCOPY CHIMENTI peculiar features have been described such as target network, target globules and target vessels.62 A careful examination of a sequential digital clinical image of the entire lesion, integrated with a dermoscopic image representative of the architectural pattern, borders, and areas of special interest, may make it possible to identify the appearance of atypical features.61 In such case, small congenital nevi should be excised, while in large or giant congenital nevi, an incisional biopsy in dermoscopically suspicious areas is recommended. Recurrent nevi (or persistent nevi) usually exhibit such bizarre and atypical features to be suspicious for melanoma, including irregular streaks and irregular dots/globules close to or in the context of the scar.63 Recurrent nevi following incomplete excision of a histopathologically non atypical lesion can be monitored over time. When anamnestic data or previous histopathologic diagnosis are not available or not clear, the lesion must be excised. Another example of the usefulness of a dermoscopic follow-up is the irritated nevi (mostly by trauma or infections), also named Meyerson nevi:72 a close digital monitoring (1-2 weeks) after local treatment can solve the diagnostic doubt. Reticulated lentigo (or ink spot lentigo) is usually located in severe sun damaged skin and may mimic a melanoma in situ. Dermoscopically it is characterized by a thickened, dark brown to black pigmented network, with irregular and asymmetric mashes which are uniformly distributed throughout the lesion.64 In equivocal lesions a biopsy is recommended. BCC especially if pigmented, may simulate a melanoma. Dermoscopic hallmarks of BCC are: arborizing vessels, leaf-like areas, large blue-gray ovoid nests, multiple blue-gray globules, spoke wheel areas and ulceration, in the absence of a pigment network.65, 66 Seborrheic keratosis, mainly the acanthotic and pigmented variants, may mimic a melanoma. Typical dermoscopic features include: milia-like cysts, comedolike openings, fingerprint-like structures and a cerebriforme pattern with fissures and ridges. Recently other dermoscopic criteria have been described such as sharp demarcation and moth-eaten borders,68 above all in the early reticulated type of seborrheic keratosis arising from a solar lentigo. A false pigment network, especially of the reticulated type, and pseudo-globular structures can sometimes be observed.68, 69 A sur- Vol. 140 - N. 4 gical excision is suggested in equivocal lesions to rule out the possibility, though rare, of a melanoma simulating a seborrheic keratosis.67 Dermoscopic examination of vascular lesions allows us to detect specific criteria and to differentiate with accuracy vascular lesions from melanoma. Haemangioma is characterized by a lacunar pattern, composed of numerous red to red-bluish ovoid, sharply circumscribed areas, called red lacunas. These structures must be distinguished from the less defined milky-red areas, that can be sometimes but specifically seen in melanoma.14 The lacunar pattern is hardly recognized in a pyogenic granuloma, therefore a biopsy and histopathologic confirmation are recommended in adult patients. Angiokeratoma exhibits red-bluish to black lacunas, associated with whitish-yellowish keratotic areas.14 Subcorneal hemorrhage dermoscopically shows a blackish homogeneous area and also a pseudo-parallel or pseudoglobular pattern. Technological standard in dermoscopy The hand-held dermatoscope and digital videodermatoscope represent the most widely used instruments for dermoscopic examination.1-3, 14 In some dermatology centers, a stereomicroscope implemented by digital system such as a high resolution digital camera (3CCD) are employed.3, 52, 53 The current standard tool for dermoscopic photographic documentation of PSL is the Dermaphot (Heine Optotechnik, Herrsching, Germany), consisting of a special designed lens on a camera with high resolution power and optimal image quality.73-75 However, a large number of instruments and systems for digital videodermoscopy are currently commercially available, achieving an image quality not always comparable to the standard.76, 77 We hope that in the near future the companies will conform to the standard, possibly through the institution of a specific committee for the evaluation and validation of videodermoscopy systems. Video-dermoscopic report controversies At the moment, there are no precise regulations nor published papers regarding this topic. In the present we would suggest a proposal of standardization of the video-dermoscopic report. Considering together GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 337 CHIMENTI GUIDELINES IN DERMOSCOPY medico-legal and deontological matters, and aiming a full statement of dermoscopy as a second-level instrumental examination, it is evidently important to issue a suitable report after providing a specialized service. Hence, in every video-dermoscopic report, we suggest including the following points (minimal criteria): — Symmetry/asymmetry of the lesion. — Global pattern. — Local features. — Diagnostic conclusion. — Management recommendation. Although the different video-dermoscopy systems have some limits in the standard of image quality and resolution, printing quality etc., we believe that a printed dermoscopic image of the lesion should be given to the patient at the dermatologist’s discretion, specifying current technological problems. References 1. Argenziano G, Soyer HP. Dermoscopy of pigmented skin lesions-a valuable tool for early diagnosis of melanoma. Lancet Oncol 2001;2:443-9. 2. Wolf IH, Smolle J, Soyer HP, Kerl H. Sensitivity in the clinical diagnosis of malignant melanoma. Melanoma Res 1998;8:425-9. 3. Kittler H, Pehamberger H, Wolff K, Binder M. Diagnostic accuracy of dermoscopy. Lancet Oncol 2002;3:159-65. 4. Binder M, Schwarz M, Winkler A, Binder M. Epiluminescence microscopy. A useful tool for the diagnosis of pigmented skin lesions for formally trained dermatologists. Arch Dermatol 1995;131:286-91. 5. Piccolo D, Smolle J, Argenziano G, Wolf IH, Braun R, Cerroni L et al. Teledermoscopy--results of a multicentre study on 43 pigmented skin lesions. J Telemed Telecare 2000;6:132-7. 6. Binder M, Puespoeck-Schwarz M, Steiner A, Kittler H, Muellner M, Wolff K et al. Epiluminescence microscopy of small pigmented skin lesions: short-term formal training improves the diagnostic performance of dermatologists. J Am Acad Dermatol 1997;36: 197-202. 7. Carli P, Quercioli E, Sestini S, Stante M, Ricci L, Brunasso G et al. Pattern analysis, not simplified algorithms, is the most reliable method for teaching dermoscopy for melanoma diagnosis to residents in dermatology. Br J Dermatol 2003;148:981-4. 8. Pagnanelli G, Soyer HP, Argenziano G, Talamini R, Barbati R, Bianchi L et al. Diagnosis of pigmented skin lesions by dermoscopy: web-based training improves diagnostic performance of non-experts. Br J Dermatol 2003;148:698-702. 9. Menzies SW, Ingvar C, Crotty KA, McCarthy WH. Frequency and morphologic characteristics of invasive melanomas lacking specific surface microscopic features. Arch Dermatol 1996;132:1178-82. 10. Kittler H, Seltenheim M, Dawid M, Pehamberger H, Wolff K, Binder M. Morphologic changes of pigmented skin lesions: a useful extension of the ABCD rule for dermatoscopy. J Am Acad Dermatol 1999;40:558-62. 11. Carli P, De Giorgi V, Giannotti B. Dermoscopy as a second step in the diagnosis of doubtful pigmented skin lesions: how great is the risk of missing a melanoma? J Eur Acad Dermatol Venereol 2001;15: 24-6. 338 12. Argenziano G, Soyer HP, Chimenti S, Ruocco V. Impact of dermoscopy on the clinical management of pigmented skin lesions. Clin Dermatol 2002;20:200-2. 13. Argenziano G, Soyer HP, Chimenti S, Talamini R, Corona R, Sera F et al. Dermoscopy of pigmented skin lesions: results of a consensus meeting via the Internet. J Am Acad Dermatol 2003;48:679-93. 14. Argenziano G, Soyer HP, De Giorgi V, Piccolo D, Carli P, Delfino M et al. Interactive atlas of dermoscopy. Milan: Edra Medical Publishing and New Media; 2000. 15. Pehamberger H, Binder M, Steiner A, Wolff K. In vivo epiluminescence microscopy: improvement of early diagnosis of melanoma. J Invest Dermatol 1993;100:356S-62S. 16. Nachbar F, Stolz W, Merkle T, Cognetta AB, Vogt T, Landthaler M et al. The ABCD rule of dermatoscopy. High prospective value in the diagnosis of doubtful melanocytic skin lesions. J Am Acad Dermatol 1994;30:551-9. 17. Argenziano G, Fabbrocini G, Carli P, De Giorgi V, Sammarco E, Delfino M. Epiluminescence microscopy for the diagnosis of doubtful melanocytic skin lesions. Comparison of the ABCD rule of dermatoscopy and a new 7-point checklist based on pattern analysis. Arch Dermatol 1998;134:1563-70. 18. Soyer HP, Argenziano G, Zalaudek I, Corona R, Sera F, Talamini R et al. Three-point checklist of dermoscopy. A new screening method for early detection of melanoma. Dermatology 2004;208:27-31. 19. Nilles M, Boedeker RH, Schill WB. Surface microscopy of naevi and melanomas--clues to melanoma. Br J Dermatol 1994;130:349-55. 20. Soyer HP, Smolle J, Leitinger G, Rieger E, Kerl H. Diagnostic reliability of dermoscopic criteria for detecting malignant melanoma. Dermatology 1995;190:25-30. 21. Steiner A, Pehamberger H, Wolff K. In vivo epiluminescence microscopy of pigmented skin lesions. II. Diagnosis of small pigmented skin lesions and early detection of malignant melanoma. J Am Acad Dermatol 1987;17:584-91. 22. Argenziano G, Fabbrocini G, Carli P, De Giorgi V, Delfino M. Epiluminescence microscopy: criteria of cutaneous melanoma progression. J Am Acad Dermatol 1997;37:68-74. 23. Pizzichetta MA, Argenziano G, Talamini R, Piccolo D, Gatti A, Trevisan G et al. Dermoscopic criteria for melanoma in situ are similar to those for early invasive melanoma. Cancer 2001;91: 992-7. 24. Soyer HP, Kenet RO, Wolf IH, Kenet BJ, Cerroni L. Clinicopathological correlation of pigmented skin lesions using dermoscopy. Eur J Dermatol 2000;10:22-8. 25. Massi D, De Giorgi V, Soyer HP. Histopathologic correlates of dermoscopic criteria. Dermatol Clin 2001;19:259-68, vii. 26. Argenziano G, Fabbrocini G, Carli P, De Giorgi V, Delfino M. Clinical and dermatoscopic criteria for the preoperative evaluation of cutaneous melanoma thickness. J Am Acad Dermatol 1999;40:61-8. 27. De Giorgi V, Carli P. Dermoscopy and preoperative evaluation of melanoma thickness. Clin Dermatol 2002;20:305-8. 28. Stante M, Carli P, Massi D, De Giorgi V. Dermoscopic features of naevus-associated melanoma. Clin Exp Dermatol 2003;28:476-80. 29. Carli P, Massi D, De Giorgi V, Giannotti B. Clinically and dermoscopically featureless melanoma: when prevention fails. J Am Acad Dermatol 2002;46:957-9. 30. Pizzichetta MA, Talamini R, Stanganelli I, Puddu P, Bono R, Argenziano G et al. Amelanotic/hypomelanotic melanoma: clinical and dermoscopic features. Br J Dermatol 2004;150:1117-24. 31. Ferrara G, Argenziano G, Soyer HP, Corona R, Sera F, Brunetti B et al. Dermoscopic and histopathologic diagnosis of equivocal melanocytic skin lesions: an interdisciplinary study on 107 cases. Cancer 2002;95:1094-100. 32. Menzies SW, Gutenev A, Avramidis M, Batrac A, McCarthy WH. Short-term digital surface microscopic monitoring of atypical or changing melanocytic lesions. Arch Dermatol 2001;137:1583-9. 33. Carli P, De Giorgi V, Giannotti B. Dermoscopy and early diagnosis of melanoma: the light and the dark. Arch Dermatol 2001;137: 1641-4. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 GUIDELINES IN DERMOSCOPY CHIMENTI 34. Hofmann-Wellenhof R, Blum A, Wolf IH, Piccolo D, Kerl H, Garbe C et al. Dermoscopic classification of atypical melanocytic nevi (Clark nevi). Arch Dermatol 2001;137:1575-80. 35. Blum A, Soyer HP, Garbe C, Kerl H, Rassner G, Hofmann-Wellenhof R. The dermoscopic classification of atypical melanocytic naevi (Clark naevi) is useful to discriminate benign from malignant melanocytic lesions. Br J Dermatol 2003;149:1159-64. 36. Pizzichetta MA, Argenziano G, Grandi G, De Giacomi C, Trevisan G, Soyer HP. Morphologic changes of a pigmented Spitz nevus assessed by dermoscopy. J Am Acad Dermatol 2002;47:137-9. 37. Peris K, Ferrari A, Argenziano G, Soyer HP, Chimenti S. Dermoscopic classification of Spitz/Reed nevi. Clin Dermatol 2002;20: 259-62. 38. Piccolo D, Ferrari A, Peris K. Sequential dermoscopic evolution of pigmented Spitz nevus in childhood. J Am Acad Dermatol 2003;49: 556-8. 39. Argenziano G, Soyer HP, Ferrara G, Piccolo D, Hofmann-Wellenhof R, Peris K et al. Superficial black network: an additional dermoscopic clue for the diagnosis of pigmented spindle and/or epithelioid cell nevus. Dermatology 2001;203:333-5. 40. Argenziano G, Scalvenzi M, Staibano S, Brunetti B, Piccolo D, Delfino M et al. Dermatoscopic pitfalls in differentiating pigmented Spitz naevi from cutaneous melanomas. Br J Dermatol 1999;141: 788-93. 41. Zalaudek I, Argenziano G, Ferrara G, Soyer HP, Corona R, Sera F et al. Clinically equivocal melanocytic skin lesions with features of regression: a dermoscopic-pathological study. Br J Dermatol 2004;150:64-71. 42. Malvehy J, Puig S. Follow-up of melanocytic skin lesions with digital total-body photography and digital dermoscopy: a two-step method. Clin Dermatol 2002;20:297-304. 43. Robinson JK, Nickoloff BJ. Digital epiluminescence microscopy monitoring of high-risk patients. Arch Dermatol 2004;140: 49-56. 44. Kittler H, Seltenheim M, Dawid M, Pehamberger H, Wolff K, Binder M. Frequency and characteristics of enlarging common melanocytic nevi. Arch Dermatol 2000;136:316-20. 45. Rhodes AR. Common acquired nevomelanocytic nevi and the fourth dimension. Arch Dermatol 2000;136:400-5. 46. Braun RP, Lemonnier E, Guillod J, Skaria A, Salomon D, Saurat JH. Two types of pattern modification detected on the follow-up of benign melanocytic skin lesions by digitized epiluminescence microscopy. Melanoma Res 1998;8:431-7. 47. Kittler H, Pehamberger H, Wolff K, Binder M. Follow-up of melanocytic skin lesions with digital epiluminescence microscopy: patterns of modifications observed in early melanoma, atypical nevi, and common nevi. J Am Acad Dermatol 2000;43:467-76. 48. Kittler H, Binder M. Risks and benefits of sequential imaging of melanocytic skin lesions in patients with multiple atypical nevi. Arch Dermatol 2001;137:1590-5. 49. Carli P, De Giorgi V, Chiarugi A, Nardini P, Weinstock MA, Crocetti E et al. Addition of dermoscopy to conventional naked-eye examination in melanoma screening: a randomized study. J Am Acad Dermatol 2004;50:683-9. 50. Hofmann-Wellenhof R, Wolf P, Smolle J, Reimann-Weber A, Soyer HP, Kerl H. Influence of UVB therapy on dermoscopic features of acquired melanocytic nevi. J Am Acad Dermatol 1997;37: 559-63. 51. Hofmann-Wellenhof R, Soyer HP, Wolf Ich, Smolle J, Reischle S, Rieger E et al. Ultraviolet radiation of melanocytic nevi: a dermoscopic study. Arch Dermatol 1998;134:845-50. 52. Stanganelli I, Rafanelli S, Bucchi L. Seasonal prevalence of digital epiluminescence microscopy patterns in acquired melanocytic nevi. J Am Acad Dermatol 1996;34:460-4. 53. Stanganelli I, Bauer P, Bucchi L, Serafini M, Cristofolini P, Rafanelli S et al. Critical effects of intense sun exposure on the expression of epiluminescence microscopy features of acquired melanocytic nevi. Arch Dermatol 1997;133:979-82. Vol. 140 - N. 4 54. Schiffner R, Schiffner-Rohe J, Vogt T, Landthaler M, Wlotzke U, Cognetta AB et al. Improvement of early recognition of lentigo maligna using dermatoscopy. J Am Acad Dermatol 2000;42: 25-32. 55. Ronger S, Touzet S, Ligeron C, Balme B, Viallard AM, Barrut D et al. Dermoscopic examination of nail pigmentation. Arch Dermatol 2002;138:1327-33. 56. Tosti A, Argenziano G. Dermoscopy allows better management of nail pigmentation. Arch Dermatol 2002;138:1369-70. 57. Baran R, Kechijian P. Hutchinson’s sign: a reappraisal. J Am Acad Dermatol 1996;34:87-90. 58. De Giorgi V, Massi D, Carli P. Dermoscopy in the management of pigmented lesions of the oral mucosa. Oral Oncol 2003;39:534-5. 59. Massi D, De Giorgi V, Carli P, Santucci M. Diagnostic significance of the blue hue in dermoscopy of melanocytic lesions: a dermoscopicpathologic study. Am J Dermatopathol 2001;23:463-9. 60. Ferrari A, Peris K, Piccolo D, Chimenti S. Dermoscopic features of cutaneous local recurrent melanoma. J Am Acad Dermatol 2000;43:722-4. 61. Braun RP, Calza AM, Krischer J, Saurat JH. The use of digital dermoscopy for the follow-up of congenital nevi: a pilot study. Pediatr Dermatol 2001;18:277-81. 62. Seidenari S, Martella A, Pellacani G. Polarized light-surface microscopy for description and classification of small and mediumsized congenital melanocytic naevi. Acta Derm Venereol 2003;83: 271-6. 63. Marghoob AA, Kopf AW. Persistent nevus: an exception to the ABCD rule of dermoscopy. J Am Acad Dermatol 1997;36:474-5. 64. Kaddu S, Soyer HP, Wolf IH, Rieger E, Kerl H. Reticular lentigo. Hautarzt 1997;48:181-5. 65. Menzies SW. Dermoscopy of pigmented basal cell carcinoma. Clin Dermatol 2002;20:268-9. 66. Peris K, Altobelli E, Ferrari A, Fargnoli MC, Piccolo D, Esposito M et al. Interobserver agreement on dermoscopic features of pigmented basal cell carcinoma. Dermatol Surg 2002;28:643-5. 67. Argenziano G, Rossiello L, Scalvenzi M, Staibano S, Ruocco E, Cicale L et al. Melanoma simulating seborrheic keratosis: a major dermoscopy pitfall. Arch Dermatol 2003;139:389-91. 68. Braun RP, Rabinovitz HS, Krischer J, Kreusch J, Oliviero M, Naldi L et al. Dermoscopy of pigmented seborrheic keratosis: a morphological study. Arch Dermatol 2002;138:1556-60. 69. De Giorgi V, Massi D, Stante M, Carli P. False “melanocytic” parameters shown by pigmented seborrheic keratoses: a finding which is not uncommon in dermoscopy. Dermatol Surg 2002;28:776-9. 70. Saida T, Oguchi S, Miyazaki A. Dermoscopy for acral pigmented skin lesions. Clin Dermatol 2002;20:279-85. 71. Gasparini S, Giovene GL, Ferranti G. Trattato di Dermoscopia. Milan: Spriger-Verlag Italia; 2003. 72. Worret WI. Halo eczema and nevus cell nevi (Meyerson nevi). Hautarzt 1990;41:262-4. 73. Carli P, de Giorgi V, Salvini C, Mannone F, Chiarugi A. The gold standard for photographing pigmented skin lesions for diagnostic purposes: contact versus distant imaging. Skin Res Technol 2002;8:255-9. 74. Kittler H, Seltenheim M, Pehamberger H, Wolff K, Binder M. Diagnostic informativeness of compressed digital epiluminescence microscopy images of pigmented skin lesions compared with photographs. Melanoma Res 1998;8:255-60. 75. Bittorf A, Fartasch M, Schuler G, Diepeng TL. Resolution requirements for digital images in dermatology. J Am Acad Dermatol 1997;37: 195-8. 76. Abramovits W, Stevenson LC. Changing paradigms in dermatology: new ways to examine the skin using noninvasive imaging methods. Clin Dermatol 2003;21:353-8. 77. Oliveria SA, Sachs D, Belasco KT, Halpern AC. Adoption of new technologies for early detection of melanoma in dermatologic practice. J Am Acad Dermatol 2003;49:955-9. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 339 CHIMENTI GUIDELINES IN DERMOSCOPY Linee guida in dermatoscopia L e linee guida proposte in questo articolo riflettono lo stato dell’arte in dermoscopia al momento in cui sono state stilate. È, quindi, probabile che i risultati di studi in corso possano determinare alcune modifiche delle definizioni e delle indicazioni che verranno di seguito riportate. L’applicazione clinica di linee guida non deve prescindere dalla prudenza e dalla coscienziosità, che vanno sempre esercitate nell’interpretare i criteri dermoscopici. Gli obiettivi finali, che sono stabilire la diagnosi preoperatoria più accurata ed effettuare la scelta terapeutica appropriata, devono essere raggiunti considerando tutte le condizioni individuali del paziente in esame. Le linee guida non possono mai sostituire le responsabilità mediche individuali. ha un buon livello di esperienza nell’utilizzo della metodica, mentre l’accuratezza della diagnosi dermoscopica può risultare anche peggiore rispetto alla sola diagnosi clinica, per i non esperti 4, 5. Pertanto, un’adeguata preparazione è fondamentale ai fini di un’applicazione diagnostica realmente efficace 6-8. Da qui si può dedurre l’importanza di corsi di insegnamento formali sulla metodica, tenuti da personale qualificato ed esperto. Sembrerebbe auspicabile anche l’inserimento di un esame di dermoscopia all’interno delle scuole di specializzazione in Dermatologia e Venereologia. Integrazione fra clinica e dermoscopia Significato della dermoscopia La dermoscopia è una metodica diffusamente utilizzata nella pratica clinica per la diagnosi precoce del melanoma 1. Studi di valutazione sull’accuratezza diagnostica del solo esame clinico hanno mostrato che il dermatologo è in grado di individuare il melanoma nel 65-80% dei casi 2. Una recente revisione sistematica della letteratura ha dimostrato che la dermoscopia è in grado di incrementare la sensibilità diagnostica del melanoma del 10-35% rispetto alla sola osservazione clinica 3. È stato, inoltre, riportato che tale miglioramento diagnostico può essere ottenuto solo se l’osservatore 100 90 80 70 % 60 50 40 30 L’integrazione della dermoscopia nel contesto della valutazione clinica globale del paziente si è dimostrata capace di migliorare ulteriormente la diagnosi preoperatoria del melanoma (Figura 1). Nel 1996, Menzies et al. hanno riscontrato che 9 (8%) di 107 melanomi inclusi nel loro studio erano privi dei caratteri diagnostici dermatoscopici specifici e per tale motivo erano definiti «featureless». Per tali lesioni, l’asportazione venne effettuata solo sulla base di un cambiamento dell’aspetto clinico riferito dal paziente 9. Proprio in considerazione dell’importanza del criterio clinico evolutivo, è stato proposto un nuovo sistema ABCD che prevede l’inserimento di un criterio ulteriore, denominato E, relativo alla storia evolutiva della lesione in esame 10. È stato, inoltre, osservato che la percentuale di melanomi diagnosticati correttamente mediante l’osservazione dermoscopica effettuata dal vivo (faccia a faccia con il paziente) risulterebbe maggiore rispetto a quella ottenuta esaminando le immagini dermoscopiche, in diapositiva, dei medesimi casi di melanoma 11. Questo significa che esistono dei parametri clinici, quali l’età del paziente, il fototipo, il numero e la tipologia degli altri nevi, la sede della lesione, la storia evolutiva, ecc., che sono in grado di aumentare l’accuratezza della diagnosi finale. In presenza di dati clinici sospetti, il dermatologo sarà più stimolato all’attenta valutazione anche di parametri dermoscopici sfumati o appena percettibili, che altrimenti potrebbero sfuggire 11. 20 10 Impatto della dermoscopia sul management clinico 0 Specificità Clinica ABCD Sensibilità Analisi di pattern Approccio integrato 7-point Figura 1. — Valori di sensibilità e specificità per la diagnosi di melanoma relativi a differenti sistemi di valutazione a confronto. 340 In uno studio recente è stato valutato il ruolo della dermoscopia come ausilio nella gestione clinica delle lesioni pigmentate cutanee e, in particolare, quanto essa permetta di ridurre il numero di escissioni chirurgiche delle lesioni benigne 12. A questo scopo, una serie di lesioni, tutte asportate e istologicamente confermate, sono state retrospettivamente GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 GUIDELINES IN DERMOSCOPY CHIMENTI Diagnosi definitiva di BENIGNITÀ ~ 65% Lesioni = ESAME CLINICO Paziente (circa 1 melanoma ogni 100 pazienti osservati) ~ 35% lesioni clinicamente EQUIVOCHE Follow-up in selezionati DERMOSCOPIA (Analisi pattern, ABCD, 7-Point) Sospetto melanoma Approccio integrato: almeno 1 diagnosi (clinica o dermoscopica) del melanoma -40% delle lesioni equivoche: diagnosi definitiva di benignità (escissione evitata) ASPORTAZIONE Follow-up in casi selezionati Figura 2. — Sistema di gestione integrata delle lesioni pigmentate, elaborato sulla base dell’attività media di un Centro di Riferimento per lo screening delle lesioni pigmentate cutanee e la diagnosi dermoscopica precoce del melanoma. valutate dal punto di vista sia clinico che dermoscopico. I risultati hanno mostrato che, pur essendo la sensibilità diagnostica del melanoma del 90%, tutte le lesioni maligne (melanomi e carcinomi basocellulari) venivano comunque giudicate tali da essere asportate. Inoltre, il 40% delle lesioni clinicamente classificate come sospette (falsi positivi) venivano invece diagnosticate come lesioni benigne all’esame dermoscopico e, quindi, da non asportare. Ne consegue che, quando la dermoscopia viene utilizzata per stabilire se la lesione debba essere asportata o meno, questa metodica consente di migliorare significativamente in termini di specificità la gestione clinica delle lesioni pigmentate 12. Un sistema di gestione di lesioni pigmentate, elaborato sulla base dell’attività media di un centro di riferimento per lo screening delle lesioni pigmentate cutanee e la diagnosi precoce del melanoma e basato sull’approccio diagnostico integrato, è schematizzato nella Figura 2. Procedura in due fasi per la diagnosi dermoscopica Il metodo diagnostico per l’esame dermoscopico delle lesioni pigmentate è stato recentemente standardizzato e proposto come riferimento nel corso del Consensus Net Meeting on Dermoscopy (CNMD). Il CNMD, tenutosi nel 2000 via internet tra 40 esperti di 14 diversi Paesi, aveva come obiettivo stabilire alcune linee guida fondamentali in dermoscopia: definizione, standardizzazione e semplifica- Vol. 140 - N. 4 zione della terminologia, e verifica della riproducibilità e validità dei diversi criteri e degli algoritmi diagnostici 13. Il metodo diagnostico dermoscopico proposto consta di una procedura in 2 fasi. La prima fase consiste nel differenziare la natura melanocitica o non melanocitica della lesione pigmentata in questione. A tale proposito, dovranno, pertanto, essere identificati i criteri che consentono di definire una lesione come melanocitica, quali: reticolo pigmentato, globuli marroni, strie, pigmentazione blu omogenea, pattern parallelo. In assenza di tali aspetti verrà esaminata la presenza di criteri per le lesioni non melanocitiche e, in particolare, i criteri per la diagnosi di cheratosi seborroica (pseudocisti cornee, sbocchi simil-comedonici, strutture a impronta digitale, aree con aspetto cerebriforme con giri e solchi), carcinoma basocellulare (vasi arboriformi, aree a foglia d’acero, grandi aree ovoidali grigio-blu, multipli globuli grigio-blu, aree a ruota di carro e ulcerazione) o di lesioni vascolari (lacune rosso-blu, aree omogenee da rosso-bluastre a rosso nerastre) 14. Nel caso in cui anche questi criteri siano assenti, il pattern viene definito aspecifico e deve far, comunque, sospettare una diagnosi di lesione melanocitica 13. La seconda fase diagnostica prevede la differenziazione tra le lesioni melanocitarie benigne e il melanoma mediante l’applicazione di differenti algoritmi diagnostici: l’analisi di pattern modificata 14, 15, l’ABCD di Stolz 16, il metodo di Menzies 9 e il seven-point check-list 17. In base ai risultati del CNMD è stato osservato che tutti questi metodi assicurano una sensibilità elevata nella diagnosi di melanoma, ma che GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 341 CHIMENTI GUIDELINES IN DERMOSCOPY TABELLA I. — Criteri dermoscopici melanoma-specifici, che hanno mostrato un valore predittivo significativamente elevato per la diagnosi di melanoma, e loro correlati istopatologici 13-15, 19-28. Criteri dermoscopici melanoma-specifici Descrizione dermoscopica Pattern polimorfo (o mul- Combinazione di 3 o più strutture dermoscoticomponente) piche distinte in una stessa lesione Reticolo pigmentato atipico Caratterizzato da maglie irregolari e trama ispessita, brusca interruzione alla periferia, e di colore marrone-nerastro Strie irregolari (o pseudopodi) Strutture lineari di spessore variabile, irregolarmente distribuite, non chiaramente associate alle maglie del reticolo Strutture di regressione Associazione di aree bianche simil-cicatriziali, e di aree grigio-blu tipo «peppering» Punti deboli irregolari Strutture rotondeggianti di forma e dimensione irregolari e disomogeneamente distribuiti nel contesto della lesione Pigmetazione irregolare Velo blu-biancastro Asimmetria strutturale Associazione con la diagnosi di melanoma (Odds ratio) 13 4.3 Rate ridges irregolare e ispessita, un suo scompaginamento correla con la progressione del melanoma Teche di melanociti confluenti alla giunzione dermo-epidermica 9,3 Associazione di fibrosi e melanofagi a livello del derma papillare ispessito 5.4 5.8 4.8 Accumuli di melanina nello strato corneo (o anche segno di invasione pagetoide dell’epidermide) i primi, e nidi di melanociti situati alla giunzione o nel derma papillare i secondi 4.1 Aree pigmentate nere, marroni o grigie di for- Intensa pigmentazione melaninica distribuita ma e/o distribuzione irregolare a tutti i livelli dell’epidermide o nel derma superficiale 2.9 Pigmentazione diffusa e confluente, di colo- Epidermide acantosica con ipergranulosi focare variabile dal grigio-blu al blu-biancale che sovrasta teche di melanociti fortestro, associata a reticolo pigmentato mente pigmentati nel derma Asimmetria nella forma della lesione (ma 13,7 (asimmetria in 2 assi anche di colori e strutture dermoscopiche), secondo l’ABCD di Stolz) calcolata sia con il metodo dell’ABCD che 43,8 (asimmetria secondo con quello di Menzies il metodo di Menzies) l’analisi di pattern mostra una più elevata specificità rispetto agli algoritmi alternativi semplificati, anche se richiede una maggiore esperienza da parte dell’osservatore 13. Recentemente, è stato proposto il three-point checklist 18 per consentire anche ai dermoscopisti meno esperti di diagnosticare il maggior numero di melanomi (anche se con una diminuzione della specificità). Si tratta di un metodo semplificato basato sulla valutazione di soli 3 criteri dermoscopici: l’asimmetria della lesione, la presenza di reticolo pigmentato atipico e di strutture bianco-blu (definite come la presenza di qualsiasi struttura di colore blu e/o bianco). Il three-point checklist potrebbe rappresentare un efficace sistema dermoscopico di screening delle lesioni pigmentate, anche per dermoscopisti non esperti 18. Criteri dermoscopici melanoma-specifici Negli ultimi anni numerosi studi hanno dimostrato la validità e la riproducibilità di criteri dermoscopici che sono più frequentemente osservati nel melanoma e, per questo, sono definiti melanoma-specifici 13-15, 19-28 (Tabella I.) Il pattern 342 Correlati istopatologici dermoscopico globale che è risultato avere un valore predittivo maggiore riguardo al melanoma è stato quello polimorfo (o multicomponente), definito come combinazione di 3 o più strutture dermoscopiche distinte in una stessa lesione 13. Al contrario, i pattern globulare, ad acciottolato, omogeneo e «a stella che esplode», sono risultati maggiormente predittivi per la diagnosi di lesioni melanocitiche benigne 13. Per quanto riguarda i criteri dermoscopici locali, il reticolo pigmentato atipico, le strie irregolari e le strutture di regressione hanno mostrato il valore predittivo più elevato nei confronti del melanoma, seguite da punti e globuli irregolari, pigmentazione irregolare e velo blu-biancastro 13. Invece, il reticolo pigmentato tipico, i punti e i globuli regolari, le strie regolari e la pigmentazione regolare, sono risultati maggiormente associati con lesioni melanocitiche benigne 13. Anche l’asimmetria strutturale della lesione (calcolata sia con il metodo dell’ABCD che con quello di Menzies) è risultata statisticamente predittiva di malignità 13. Questi criteri melanoma specifici, espressione di alterazioni istopatologiche ben definite (Tabella I), vanno sempre accuratamente ricercati nel contesto di una lesione pigmentata e la loro osservazione giustifica il più delle volte l’escissione chirurgica e l’esame istologico. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 GUIDELINES IN DERMOSCOPY CHIMENTI Il problema dei falsi negativi e delle lesioni equivoche In dermoscopia, i falsi negativi comprendono i melanomi che non vengono diagnosticati e, quindi, non asportati chirurgicamente, e includono essenzialmente i melanomi che mimano lesioni melanocitiche benigne quali il nevo di Clark e il nevo di Spitz/Reed, oppure i melanomi «featureless», ossia lesioni che non mostrano criteri dermoscopici specifici o sono apigmentate 9, 14, 29. Dal punto di vista pratico, nel caso di lesioni clinicamente sospette ma dermoscopicamente apparentemente benigne, è importante esaminare accuratamente la lesione al fine di individuare possibili aspetti dermoscopici atipici. La diagnosi di melanoma deve, comunque, essere sempre sospettata quando la lesione mostra un pattern aspecifico. In questi casi, è, comunque, fondamentale integrare i dati dermoscopici con la storia clinica della lesione 10. In particolare, per le lesioni poco pigmentate, la ricerca di un pattern vascolare atipico (essenzialmente aree/globuli rossolattescenti, vasi lineari-irregolari o la combinazione vasi puntiformi e lineari-irregolari) 29, 30 può suggerire la diagnosi di melanoma se associato ai criteri quali pigmentazione irregolare, globuli/punti irregolari, strutture di regressione e velo bianco-bluastro 30. Per le lesioni «rosa» o nulla pigmentate, i pattern vascolari possono da soli non essere sufficienti a porre diagnosi di melanoma e vanno integrati con le informazioni cliniche quali età, sesso, familiarità per melanoma, numero delle lesioni, sede, epoca d’insorgenza e modificazioni nel tempo della lesione. L’approccio integrato (indagine dermoscopica-informazioni cliniche) può consentire di diagnosticare un melanoma amelanotico in uno stadio più precoce 30. Vi è, infine, una percentuale di lesioni melanocitiche per le quali è difficile o impossibile stabilire una diagnosi di benignità o malignità, dal punto di vista sia clinico sia dermoscopico, e, in alcuni casi, anche istopatologico 31. Appartengono a questo gruppo le lesioni melanocitiche in cui la diagnosi differenziale tra nevo di Clark di tipo giunzionale e melanoma in situ, o tra nevo di Spitz/Reed e melanoma spitzoide è particolarmente difficile. In questi casi, le lesioni dovrebbero essere asportate chirurgicamente o sottoposte a uno stretto monitoraggio dermoscopico (1-3 mesi) che permette di apprezzare un eventuale accrescimento asimmetrico della lesione stessa o modificazioni delle strutture dermoscopiche 32, 33. Recentemente è stata proposta una nuova classificazione dermoscopica dei nevi di Clark, utile nella selezione delle lesioni da sottoporre a escissione chirurgica 1. È stato osservato, infatti, che un’iperpigmentazione eccentrica (periferica) e la coesistenza di 3 strutture nell’ambito della stessa lesione (reticolare, globulare e omogenea) sono caratteristiche significativamente più frequenti nel melanoma; per tale motivo, queste lesioni dovrebbero essere asportate 35. Per le lesioni con aspetto spitzoide, è stato descritto un modello di possibile evoluzione naturale nel tempo: da un pattern globulare a un pattern a stella che esplode («starburst») 36, per poi andare incontro a una graduale scomparsa delle strie periferiche e a una pigmentazione centrale più diffusa e omogenea 37, 38. In una percentuale di casi, il riscon- Vol. 140 - N. 4 tro in una lesione spitzoide di un reticolo nero superficiale (che, istopatologicamente, corrisponde a focali aree di paracheratosi pigmentata, che producono un aspetto reticolato e nero sul piano orizzontale) può essere di ausilio nel porre diagnosi di benignità 39. Per le lesioni con aspetto spitzoide, l’età del paziente risulta, comunque, di fondamentale importanza: nei pazienti adulti esse vanno senz’altro asportate; nei pazienti pediatrici, se presentano pattern dermoscopici tipici, possono essere monitorate nel tempo 40. Infine le lesioni melanocitiche che dermoscopicamente possono essere di difficile interpretazione sono quelle che presentano strutture di regressione, quali aree bianche similcicatriziali e/o aree blu tipo peppering. Un recente studio di correlazione dermoscopico-patologica su lesioni melanocitiche clinicamente equivoche con caratteristiche di regressione ha evidenziato che la maggioranza dei nevi con regressione mostra aree blu che coinvolgono <50% della lesione e hanno una distribuzione prevalentemente centrale, mentre le lesioni istologicamente equivoche mostrano una combinazione di aree bianche e aree blu, irregolarmente distribuite, che coinvolgono >50% della lesione 41. In base a tali risultati, è stato proposto un algoritmo per la gestione clinica delle lesioni che dermoscopicamente presentano regressione: le lesioni che mostrano un basso grado di strutture di regressione (<10%) potrebbero essere sottoposte a monitoraggio dermoscopico, al contrario, l’escissione andrebbe sempre effettuata per le lesioni che mostrano un alto grado di regressione (>50%) o che presentano un grado moderato di strutture di regressione (compreso tra 10% e 50%) ma con la presenza contemporanea di aree bianche e di aree blu 41. Follow-up dermoscopico e modificazioni nel tempo delle lesioni pigmentate La necessità di sottoporre un paziente a esami clinici periodici deriva dalla duplice esigenza di monitorare un soggetto che presenta significativi fattori di rischio per lo sviluppo di un melanoma (e.g. storia personale o familiare di melanoma, elevato numero totale di nevi ecc.) e di osservare l’evoluzione nel tempo di singole lesioni melanocitiche moderatamente atipiche, ma non tali da sospettare un melanoma. Inoltre, il monitoraggio dermoscopico è particolarmente utile nei soggetti che presentano un elevato numero di nevi, molti dei quali clinicamente atipici, la cui asportazione contemporanea sarebbe praticamente impossibile 1, 42, 43. In uno studio recente sono state descritte le caratteristiche dermoscopiche dei nevi in accrescimento: su una casistica di 1 612 nevi melanocitici comuni, il 5% dei nevi ha mostrato un aumento delle dimensioni in un periodo di 12 mesi. Nel 50% di queste lesioni era possibile evidenziare un anello di globuli marroni simmetricamente distribuito alla periferia della lesione, espressione dell’attività proliferativa delle cellule neviche 44. Sebbene questo fenomeno fosse riscontrabile più comunemente in soggetti di età inferiore ai 20 anni, l’accrescimento simmetrico di una lesione melanocitaria (in GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 343 CHIMENTI GUIDELINES IN DERMOSCOPY assenza di altri segni di atipia) non è di per sé indicativo di malignità, come dimostrato dall’esame istologico condotto su queste lesioni 44. Lesioni in accrescimento in soggetti adulti, soprattutto se in presenza dell’anello globulare periferico, devono tuttavia essere attentamente seguite attraverso un monitoraggio digitale stretto (3 mesi) o asportate 45. Altri studi hanno dimostrato l’efficacia del monitoraggio digitale nell’individuare i pattern di modificazione delle lesioni 46. I nevi atipici hanno mostrato essenzialmente un accrescimento focale in assenza di importanti modifiche strutturali, mentre nei melanomi è stato rilevato un accrescimento focale, associato a modifiche della forma e comparsa di caratteri dermoscopici quali punti neri irregolari, rete pigmentaria irregolare, strutture di regressione, strie irregolari, velo blu-biancastro 47. Quando a un esame dermoscopico di follow-up si riscontrano modificazioni di criteri dermoscopici, quali espansione o riduzione del reticolo pigmentato, distribuzione o numero di punti neri, e/o aree di ipopigmentazione o di regressione, è sempre consigliabile l’asportazione chirurgica. Analogamente, la comparsa di ulteriori caratteri dermoscopici atipici, quali reticolo pigmentato atipico, punti neri irregolari, strutture di regressione, strie irregolari, velo blu-biancastro e pattern vascolare atipico, sono indicativi di lesione sospetta che deve essere escissa chirurgicamente. Inoltre Menzies et al. hanno dimostrato che, effettuando un follow-up a breve termine (in media 3 mesi) su 318 lesioni solo moderatamente atipiche, è stato possibile identificare ben 7 melanomi in stadio iniziale dermoscopicamente «featureless», cioè che non mostravano criteri dermoscopici atipici ma che la sola modificazione in un intervallo stretto di tempo ha permesso di diagnosticare 32. Quando si effettua un follow-up di lesioni atipiche, vi sono, comunque, alcuni rischi da non sottovalutare. A tale proposito è stato recentemente dimostrato che il ricorso indiscriminato al monitoraggio dermoscopico non è raccomandabile, in quanto la sua efficacia clinica dipende dall’esperienza dell’osservatore e dalla compliance del paziente e, quindi, la sua adesione a un programma di monitoraggio nel tempo 48. La scelta delle lesioni e dei pazienti da sottoporre a follow-up digitale va, dunque, sempre valutata attentamente per non rischiare di perdere un melanoma 33. Recentemente Carli et al. Hanno, infatti, dimostrato, in uno studio randomizzato effettuato su 938 pazienti, che l’archiviazione delle immagini dermoscopiche di lesioni equivoche si associa, da un lato, a una diminuzione di casi sottoposti a escissione chirurgica, dall’altro, a un rischio non trascurabile di melanomi iniziali non asportati 49. Infine, nel contesto delle variazioni dermoscopiche osservabili nelle lesioni melanocitiche, bisogna tener presenti quelle dovute a esposizioni alle radiazioni ultraviolette, che consistono in una maggiore pigmentazione e irregolarità nella distribuzione del pigmento, un incremento delle dimensioni dei globuli marroni, una diminuzione delle aree ipopigmentate e una minore visibilità del reticolo pigmentario 50-53. Questi cambiamenti morfologici sono, tuttavia, transitori e legati presumibilmente a un’attivazione reversi- 344 bile delle cellule neviche 50-53. Risulta necessario, quindi, esaminare nuovamente queste lesioni 4-6 settimane dopo l’esposizione solare, a causa della loro difficile differenziazione dal melanoma nel periodo immediatamente successivo alle esposizioni solari stesse 50-53. In generale, le lesioni che possono essere sottoposte a un monitoraggio digitale nel tempo sono quelle solo lievemente atipiche, piane e non rilevate e non devono avere una storia di variazioni morfologiche né presentare criteri melanoma-specifici. Il follow-up non dovrebbe mai essere eseguito nelle lesioni nodulari che presentano caratteri di atipia, data l’impossibilità di escludere con certezza una diagnosi di melanoma nodulare. In questi casi, infatti, è sempre consigliata l’asportazione chirurgica. Aspetti morfologici salienti e indicazioni per la gestione clinica delle lesioni pigmentate di più difficile interpretazione Precedentemente sono stati esaminati in dettaglio i criteri dermoscopici melanoma specifici, che, quando osservati in una lesione, devono far procedere a un’asportazione chirurgica. In Tabella II 13, 14, 32, 34-36, 38, 40, 41, 54-70 sono riportati gli aspetti dermoscopici delle lesioni pigmentate che più comunemente possono costituire dei falsi positivi o dei falsi negativi, nonché alcuni suggerimenti per un più accurato inquadramento diagnostico e per una migliore gestione delle lesioni che generano problemi di diagnosi differenziale. Per i nevi di Clark, come già accennato, è importante l’individuazione delle lesioni che presentano un’iperpigmentazione eccentrica (periferica) e delle lesioni in cui vi è la coesistenza di strutture reticolari, globulari e omogenee. Questi nevi dovrebbero, infatti, essere asportati chirurgicamente, in quanto le stesse caratteristiche possono riscontrarsi anche nel melanoma 34, 35. Quando un paziente presenta lesioni multiple con aspetti atipici si può ricorrere all’escissione di quella/e maggiormente sospetta/e ed effettuare un follow-up dermoscopico stretto (3 mesi) per le altre 32, 34. Un discorso a parte meritano le lesioni con regressione di cui viene proposto un modello di gestione in Tabella II 41. I nevi di Spitz/Reed (nevi a cellule epitelioidi e/o fusate, compresa la variante pigmentata, prima considerata distinta, detta nevo di Reed), come precedentemente accennato, possono presentarsi dermoscopicamente con una varietà di pattern: a stella che esplode, globulare, reticolare, omogeneo, ipopigmentato e atipico 37. Queste lesioni pongono spesso problemi di diagnosi differenziale con un melanoma spitzoide, dal punto di vista sia clinico-dermoscopico sia istologico. Per questo si consiglia l’escissione di qualsiasi lesione che nell’adulto mostri un aspetto spitzoide 40. Nei pazienti pediatrici, un nevo di Spitz/Reed che presenta aspetti tipici può essere sottoposto a uno stretto follow up, in modo da poter monitorare i diversi pattern di modificazione e evitare inutili asportazioni di lesioni benigne 36, 38. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 GUIDELINES IN DERMOSCOPY CHIMENTI TABELLA II. — Aspetti dermoscopici salienti delle lesioni pigmentate che più comunemente possono costituire dei falsi positivi o dei falsi negativi, e suggerimenti per una migliore gestione clinica. Lesioni pigmentate Falsi positivi 1) Nevo di Clark con iperpigmentazione eccentrica 2) Nevo di Clark con regressione 3) Nevo di Spitz Falsi negativi 1) Melanoma che simula una delle seguenti lesioni pigmentate: — Nevo di Clark — Nevo di Spitz — Nevo dermico — Lesioni pigmentate del volto — Lesioni in sede acrale — Lesioni in sede ungueale — Melanosi delle mucose — Nevo blu — Nevo congenito — Nevo ricorrente — Nevo irritato — Lentigo reticolare — Carcinoma basocellulare — Cheratosi seborroica — Lesioni vascolari 2) Melanoma con pattern aspecifico 3) Melanoma amelanotico 4) Metastasi di melanoma Vol. 140 - N. 4 Aspetti morfologici salienti e indicazioni Escissione se unico elemento; follow-up stretto (3 mesi) se più lesioni con aspetto simile nello stesso paziente 32, 34, 35 Escissione se regressione >50% della superficie lesionale; follow-up se regressione <10% lesione; escissione se regressione 10-50% ma presenza contemporanea di aree bianche e di aree blu 41 Escissione nell’adulto 40, nel bambino se aspetti tipici, stretto follow-up dermoscopico 36, 38 Follow-up a 3 mesi se in paziente con multiple lesioni atipiche 32, 34, 35; escissione se unico elemento anche se lievemente atipico. Escissione se contemporanea presenza di strutture reticolari, globulari e omogenee 35 Escissione di qualsiasi lesione spitzoide nell’adulto 40 nel bambino stretto follow-up di lesioni con aspetti tipici 36, 38 Criteri differenziali da valutare: — pattern ad acciottolato, vasi a virgola e peli sono in favore di un nevo dermico 14 — asimmetria, velo blu, punti/globuli irregolari e pattern vascolare atipico sono in favore di un melanoma anche in presenza di un pattern ad acciottolato 13. Mai follow-up in lesioni nodulari sospette Criteri diagnostici da valutare 54: — follicoli pigmentati asimmetrici — strutture anulari-granulari — strutture romboidali. Escissione chirurgica o biopsia incisionale in aree sospette (grigio-blu) se lesioni estese Escissione chirurgica delle lesioni che mostrano 70: — pattern a creste parallele — pattern atipici o multicomponenti Biopsia incisionale se si rinviene:55-57 — pigmentazione marrone di fondo e linee longitudinali da marroni a nere, irregolari — micro-segno di Hutchinson Follow-up negli altri casi meno dubbi 56 Biopsia incisionale se lesioni con distribuzione irregolare del pigmento e colore variegato 58 Asportazione delle lesioni nodulari Escissione se storia clinica e aspetti dermoscopici dubbi 59, mai follow-up in lesioni nodulari sospette Escludere una metastasi da melanoma 60 Follow-up attraverso immagini dermoscopiche di:61, 62 — intera lesione se possibile — zone rappresentative del pattern architetturale — bordi della lesione — zone di particolare interesse 61, 62 Escissione chirurgica delle lesioni sospette o biopsia incisionale Aspetti dermoscopici spesso atipici, fondamentale la storia clinica nel decidere se asportare o monitorare nel tempo 63 Follow-up stretto (anche 1-2 settimane). Escissione chirurgica nei casi che non si risolvono Reticolo pigmentato, prominente e molto scuro (anche nerastro) a maglie irregolari 64. Escissione chirurgica nelle lesioni sospette Ricercare la presenza di: vasi arboriformi, aree a foglia d’acero, grandi aree ovoidali grigio-blu, multipli globuli grigio-blu, aree a ruota di carro ed ulcerazione 65, 66 Asportazione chirurgica in ogni caso — valutare il numero di pseudocisti cornee e sbocchi simil-comedonici (numerosi nella cheratosi, pochi nel melanoma) 67 — escissione delle lesioni sospette che mostrano caratteristiche quali falso reticolo pigmentario e strutture pseudo-globulari 67-69 — Emangioma: differenziare le lacune blu-rosse (ben circoscritte nell’angioma) dalle milky red areas (non ben circoscritte nel melanoma) 14 — Granuloma piogenico: escissione ed esame istologico nell’adulto Escissione in assenza di criteri definiti per la diagnosi 14 Criteri diagnostici da valutare nelle lesioni rosa, che devono far propendere per un’escissione 29, 30 — lesione nodulare e/o ulcerata — residui di pigmento, specialmente se di colore blu-grigio — milky red areas (aree rosso lattescenti) — pattern vascolare atipico (vasi punteggiati e/o lineari-irregolari) — pigmentazione omogenea bluastra — pigmentazione diffusa non omogenea marrone-bluastra — pattern a globuli rosso-bluastri 60 GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 345 CHIMENTI GUIDELINES IN DERMOSCOPY I nevi dermici (Unna e Miescher) dermoscopicamente sono caratterizzati frequentemente da una pigmentazione omogenea (che a livello del volto assume l’aspetto di pseudoreticolo pigmentato), da un pattern ad acciottolato e da vasi disposti a virgola 14. Spesso presentano i caratteri dermoscopici tipici delle lesioni esofitiche: strutture papillari esofitiche e cripte irregolari 14. Tuttavia, il riscontro di asimmetria, velo blu-biancastro, punti e globuli irregolari e pattern vascolare atipico, deve far sospettare un melanoma 13. Non è mai consigliabile ricorrere a un follow-up in lesioni nodulari quando si ha anche solo un minimo dubbio diagnostico. Le lesioni pigmentate del volto si presentano all’esame dermoscopico con il caratteristico pseudoreticolo pigmentato, dovuto alla distribuzione del pigmento attorno agli osti follicolari 14. I criteri che permettono di sospettare una lentigo maligna sono la presenza di strutture anulari-granulari (di colorito grigio-bluastro) e la pigmentazione asimmetrica degli sbocchi follicolari 54. Le strutture romboidali e le aree omogenee di invasione dei follicoli indicano, invece, una fase più avanzata di progressione del melanoma (lentigo maligna melanoma) 54. Quando si osservano questi caratteri, è bene procedere all’asportazione chirurgica; in caso di lesioni molto estese, si può procedere a una biopsia incisionale, preferibilmente a livello delle zone che mostrano una pigmentazione bluastra. I nevi in sede acrale (a livello palmo-plantare) mostrano un caratteristico pattern parallelo dovuto alla disposizione del pigmento lungo i solchi. In particolare, nei nevi acrali, si osservano comunemente il pattern a solchi paralleli, a rete di metallo e fibrillare, mentre il pattern a creste parallele è altamente suggestivo di un melanoma acrale-lentigginoso 70. Le lesioni che mostrano dei pattern atipici o multicomponente vanno comunque sottoposte a escissione chirurgica. Tra le lesioni a livello ungueale la diagnosi differenziale si pone essenzialmente con l’emorragia subungueale (storia clinica di trauma e aspetto dermoscopico caratterizzato dalla presenza di aree tondeggianti ben circoscritte di colore nero-rossastro e da «blood spots» o punti di colorito rossonerastro), con i nevi melanocitici (linee longitudinali regolari per spessore e parallelismo, su di un fondo marrone omogeneo) e con la melanonichia indotta da farmaci (pigmentazione omogenea di colorito grigiastro con linee longitudinali regolari) 55. Appare utile, in questi casi, il followup dermoscopico a conferma della diagnosi 56. Il melanoma subungueale, invece, si presenta dermoscopicamente con pigmentazione marrone di fondo e linee longitudinali da marroni a nere, irregolari per spessore, parallelismo e colorazione. È importante anche ricercare il micro-segno di Hutchinson (pigmentazione a livello della cuticola e della cute periungueale, visibile solo all’esame dermoscopico) che, anche se raro, deve far sospettare un melanoma 55, 57. Nei casi sospetti si deve ricorrere a una biopsia incisionale che coinvolga anche la matrice ungueale. Le melanosi labiali e genitali sono lesioni pigmentate benigne che si presentano dermoscopicamente con una pigmentazione diffusa di fondo con rinforzi del pigmento di tipo 346 granulare, globulare (con globuli spesso allineati) o linearecurvilineo, sovente parallelo, di colore marrone chiaro, bruno o grigiastro 71. Appare, comunque, utile un follow-up di queste lesioni e di quelle che mostrano una pigmentazione lievemente irregolare. Nel caso di lesioni sospette, caratterizzate da colore variegato e distribuzione irregolare del pigmento, la dermoscopia può permettere di identificare la zona più atipica dove praticare una biopsia incisionale 58. I nevi blu sono facilmente diagnosticabili quando mostrano la tipica pigmentazione omogenea bluastra, si riscontrano nelle sedi caratteristiche e senza storia di modificazioni. Tuttavia qualche dubbio di diagnosi differenziale con il melanoma può insorgere nelle lesioni che presentano aree bianco-giallastre, indice di una fibrosi associata. Data l’importanza delle strutture blu in dermoscopia, è sempre richiesta un’attenta valutazione di tali aspetti, mentre l’asportazione è consigliabile nei casi in cui la storia clinica di queste lesioni sia dubbia o la dermoscopia anche solo lievemente sospetta 59. Inoltre, bisogna tenere presente che le metastasi da melanoma possono simulare un nevo blu 60. La dermoscopia risulta molto utile nello studio e nel follow-up dei nevi congeniti. La presentazione dermoscopica di tali lesioni è eterogenea: il pattern più comune è quello ad acciottolato, ma, frequentemente, è possibile osservare anche un pattern multicomponente, per la presenza di colori multipli, punti e globuli marroni, zone di reticolo pigmentato, aree omogenee ipopigmentate e aree omogenee bluastre 61. Sono stati recentemente descritti anche peculiari aspetti dermoscopici come il reticolo a bersaglio, i globuli a bersaglio e i vasi a bersaglio 62. In ogni caso, un monitoraggio attento delle immagini cliniche digitali dell’intera lesione, integrate dalle immagini dermoscopiche rappresentative del pattern architetturale, dei bordi della lesione e di zone di particolare interesse, può permettere di seguire nel tempo queste lesioni congenite e di individuare l’eventuale comparsa di caratteri atipici 61. In questi casi, se la lesione è di piccole dimensioni è consigliabile l’asportazione chirurgica mentre in caso di nevi grandi o giganti è possibile effettuare una biopsia incisionale nelle aree dubbie. I nevi ricorrenti (o persistenti), spesso mostrano caratteristiche dermoscopiche talmente bizzarre e atipiche da far sospettare un melanoma, quali strie irregolari, punti e globuli irregolari in prossimità o nel contesto dell’area cicatriziale 63. Se i nevi ricorrenti compaiono dopo un’escissione incompleta di un nevo istologicamente non atipico, possono essere monitorati nel tempo. Quando non vi è certezza sulla precedente diagnosi clinico-istopatologica, la lesione deve essere asportata chirurgicamente. Un altro utile campo di applicazione del monitoraggio digitale riguarda i nevi irritati (da traumatismi, infezioni) o nevi di Meyerson 72 in cui un follow-up molto stretto (anche di 1-2 settimane) dopo trattamento locale può permettere di dirimere il dubbio diagnostico. Un’altra lesione pigmentata che può mimare un melanoma (in situ) è la lentigo reticolare (o «ink spot lentigo»), che si riscontra frequentemente su cute intensamente foto-danneggiata. Dermoscopicamente la lesione è caratterizzata da GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 GUIDELINES IN DERMOSCOPY CHIMENTI un reticolo pigmentato, prominente e di colore marrone scuro-nerastro, a maglie irregolari e sfrangiate, che tuttavia è uniformemente distribuito su tutta la lesione 64. Nelle lesioni dubbie è bene ricorrere all’escissione chirurgica. Il carcinoma basocellulare, specie se pigmentato, può porre problemi di diagnosi differenziale con il melanoma. Gli aspetti dermoscopici tipici del BCC sono i vasi arboriformi, le aree a foglia d’acero, le grandi aree ovoidali grigio-blu, i globuli multipli grigio-blu, le aree a ruota di carro e l’ulcerazione, in assenza di reticolo pigmentato 65, 66. La cheratosi seborroica, specie nella sua variante acantotica e pigmentata, può simulare un melanoma. Tipicamente, le caratteristiche dermoscopiche includono le pseudocisti cornee e gli sbocchi simil-comedonici, ma anche strutture a impronta digitale, e aree con aspetto cerebriforme con giri e solchi. Recentemente sono stati descritti come caratteristici delle cheratosi seborroiche i limiti nettamente demarcati e i bordi «a morsicatura concava» («moth-eaten borders») 68 osservabili soprattutto nelle cheratosi seborroiche in fase iniziale e/o lentigo solari. Talvolta si possono, però, osservare anche un falso reticolo pigmentato (nelle cheratosi seborroiche di tipo reticolare) e strutture pseudo-globulari 68, 69. Nelle lesioni dubbie è consigliabile l’asportazione, nell’evenienza, sebbene rara, di un melanoma che simula una cheratosi seborroica 67. L’esame dermoscopico delle lesioni di natura vascolare, attraverso la ricerca di criteri specifici, consente di escludere il melanoma con elevata accuratezza. Gli emangiomi sono caratterizzati da un pattern lacunare, per la presenza di numerose aree ovoidali ben circoscritte, di colore dal rosso al rosso bluastro, denominate lacune rosse. Queste strutture vanno differenziate dalle aree rosso-lattescenti, meno ben definite, che possono talvolta, ma in maniera specifica, essere osservate nel melanoma 14. Nel granuloma piogenico il pattern lacunare può non essere facilmente riconoscibile, quindi è consigliabile l’escissione con esame istologico nell’adulto. Gli angiocheratomi sono caratterizzati da lacune di colore dal rosso-bluastro al nero, associate ad aree cheratosiche biancogiallastre 14. Infine gli ematomi subcornei mostrano dermoscopicamente un’area omogenea di colorito nerastro ma anche un aspetto pseudo-parallelo o pseudo-globulare. Standard tecnologici in dermoscopia Il dermatoscopio e il videodermatoscopio rappresentano gli strumenti più utilizzati per eseguire l’esame dermosco- Vol. 140 - N. 4 pico 1-3, 14. In alcuni centri si impiega anche lo stereomicroscopio, implementato da sistemi digitali con telecamere ad alta risoluzione (3 CCD) 3, 52, 53. L’attuale standard di riferimento, per quanto riguarda la fotografia dermoscopica, è costituito dal sistema di acquisizione Dermaphot (Heine Optotechnik, Herrsching, Germany) che garantisce un ottimo potere risolutivo e un’elevata qualità di immagine 73-75. Attualmente è disponibile in commercio una serie di sistemi e strumenti di videodermoscopia digitale, che consente di ottenere, in alcuni casi, un’immagine di qualità sovrapponibile allo standard 76, 77. È auspicabile che, in un futuro prossimo, le aziende si adeguino a tali standard, eventualmente attraverso l’istituzione di una commissione ad hoc per la valutazione e la validazione dei sistemi di videodermoscopia. Le problematiche della refertazione dell’esame videodermoscopico Anche se attualmente non esistono a riguardo né una normativa precisa né dati in letteratura, vogliamo farci portavoci di una proposta di unificazione e standardizzazione del referto che è opportuno rilasciare a seguito di un esame videodermoscopico. Alla luce delle responsabilità di ordine medico-legale, di deontologia professionale e, non ultimo, anche per un riconoscimento completo della demoscopia quale esame strumentale di secondo livello, appare fondamentale il rilascio di una refertazione idonea a seguito della prestazione specialistica effettuata. Pertanto, nel referto di un esame videodermoscopico, suggeriamo di includere sempre i seguenti punti essenziali (criteri minimi): — simmetria/asimmetria della lesione; — aspetto globale; — aspetti locali; — conclusione diagnostica; — indicazione sul trattamento. Considerando i limiti attuali nella standardizzazione dei diversi sistemi videodermoscopici (differente qualità dell’immagine e diversa risoluzione e qualità di stampa), riteniamo per il momento che l’immagine dermoscopica stampata possa essere rilasciata al paziente, a discrezione del dermatologo, specificando le attuali problematiche tecnologiche. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 347 ORIGINAL ARTICLES G ITAL DERMATOL VENEREOL 2005;140:349-58 The problem of clinically atypical nevi submitted to verification biopsy: can dermoscopy help excluding histologically common lesions? A. CHIARUGI, P. NARDINI, V. DE GIORGI, P. CARLI Aim. Early diagnosis of cutaneous melanoma is associated with costs in term of false positive diagnosis, i.e. benign pigmented lesions defined suspicious or equivocal by clinical examination and submitted to verification biopsy. The aim of the study is to investigate the possible role of dermoscopy in selecting histologically common nevi from those with histologic atypia. Methods. Two hundred and sixthy-four clinically atypical melanocytic nevi were classified by experienced pathologists as nevi with histologic atypia and without histologic atypia. Two observers analysed dermoscopic features with classic pattern analysis and simplified algorithms. The study population was divided in a training set and in a test set (observers aware of histologic classification and blinded as to histologic classification respectively). Results. In the training set, nevi with histologic atypia significantly differed from common nevi since they showed with higher frequency the following dermoscopic criteria: atypical pigment network, regression features, ABCD score ≥4 and seven point check list ≥2. In the test set, however, only atypical pigment network and seven point score ≥2 were still more frequent in atypical nevi than in common nevi. The negative predictive value was less than 70% for any of selected dermoscopic features. This means that when an observer predict that a nevus lacking the above-mentioned criteria of atypia is histologicaly common, it will be true in less than 70% of cases. Conclusion. Dermoscopy can play a role in detecting banal melanocytic lesions, ie. nevi without histologic atypia, within the pool of clinically equivocal lesions submitted to verification biopsy. Further study with evidence-based design (prospective, randomised study) are needed to investigate the impact of Address reprint requests to: P. Carli, Department of Dermatology, University of Florence, Via della Pergola 58/60, 50121 Firenze, Italy. E-mail: [email protected] Vol. 140 - N. 4 Department of Dermatology University of Florence, Florence, Italy dermoscopy in a better selection of lesions to remove in real practice. KEY WORDS: Dermoscopy - Pigmented lesions - Atypical nevi False positive. E arly diagnosis of cutaneous melanoma is associated with costs in term of false positive diagnosis, i.e. benign pigmented lesions defined suspicious or equivocal by clinical examination and submitted to verification biopsy. This leads to inevitable scarring and morbidity. The frequency of verification biopsies is not negligible: according to recent australian data, the ratio of melanomas to benign lesions excised is 1:17 (1:26 including seborrheic keratosis) among family doctors.1 When selection of lesions to be removed is made by dermatologists at specialised pigmented lesion clinics, less false positive excisions are made (about 1:67 according to italian and British data).2, 3 It is likely that only small improvement of the malignant: benign ratio is attainable without increasing the risk of leaving a melanoma unexcised. Dermoscopy (dermatoscopy, epiluminescence microscopy, surface microscopy), a non invasive technique able to improve diagnostic performance of melanoma diagnosis, may GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 349 CHIARUGI THE PROBLEM OF CLINICALLY ATYPICAL NEVI SUBMITTED TO VERIFICATION BIOPSY play a key role in a better preselection of lesions to be removed. An improvement of specificity of melanoma screening by use of dermoscopy has been recently shown.4 We investigated the possible role of dermoscopy in selecting, within the pool of lesions submitted to verification biopsy according to their clinical features, histologically banal nevi from those with histologic atypia. Many studies have demonstrated that clinical atypia and histologic atypia in nevi have poor correlation.5 This means that a clinically atypical nevi can be histologic common and viceversa.5 Since the risk of histopathologic misclassification between nevi with histologic atypia and early melanoma is likely to occur,6 the exclusion of histologically atypical nevi from verification biopsy, once judged equivocal from a clinical point of view, could be hazardous. Conversely, an histopathologic misclassification between early melanoma and common nevi, even if equivocal by clinical examination, is unlikely. The latter lesions are those who would mostly benefit of a non invasive classification by dermoscopy in order to be left in place without risk for the patient. Materials and methods This study included 264 clinically atypical melanocytic nevi, consecutively excised for diagnostic verification in the period January 2001-December 2002 at the First Dermatology Unit, Florence. A staff of pathologists with experience in diagnosis of melanocytic lesions classified the lesions in accordance with the criteria of the NIH Consensus Conference on Diagnosis and Treatment of Early Melanoma 7 as common melanocytic nevi (compound and junctional nevi, n=133) and melanocytic nevi with histologic atypia (architectural disorders and cytological atypia, n=131). Therefore, in this set of lesions, nevi with histologic atypia represented 131/264 (49.6%) of cases. Before excision, clinical and dermoscopic images were obtained using an F50 Nikon camera with objective AF micro Nikkor 60. For dermoscopy, images were obtained after oil application on the surface of the lesions using a Dermaphot objective (magnification ×10). We randomly divided the series of lesions in a training set and a test set. The same two experienced observers (AC and PN) examined both training and test sets. 350 TABLE I.—Training set. Clinical features of histologically atypical nevi and common nevi (N=114). The two subsets of nevi (common and atypical on histology) are largely comparable as to clinical characteristics. Clinical feature Atypical nevi (N=68) Palpability 27/68 (40%) Macular-papular aspect 22/68 (32%) Asimmetry N 3/68 (4%) 1 axis 30/68 (44%) 2 axis 35/68 (51%) N Colours 1 colours 14/68 (20.5%) 2 colours 46/68 (68%) 3 colours 8/68 (12%) Type of predominant colour Black 3/68 (4%) Dark brown 34/68 (50%) Light brown 27/68 (40%) Blue/grey 1/68 (1.5%) Pink/reddish 3/68 (4%) Border irregularity 51/68 (75%) Common nevi (N= 46) Pearson’s χ2 test 23/46 (50%) 10/46 (22%) 0.277 0.216 2/46 (4%) 22/46 (48%) 22/46 (48%) NS 14/46 (30%) 28/46 (61%) 4/46 (9%) NS 2/46 (4%) 25/46 (54%) 15/46 (33%) 2/46 (4%) 2/46 (4%) 31/46 (67%) NS NS The training set (68 atypical nevi and 46 common nevi) was aimed to identify dermoscopy features associated with histologic atypia. In this study phase the observers were aware of the histologic diagnosis. Since the criteria for entry the study was the selection of the lesion for diagnostic verification, clinical features of nevi, either histologically common or atypical, were those of clinical atypical lesions, i.e. showing some or all the ABCD features of melanoma. Table I shows in details clinical features of common and atypical nevi included in the training set. No statistically significant difference in the frequency of selected clinical parameters was found. Therefore, the two subsets of nevi (common and atypical on histology) were largely comparable as to clinical characteristics. The training set analysis should enable us in identifying dermoscopic features significantly associated with histologic atypia. The possible role of these features in removing nevi without histologic atypia from the pool of lesions selected for excision on the basis of their clinical features will be investigated by means of test set analysis, with observers blinded as to histologic diagnosis. Test set included 63 nevi with histologic atypia and 87 nevi without histological atypia. Dermoscopy terminology adopted in the study was GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 THE PROBLEM OF CLINICALLY ATYPICAL NEVI SUBMITTED TO VERIFICATION BIOPSY CHIARUGI TABLE II.—Training set. Difference in the frequency of selected dermoscopic features between histologically common and atypical nevi (only parameters with significant difference are shown). Dermoscopic feature Atypical nevi (N=68) Common nevi (N=46) Pearson’s χ2 test Irregular/prominent pigment network Regression structures 40/68 (59%) 29/68 (43%) 18/46 (39%),3 9/46 (19,5%) P=0.039 P=0.010 that of the Internet consensus meeting, held in 20002001.8 We also tested the diagnostic algorithms: ABCD rule of dermoscopy as published by Stolz et al.9 and 7Point Checklist according to Argenziano et al.10 The TDS derived from ABCD rule signifies a benign lesion if the value is <4.75, malignant lesion if it is >5.45 and doubtful lesion for intermediate values; the TDS for histologically atypical nevi is frequently between 4.5 and 5.8. We tested our series of melanocytic lesions using different cut-off values (4.75 and 4) of TDS. The 7- Point Checklist is a method based on simplified ELM pattern analysis; a total score ≥3 indicates a malignant lesion. Also for this algorithm we used two different cut off values : 3 and 2 points. Additionally, both training set and test set were classified in accordance with the dermoscopic classification of Clark’s nevi suggested by HofmannWellenhof et al.11 including the following global patterns: reticular, globular, homogeneous, or reticularglobular, reticular-homogeneous, globular-homogeneous if two components were dominant. Concerning the distribution of pigmentation the nevi were classified with central hyperpigmentation/hypopigmentation, eccentric peripheral hyperpigmentation/hypopigmentation, multifocal hyperpigmentation/hypopigmentation. Statistical analysis For statistical analysis non parametric test were used (χ2 test, Fisher’s exact test when appropriate). In order to evalute the power of selected dermoscopic methods in the identification of histologic common nevi (i.e. without histologic atypia), the negative predictive value (NPV) was calculated (true negative/true negative + false negative). The NPV represents the probability that a nevus defined “without histologic atypia” by dermoscopy will be eventually confirmed by histologic examination. Vol. 140 - N. 4 Results Training set As expected, nevi with and without histologic atypia were not dissimilar from a clinical point of view (Table I). To the contrary, these two subsets of nevi showed significant difference about dermoscopic features. Concerning the frequency of major features selected on the basis of the Consensus Conference 2001, an irregular/prominent pigment network, i.e. a network with dark or thick lines and large holes, was found in 59% of nevi with histologic atypia and in 39% of those without atypia (P=0.039). Dermoscopic features of regression, i.e. blue and with areas, were found in 43% of nevi with atypia and in 19.5% of those without atypia (P=0.010) (Table II). No difference was found for other features. Figures 1 and 2 show major dermoscopic features of nevi with histological atypia. Concerning simplified algorithms, nevi with histologic atypia more frequently than common nevi reached a value >4.75 (43% vs 19.5%) (P=0.02) according to ABCD rule (threshold of suspicion) and ≥3 according to the 7- Point Checklist rule (threshold for malignancy) (20.6 vs 6.5%) (P=0.039) (Table III). A statistically significant difference between nevi with and without histologic atypia was also found concerning the classification of Clark’s nevi reported by Hofmann-Wellenhof, both concerning global features (Table IV) and distribution of pigmentation (Table V). Test set Dealing with test set, the presence of an atypical pigment network, but no that of regression features, was significantly associated with histologic atypia (60.3% vs 42.5%)(Table VI). According to the NPV associated with this parameter (66.6%), the use of this feature as a marker for excision, would have resulted in GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 351 CHIARUGI THE PROBLEM OF CLINICALLY ATYPICAL NEVI SUBMITTED TO VERIFICATION BIOPSY TABLE III.—Training set. Distribution of the SCORE according to 7 - Point Check List and TDS according to ABCD rule of dermoscopy in histologically atypical and common nevi. 7-Point check list <3 ≥3 <2 ≥2 ABCD score <4.75 ≥4.75 <4 ≥4 Figure 1.—Dermoscopic image (10×) of a nevus with histologic atypia. The arrow indicates atypical pigment network. Atypical nevi (N=68) Common nevi (N= 46) 54/68 (79.4%) 14/68 (20.6%) 22/68 (32.4%) 46/68 (67.6%) 43/46 (93.5%) 3/46 (6.5%) 32/46 (69.6%) 14/46 (30.4%) 39/68 (57%) 29/68 (43%) 26/68 (38%) 42/68 (62%) 37/46 (80.5%) 9/46 (19.5%) 28/46 (61%) 18/46 (39%) Pearson’s χ2 test P=0.039 P=0.000 P=0.020 P=0.018 TABLE IV.—Training set. Dermoscopic classification of histologically atypical and common nevi according to “Hofmann-Wellenhof”: global dermoscopic pattern. Global dermoscopic pattern Atypical nevi (N=68) Common nevi (N=46) Reticular Globular Homogeneous Reticular-globular Reticular-homogeneous Globular-homogeneous Unclassified 32/68 (47%) 1/68 (1.5%) 2/68 (3%) 10/68 (15%) 18/68 (26%) 4/68 (6%) 1/68 (1.5%) 19/46 (41%) 8/46 (17%) 1/46 (2%) 7/46 (15%) 10/46 (21%) 1/46 (2%) 0/46 (0%) Exact test (Montecarlo) P=0.011 TABLE V.—Training set. Dermoscopic classification of histologically atypical and common nevi according to “Hofmann-Wellenhof”: distribution of pigmentation. Figure 2.—Dermoscopic image (10×) of a nevus with histologic atypia. The arrows indicate regression structures. Blue area (black arrow), white area (empty arrow). about 33% of false negative diagnosis, ie. nevi with histologic atypia left unexcised. Concerning semiquantitative algorithms, both the ABCD rule and the seven point check-list confirmed their possible role in selecting nevi with from those without atypia (Table VII). As occurred in the training set, even in the test set nevi with histologic atypia more frequently reached the threshold value of suspicion compared to common nevi. The NPV ranged from 59.2% with a threshold point of 4 (instead of 4-75) for the ABCD rule to 66.6% for 2 352 Distribution of pigmentation Atypical nevi (N=68) Common nevi (N=46) Uniform pigmentation Central hyperpigmentation Central hypopigmentation Eccentric peripheral hyperpigmentation Eccentric peripheral hypopigmentation Multifocal hyperpigmentation and hypopigmentation 3/68 (4%) 13/68 (19%) 6/68 (9%) 9/68 (13%) 12/46 (26%) 6/46 (13%) 7/46 (15%) 7/46 (15%) 13/68 (19%) 3/46 (6.5%) 24/68 (35%) 11/46 (24%) Exact test (Montecarlo) P=0.011 (instead of 3) as threshold value for the seven point check list (Table VII). According to the dermoscopic classification of Clark nevi, no significant difference neither in the frequen- GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 THE PROBLEM OF CLINICALLY ATYPICAL NEVI SUBMITTED TO VERIFICATION BIOPSY CHIARUGI TABLE VI.—Test set. Difference in frequency of selected dermoscopic features between histologically atypical and common nevi. Dermoscopic feature Atypical nevi (N=63) Common nevi (N=87) Pearson’s χ2 test NPV Irregular/prominent pigment network Regression structures 38/63 (60,3%) 19/63 (30%),3 37/87 (42,5%) 24/87 (27,5%) P=0.032 P=0.700 66.6% 58.8% TABLE VII.—Training set. Distribution of the score according to 7 Point Check List and TDS according to ABCD rule dermoscopy in histologically atypical and common nevi. Atypical nevi (N=63) 7-Point check list <3 ≥3 <2 ≥2 ABCD score <4.75 ≥4.75 <4 ≥4 Common nevi (N=87) Pearson’s χ2 test 46/63 (73%) 17/63 (27%) 27/63 (42.9%) 36/63 (57.1%) 76/87 (87.4%) 11/87 (12.6%) P=0.026 56/87 (64.4%) 31/87 (35.6%) P=0.009 44/63 (69.8%) 19/63 (30.2%) 33/63 (52.4%) 30/63 (46.6%) 71/87 (81.6%) P=0.093 16/87 (18.4%) 58/87 (66.7%) P=0.077 29/87 (33.3%) NPV 62.2% 66.6% TABLE VIII.—Test set. Dermoscopic classification of histologically atypical and common nevi according to “Hofmann-Wellenhof”: global dermoscopic pattern. Global dermoscopic pattern Atypical nevi (N=63) Reticular Globular Homogeneous Reticular-globular Reticular-homogeneous Unclassified 360/63 (57.1%) 1/63 (1.6%) 7/63 (11.1%) 6/63 (9.5%) 13/63 (20.6%) 0/63 (0%) Common nevi (N=87) Exact test (Montecarlo) 39/87 (41%) 15/87 (17.2%) 8/87 (9.2%) 5/87 (6%) 18/87 (21.7%) 0/87 (0%) NS 61.7% 59.2% TABLE IX.—Training set. Dermoscopic classification of histologically atypical and common nevi according to “Hofmann-Wellenhof”: distribution of pigmentation. cy of global pattern nor in that of distribution of pigmentation between common and atypical nevi was found dealing with test set (Table VIII, IX). Discussion and conclusions In recent years, dermoscopy proved to be able to increase the accuracy of melanoma diagnosis compared to that achieved by visual examination alone.12, 13 Although further studies are still needed, it is conceivable that dermoscopy reduce the false positive rate in melanoma screening.4, 14 Among benign melanocytic lesions, verification biopsy to exclude melanoma is currently undertaken not only for Spitz nevi and nevi with histologic atypia but also for histologically common nevi.4 As reported by many studies, clinical features of a nevus do not correlate with histologic atypia.5 Therefore, even an histologically common nevus may show some or all the ABCD signs of melanoma sometimes representing a cause of concern both for patient and physician. We sought to evaluate if dermoscopy may help to achieve—compared to visual examination alone—a better selection of melanocytic lesions to be excised for Vol. 140 - N. 4 Distribution of pigmentation Atypical nevi (N=63) Common nevi (N=87) Uniform pigmentation Central hyperpigmentation Central hypopigmentation Eccentric peripheral hyperpigmentation Eccentric peripheral hypopigmentation Multifocal hyperpigmentation and hypopigmentation 19/63 (30.2%) 9/63 (14.3%) 7/63 (11.1%) 15/63 (23.8%) 28/87 (32.2%) 12/87 (13.8%) 12/87 (13.8%) 19/87 (21.8%) Exact test (Montecarlo) 7/63 (11.1%) 12/87 (13.8%) 6/63 (9.5%) 4/87 (4.5%) NS diagnostic verification, excluding histologically common nevi. Since the risk of misclassification between nevi with atypia and early melanoma by pathologist has been demonstrated by several authors,6, 15 a further reduction of false positives by means of avoid excision of nevi with histologic atypia, when clinically doubtful, seems to the contrary less desirable. According to training set analysis, two dermoscopic features were more frequently found in nevi with histologic atypia than in common nevi: atypical /prominent pigment network and regression features. This finding is in perfect agreement with the data provided GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 353 CHIARUGI THE PROBLEM OF CLINICALLY ATYPICAL NEVI SUBMITTED TO VERIFICATION BIOPSY by a previous study of our group conducted on a different series of lesions examined by another group of observers.16 Also the semiquantitative score assigned in accordance with two of the most popular simplified algorithms for melanoma diagnosis, the ABCD rule of dermoscopy 9 and the new seven-point check-list,10 significantly differed between the two subset of nevi for both the threshold values adopted for each method (4 and 4.75 for ABCD, 2 and 3 for seven point). This finding disagree with a previous study where no significant difference between nevi with atypia and those without was found concerning ABCD score.16 Possible explainations include a different source of lesions (clinically equivocal and unequivocal lesion in the previous study), and a different procedure adopted in the analysis of the data (comparison of median values in the previous study, comparison among frequencies in pre-established categories in the present study). However, only the seven point score confirmed a statistically significant difference between the two subsets of nevi according to test set analysis. No difference in the frequency of dermoscopic subtypes of Clark nevi 11 was found. In this context one should take into account that our series was small and the data have to be interpreted cautiously. Since the large number of categories proposed for the classification of nevi according to Hofman-Wellenhof et al.,11 our small series of lesions may not reach a sufficient statistical power. Our data strongly cool down the expectations of this new classification of Clark’s nevi: In the opinion of the proposers “this classification should be regarded not just as an academic morphologic exercise but as a classification system that will lead to a better understanding of the biological characteristics of these melanocytic nevi”.11 The fact that no statistical difference in the frequency of the Hofmann’s classification subtypes has been found between nevi with atypia and common nevi greatly limits its validity in the study of biological features of benign melanocytic lesions. In sum, according to training set, observers may be able to identify—within a pool of nevi indistiguinshable among them as to clinical features—nevi with histologic atypia from those without atypia. The “prototypic” nevus with atypia would therefore be a nevus showing atypical network and/or regression structures, that scores >2 in accordance with seven point check list and >4 in accordance with ABCD rule of dermoscopy. 354 This scenario has been in part confirmed by test set analysis: only atypical pigment network and seven point score ≥2 or ≥3 confirmed their different distribution between the two subset of nevi with observers blinded as to histologic diagnosis. Neither regression features nor the ABCD score were to the contrary longer associated with histologic atypia in the test set analysis. Among the points of strength of this study we mention the design of the study, with a training set and a test set; this allows to verify prospectively to what extent what found in the training set by open observers is confirmed in a new series of lesions by blinded examiners. Moreover, this study includes only nevi excised for diagnostic verification decided on the basis of clinical features, thus approaching as far as possible the diagnostic setting found in practice. Being in this study the clinical characteristics of nevi with histologic atypia not dissimilar to those without atypia we minimized the risk of lesion’s classification influenced by clinical factors acting as confounders. Among the study’s weaknesses, we mention that fact that this study is based on lesion’s classification on photographic images, in a posteriori diagnostic setting In order to investigate what consequences this finding may have in clinical practice, we calculated the negative predictive value associated with each of the above-mentioned feature, i.e. the probability that a nevus lacking these features eventually be histologically common. Unfortunately, the NPV was not higher than 60% as average for any of the selected dermoscopic features. This means that when an observer predict that a nevus lacking the above mentioned criteria of atypia is histologically common, it will be true in 60% only of cases. Further prospective, randomized studies should confirm if the above-mentioned dermoscopic criteria associated with histologic atypia in nevi, i.e. atypical network, regression features, ABCD score and Seven point score can help dermatologist in change the lesion’s management with fewer excision of clinically equivocal but histologically common nevi. References 1. English DR, Burton RC, del Mar CB, Donovan RJ, Ireland PD, Emery G. Evaluation of aid to diagnosis of pigmented skin lesions in general practice: controlled trial randomised by practice. BMJ 2003;327:375. 2. Carli P, De Giorgi V, Betti R, Vergani R, Catricala C, Mariani G et al. Relationship between cause of referral and diagnostic outcome in GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 THE PROBLEM OF CLINICALLY ATYPICAL NEVI SUBMITTED TO VERIFICATION BIOPSY 3. 4. 5. 6. 7. 8. 9. pigmented lesion clinics: a multicenter survey of the Italian Multidisciplinary Group on Melanoma (GIPMe). Melanoma Res 2003;13:207-11. Gibbon KL. Pigmented lesion clinics – are they a waste of resources? Clin Exper Dermatol 1998;23:3-8. Carli P, De Giorgi V, Crocetti E, Mannone F, Massi D, Chiarugi A et al. Improvement of malignant/benign ratio in excised melanocytic lesions in the “dermoscopy era”: a retrospective study 1997-2001. Br J Dermatol 2004;150:687-92. Annessi G, Cattaruzza MS, Abeni D, Baliva G, Laurenza M, Macchini V et al. Correlation between clinical atypia and histologic dysplasia in acquired melanocytic nevi. J Am Acad Dermatol 2001;45:77-85. Corona R, Mele A, Amini M, De Rosa G, Coppola G, Piccardi P et al. Interobserver variability of the histopathologic diagnosis of cutaneous melanoma and other pigmented skin lesions. J Clin Oncol 1996;14:1218-23. National Institute of Health Consensus Conference. Diagnosis and treatment of early melanoma. JAMA 1992;268:1314-9. Argenziano G, Soyer HP, Chimenti S, Talamini R, Corona R, Sera F et al. Dermoscopy of pigmented skin lesions: results of a consensus meeting via the Internet. J Am Acad Dermatol 2003;48:679-93. Stolz W, Riemann A, Cognetta AB, Pillet l, Abmayr W, Holzel D et al. ABCD rule of dermatoscopy: a new practical method for early recognition of melanoma. Eur J Dermatol 1994;4:521-7. CHIARUGI 10. Argenziano G, Fabbrocini G, Carli P, De Giorgi V, Sammarco E, Delfino M. Epiluminescence microscopy for the diagnosis of doubtful melanocytic skin lesions: comparison of the ABCD rule of dermatoscopy and a new 7-point check list based on pattern analysis. Arch Dermatol 1998;134:1563-70. 11. Hofmann-Wellenhof R, Blum A, Wolf IH, Piccolo D, Kerl H, Garbe C et al. Dermoscopic classification of atypical melanocytic nevi (Clark nevi). Arch Dermatol 2001;137:1575-80. 12. Pehamberger H, Steiner A, Wolff K. In vivo epiluminescence microscopy of pigmented skin lesions. I. Pattern analysis of pigmented skin lesions. J Am Acad Dermatol 1987;17:571-83. 13. Carli P, De Giorgi V, Giannotti B. Dermoscopy and early diagnosis of melanoma. The Light and the Dark. Arch Dermatol 2001;137:1641-4. 14. Carli P, De Giorgi V, Chiarugi A, Nardini P, Weinstock MA, Crocetti E et al. Addition of dermoscopy to conventional naked-eye examination in melanoma screening: a randomized study. J Am Acad Dermatol 2004;50:683-9. 15. Ferrara G, Argenziano G, Soyer HP, Corona R, Sera F, Brunetti B et al. Dermoscopic and histopathologic diagnosis of equivocal melanocytic skin lesions: an interdisciplinary study on 107 cases. Cancer 2002;95:1094-100. 16. Carli P, De Giorgi V, Massi Giannotti B. The role of pattern analysis and the ABCD rule of dermoscopy in the detection of histologic atypia in melanocytic naevi. Br J Dermatol 2000;143:290-7. Il problema dei nevi clinicamente atipici sottoposti a verifica diagnostica: la dermoscopia può essere di aiuto per escludere nevi istologicamente comuni? L a diagnosi precoce del melanoma cutaneo è associata a dei costi in termini di diagnosi di falsi positivi, vale a dire lesioni pigmentate benigne definite dubbie o sospette all’esame clinico e sottoposte a verifica bioptica. Questo porta inevitabilmente ad una serie di conseguenze per il paziente (stato di malattia, formazione di cicatrici). La frequenza delle verifiche bioptiche non è trascurabile: secondo recenti dati australiani il rapporto fra melanomi e lesioni benigne asportate è 1:17 (1:26 se includiamo anche le cheratosi seborroiche) quando la diagnosi viene effettuata dai medici di medicina generale 1. Quando la selezione delle lesioni da asportare avviene ad opera di dermatologi in cliniche specializzate nella diagnosi delle lesioni pigmentate, risultano effettuate un minor numero di escissioni di falsi positivi (circa 1:6-7 secondo dati italiani ed anglosassoni) 2, 3. È probabile che si possa ottenere solo un piccolo miglioramento del rapporto lesioni maligne/lesioni benigne senza aumentare il rischio di lasciare in sede un melanoma. La dermoscopia (o dermatoscopia, microscopia a epiluminescenza, microscopia di superficie), una tecnica non invasiva capace di migliorare la performance nella diagnosi del melanoma, può giocare un ruolo chiave nel migliorare la selezione delle lesioni pigmentate che devono essere escisse. Dati recenti hanno mostrato che l’uso della dermoscopia può aumentare la specificità nello screening del melanoma 4. Abbiamo indagato il possibile ruolo della dermoscopia nel selezionare, all’interno di un pool di lesioni sottoposte a Vol. 140 - N. 4 verifica bioptica per le loro caratteristiche cliniche, nevi istologicamente banali da quelli con atipia istologica. Molti studi hanno dimostrato che nei nevi l’atipia clinica e l’atipia istologica hanno una scarsa correlazione 5. Questo significa che un nevo clinicamente atipico può essere istologicamente comune e viceversa 5. Poiché il rischio di un errore di classificazione istopatologica fra nevi con atipia istologica e melanoma iniziale si può verificare con una certa frequenza 6, escludere i nevi istologicamente atipici da una verifica bioptica, una volta giudicati equivoci da un punto di vista clinico, può essere rischioso. Al contrario, una misclassificazione istopatologica fra melanoma iniziale e nevi comuni, anche se valutati equivoci all’esame clinico, risulta poco probabile. Sono queste ultime lesioni che potrebbero beneficiare di una classificazione non invasiva da parte della dermoscopia ed essere quindi lasciate in sede senza rischi per il paziente. Materiali e metodi Questo studio ha incluso 264 nevi melanocitici clinicamente atipici, consecutivamente escissi per la verifica diagnostica nel periodo gennaio 2001-dicembre 2002 presso la I Clinica Dermatologica, Università di Firenze. Uno staff di patologi esperti nella diagnosi delle lesioni GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 355 CHIARUGI THE PROBLEM OF CLINICALLY ATYPICAL NEVI SUBMITTED TO VERIFICATION BIOPSY melanocitiche ha classificato le lesioni in accordo con i criteri della NIH Consensus Conference per la Diagnosi e il Trattamento del Melanoma Precoce 7 come nevi melanocitici comuni (nevi giunzionali e composti, n=133) e nevi melanocitici con atipia istologica (nevi con disordine architetturale e atipia citologica, n=131). Quindi, in questo gruppo di lesioni, i nevi con atipia istologica rappresentavano 131/264 (49,6%) dei casi. Prima dell’escissione, sono state ottenute le immagini cliniche e dermoscopiche utilizzando una macchina fotografica Nikon F50 con obiettivo micro Nikkor 60. Per la dermoscopia, le immagini sono state ottenute, dopo applicazione di olio sulla superficie delle lesioni, usando un obiettivo Dermaphot (ingrandimento 10×). Abbiamo suddiviso casualmente la serie delle lesioni in un training set e in un test set. Gli stessi due osservatori esperti (AC e PN) hanno esaminato sia il training che il test set. Il training set (68 nevi atipici e 46 nevi comuni) aveva lo scopo di identificare le caratteristiche dermoscopiche associate con atipia istologica. In questa fase dello studio gli osservatori erano a conoscenza della diagnosi istologica. Poiché il criterio per l’entrata nello studio era la selezione della lesione per verifica diagnostica, le caratteristiche cliniche dei nevi, sia istologicamente atipici sia comuni, erano quelle di lesioni clinicamente atipiche, quindi che mostravano alcuni o tutti i criteri ABCD del melanoma. La Tabella I mostra in dettaglio le caratteristiche cliniche dei nevi comuni ed atipici inclusi nel training set: non sono state trovate differenze statisticamente significative nella frequenza dei parametri clinici selezionati. Quindi i due sottogruppi di nevi (istologicamente comuni ed atipici) erano ampiamente comparabili dal punto di vista delle caratteristiche cliniche. L’analisi del training set dovrebbe permetterci di identificare le caratteristiche dermoscopiche significativamente associate all’atipia istologica. Il possibile ruolo di queste caratteristiche nell’escludere i nevi senza atipia istologica dal pool di lesioni selezionate per l’escissione chirurgica sulla base delle loro caratteristiche cliniche sarà valutato per mezzo dell’analisi del test set, in cui gli osservatori non sono a conoscenza della diagnosi istologica. Il test set comprendeva 63 nevi con atipia istologica e 87 nevi senza atipia istologica. La terminologia dermoscopica adottata nello studio è stata quella dell’Internet Consensus Meeting, svoltosi nel 20002001 9. Sono stati anche testati gli algoritmi diagnostici (regola dell’ABCD della dermoscopia come pubblicata da Stolz et al. 8) e la 7 – Point Checklist in accordo con Argenziano et al. 10. Il TDS (Total Dermoscopy Score) ricavato dalla regola dell’ABCD indica che la lesione è benigna se il valore è <4,75, che la lesione è maligna se il valore è >5,45 e che la lesione è dubbia se il valore è intermedio; il TDS per i nevi istologicamente atipici risulta frequentemente compreso fra 4,5 e 5,8. Abbiamo testato le nostre serie di lesioni melanocitiche usando due differenti valori di cut-off del TDS (4,75 e 4). 356 La 7 – Point Checklist è un metodo basato sull’analisi di pattern semplificata; un punteggio totale ≥3 indica una lesione maligna. Anche per questo algoritmo abbiamo usato due differenti valori di cut-off: 3 e 2 punti. Infine sia il training set che il test set sono stati classificati in accordo con la classificazione dei nevi di Clark suggerita da Hofmann-Wellenhof et al. 11 che comprende i seguenti pattern globali: reticolare, globulare, omogeneo, o reticolare-globulare, reticolare-omogeneo, globulare-omogeneo se ci sono due componenti dominanti. Riguardo la distribuzione della pigmentazione i nevi sono stati classificati con iperpigmentazione/ipopigmentazione centrale, iperpigmentazione/ipopigmentazione eccentrica periferica, iperpigmentazione/ipopigmentazione multifocale 11. Analisi statistica Per l’analisi statistica sono stati utilizzati test non parametrici (test del χ2, test esatto di Fisher quando appropriati). Al fine di valutare il potere dei metodi dermoscopici selezionati nell’identificazione dei nevi istologicamente comuni (senza atipia istologica), è stato calcolato il valore predittivo negativo (NPV) (vero negativo/vero negativo+falso negativo). Il NPV rappresenta la probabilità che un nevo definito «senza atipia istologica» mediante la dermoscopia, sarà eventualmente confermato tale dall’esame istologico. Risultati Training set Come atteso, i nevi con e senza atipia istologica non sono risultati differenti dal punto di vista clinico (Tabella I). Al contrario, questi due sottogruppi hanno mostrato differenze significative per quanto riguarda le caratteristiche dermoscopiche. Riguardo alla frequenza dei maggiori criteri selezionati sulla base della Consensus Conference del 2001, è stato evidenziato un reticolo pigmentario irregolare/prominente (per esempio un reticolo con linee scure o spesse e maglie larghe) nel 59% dei nevi con atipia istologica e nel 39% di quelli senza atipia (P=0,039). I caratteri dermoscopici di regressione (per esempio aree blu ed aree bianche) sono stati trovati nel 43% dei nevi con atipia istologica e nel 19,5% di quelli senza atipia (P=0,010) (Tabella II). Nessuna differenza è stata trovata per gli altri caratteri. Le Figure 1 e 2 mostrano le maggiori caratteristiche trovate in nevi con atipia istologica. Riguardo agli algoritmi semplificati, i nevi con atipia istologica hanno raggiunto più frequentemente dei nevi comuni il valore >4,75 (soglia di sospetto) secondo la regola dell’ABCD (43% vs 19,5%) (P=0,02) e il valore ≥3 (soglia di malignità) in accordo alla 7 – Point Checklist (20,6% vs 6,5%) (P=0,039) (Tabella III). Anche per quanto riguarda la classificazione dei nevi di Clark riportata da Hofmann-Wellenhof è stata trovata una dif- GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 THE PROBLEM OF CLINICALLY ATYPICAL NEVI SUBMITTED TO VERIFICATION BIOPSY ferenza statisticamente significativa fra nevi con e senza atipia istologica relativa sia ai caratteri globali (Tabella IV) sia alla distribuzione della pigmentazione (Tabella V). Test set Nel test set la presenza di un reticolo pigmentario atipico è risultata significativamente associata con l’atipia istologica (60,3% vs 42,3%), mentre non lo è stata la presenza delle strutture di regressione (Tabella VI). Secondo il NPV associato al reticolo pigmentario atipico (66,6%), l’uso di questo parametro come marker per l’escissione, potrebbe comportare una diagnosi di falsi negativi in circa il 33%, cioè nevi con atipia istologica non asportati. Riguardo agli algoritmi semiquantitativi, sia la regola dell’ABCD sia la 7 – Point Checklist, hanno confermato il loro possibile ruolo nel selezionare nevi con atipia da quelli comuni (Tabella VII). Come si è verificato nel training set, anche nel test set i nevi con atipia istologica hanno raggiunto più frequentemente il valore soglia di sospetto se comparati con i nevi comuni. Il NPV oscillava fra il 59% per un valore soglia di 4 (invece di 4,75) con la regola dell’ABCD ed il 66,6% per un valore soglia di 2 (anziché 3) con la 7 – Point Checklist (Tabella VII). Nel test set, seguendo la classificazione dermoscopica dei nevi di Clark secondo Hofmann-Wellenhof non sono emerse differenze significative fra i due sottogruppi di nevi né nella frequenza del tipo di pattern globale né in quella della distribuzione della pigmentazione (Tabella VIII, IX). Discussione e conclusioni In anni recenti, la dermoscopia ha dimostrato di poter incrementare l’accuratezza della diagnosi del melanoma rispetto a quella ottenuta con il solo esame visivo 12, 13. Sebbene ulteriori studi siano ancora necessari, è concepibile che la dermoscopia riduca il tasso di falsi positivi nello screening del melanoma 4, 14. Nell’ambito delle lesioni melanocitiche, la verifica bioptica per escludere il melanoma, viene eseguita correntemente non solo per i nevi di Spitz e per i nevi con atipia istologica, ma anche per i nevi istologicamente comuni 4. Come riportato da numerosi studi, i caratteri clinici di un nevo non correlano con i caratteri istologici 5; quindi anche un nevo istologicamente comune può mostrare alcuni o tutti i segni dell’ABCD del melanoma, rappresentando talvolta una causa di ansietà sia per il paziente sia per il medico. Abbiamo cercato di valutare se la dermoscopia può aiutare a raggiungere, rispetto al solo esame visivo, una miglior selezione delle lesioni melanocitarie da sottoporre ad escissione per verifica diagnostica, escludendo i nevi istologicamente comuni. Poiché il rischio di misclassificazione da parte dei patologi fra nevi con atipia e melanoma iniziale è stato dimostrato da parecchi Autori 6, 15, una riduzione ulteriore di falsi positivi evitando l’ escissione di nevi con atipia istologi- Vol. 140 - N. 4 CHIARUGI ca, quando siano dubbi clinicamente, sembra, al contrario, meno auspicabile. Analizzando il training set è stato evidenziato che due caratteristiche dermoscopiche ricorrono più frequentemente nei nevi con atipia istologica piuttosto che nei nevi comuni: il reticolo pigmentario atipico/prominente e le strutture di regressione. Questa evidenza è in perfetto accordo con i dati forniti da un precedente studio del nostro gruppo condotto su una serie diversa di lesioni esaminate da un altro gruppo di osservatori 16. Anche il punteggio semiquantitativo, assegnato in accordo con i due più popolari algoritmi semplificati, la regola dell’ABCD della dermoscopia 9 e la nuova 7 – Point Checklist 10, differiva significativamente fra i due sottogruppi di nevi per entrambe i valori soglia utilizzati per ciascun metodo (4 e 4,75 per l’ABCD, 2 e 3 per la 7 – Point Checklist). Questo risultato è in disaccordo con un precedente studio dove non è stata rilevata nessuna differenza significativa, utilizzando la regola dell’ABCD, fra nevi con atipia e nevi senza atipia 16. Fra le possibili spiegazioni emerge la differente provenienza delle lesioni (nel precedente studio erano incluse sia lesioni clinicamente equivoche che lesioni non equivoche), e una diversa procedura adottata nell’analisi dei dati (confronto della mediana nello studio precedente, confronto fra frequenze in categorie prestabilite nel presente studio). Tuttavia, analizzando il test set, solamente il punteggio della 7 – Point Checklist ha confermato una differenza statisticamente significativa fra i due sottogruppi di nevi. Analizzando i sottotipi dermoscopici dei nevi di Clark 11 nel test set non è emersa nessuna differenza significativa. In questo contesto si può pensare che la nostra serie di lesioni fosse troppo piccola e che quindi i dati debbano essere interpretati con cautela. È possibile che la nostra serie di lesioni sia piccola per poter raggiungere un potere statistico sufficiente dato il grande numero di categorie proposte per la classificazione dei nevi da Hofmann-Wellenhof et al. 11. I nostri dati attenuano molto le aspettative di questa classificazione dei nevi di Clark: nell’opinione dei proponenti «questa classificazione dovrebbe essere guardata non solo come un esercizio morfologico accademico ma come un sistema classificativo che condurrà ad una miglior conoscenza delle caratteristiche biologiche di questi nevi melanocitici» 11. Il fatto che non sia stata trovata nessuna differenza statisticamente significativa nella frequenza dei sottotipi della classificazione di Hofmann-Wellenhoff fra nevi con atipia e nevi comuni limita fortemente la sua validità nello studio delle caratteristiche biologiche delle lesioni melanocitiche benigne. Riassumendo, secondo il training set, gli osservatori possono essere in grado di identificare, all’interno di un pool di nevi non distinguibili fra loro per le caratteristiche cliniche, nevi con atipia istologica e nevi senza atipia. Il «prototipo» di nevo con atipia dovrebbe quindi essere un nevo che presenta un reticolo pigmentario atipico e/o strutture di regressione, che ha un punteggio >2 secondo la 7 – Point Checklist e >4 secondo la regola dell’ABCD della dermoscopia. Questo scenario è stato in parte confermato dall’analisi del test set: con i due osservatori non a conoscenza della diagnosi GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 357 CHIARUGI THE PROBLEM OF CLINICALLY ATYPICAL NEVI SUBMITTED TO VERIFICATION BIOPSY istologica, solamente il reticolo pigmentario atipico e il punteggio secondo la 7 – Point Checklist ≥2 o ≥3 hanno confermato la loro differente distribuzione fra i due sottogruppi di nevi. Nell’analisi del test set né le strutture di regressione né il punteggio secondo l’ABCD si sono mantenuti associati all’atipia istologica. Fra i punti di forza di questo studio vogliamo sottolineare il disegno dello studio, strutturato con un training set ed un test set; questo porta a verificare prospetticamente fino a che punto ciò che è stato trovato nel training set in aperto dagli osservatori viene confermato in una nuova serie di lesioni esaminate in cieco dai medesimi osservatori. Inoltre, questo studio include solo nevi asportati per verifica diagnostica sulla base delle caratteristiche cliniche, avvicinandosi quindi quanto più possibile alla seduta diagnostica che avviene nella pratica. Poiché in questo studio le caratteristiche cliniche dei nevi con atipia istologica sono simili a quelle dei nevi senza atipia abbiamo ridotto al minimo il rischio di fare una classificazione influenzata da fattori clinici che agiscano da confondenti. Come punti deboli dello studio menzioniamo il fatto che questo studio è basato su una classificazione delle lesioni fatta su immagini fotografiche, in una sessione diagnostica a posteriori. Al fine di capire quali conseguenze possano avere nella pratica clinica queste evidenze, abbiamo calcolato il valore predittivo negativo associato a ciascuno dei caratteri precedentemente citati, vale a dire la probabilità che un nevo che non abbia tali caratteri sia istologicamente comune. Sfortunatamente il NPV non è risultato mediamente più del 60% per ogni carattere dermoscopico selezionato; ciò significa che quando un osservatore predice che un nevo mancante dei sopra citati criteri di atipia è istologicamente comune, ciò sarà vero solamente nel 60% dei casi. In futuro, studi randomizzati potrebbero confermare se i criteri dermoscopici associati ad atipia istologica sopra menzionati, come il reticolo pigmentario atipico, le strutture di regressione, il punteggio della regola dell’ABCD e della 7 – Point Checklist possono aiutare i dermatologi a modificare la gestione delle lesioni pigmentate, con un minor numero di escissioni di nevi istologicamente comuni, ma clinicamente equivoci. 358 Riassunto Obiettivo. La diagnosi precoce del melanoma cutaneo è associata a dei costi in termini di diagnosi di falsi positivi, cioè lesioni pigmentate benigne definite sospette o equivoche all’esame clinico e sottoposte a verifica bioptica. Scopo dello studio è indagare il possibile ruolo della dermoscopia nel selezionare i nevi istologicamente comuni da quelli con atipia istologica. Metodi. Duecentosessantaquattro nevi clinicamente atipici sono stati classificati da patologi esperti come nevi con atipia istologica e nevi senza atipia istologica. Due osservatori hanno analizzato le caratteristiche dermoscopiche utilizzando la classica analisi di pattern e gli algoritmi semplificati. La popolazione oggetto dello studio è stata divisa in un training set e in un test set (osservazione in aperto e in cieco rispettivamente riguardo alla classificazione istologica). Risultati. Nel training set i nevi con atipia istologica differivano significativamente dai nevi comuni, avendo mostrato con frequenza maggiore i seguenti criteri dermoscopici: reticolo pigmentario atipico, strutture di regressione, ABCD score ≥4 e seven point check list ≥2. Nel test set, tuttavia, solamente il reticolo pigmentario atipico e il seven point score ≥2 si sono mantenuti più frequenti nei nevi atipici che nei nevi comuni. Il valore predittivo negativo è risultato inferiore al 70% per ciascun carattere dermoscopico selezionato. Questo significa che quando un osservatore predice che un nevo mancante dei suddetti criteri di atipica è istologicamente comune, ciò sarà vero in meno del 70% dei casi. Conclusioni. La dermoscopia può giocare un ruolo nel rilevare lesioni melanocitiche banali, come i nevi senza atipie istologiche, all’interno di un pool di lesioni clinicamente equivoche sottoposte a verifica bioptica. Studi ulteriori (studi prospettici randomizzati) sono necessari per indagare l’impatto della dermoscopia nella pratica reale per una miglior selezione delle lesioni da asportare. Parole chiave: Demoscopia - Lesioni pigmentate - Nevi atipici - Falsi positivi. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 G ITAL DERMATOL VENEREOL 2005;140:359-72 The Italian Registry of Hereditary Epidermolysis Bullosa G. TADINI 1, L. GUALANDRI 2, M. COLOMBI 3, M. PARADISI 4, C. ANGELO 4, G. ZAMBRUNO 4, L. CASTIGLIA 4, G. ANNICCHIARICO 5, M. EL HASHEEM 6, S. BARLATI 3, R. GARDELLA 3, L. NALDI 7, E. BONIFAZI 8, L. GAROFALO 8, G. MORETTI 9, R. CAVALLI 1, S. CAMBIAGHI 1, S. PERCIVALLE 1, M. BELLINVIA 1, A. DI BENEDETTO 1, A. LOCATELLI 1, L. LUNARDON 1, E. BRUNI 1, A. PATRIZI 10, G. LEMBO 11, T. CAINELLI 12 Aim. At present, in Italy no exhaustive epidemiological study exists on inherited epidermolysis bullosa (EB). The necessity to have an exact evaluation of Italian cases encouraged the setting up of the national Registry, in order to collect all notifiable cases of this disease, with important implications in clinical knowledge, the development of prenatal diagnosis instruments and the start of epidemiological genetic studies. Methods. A hospital registry has been prepared: initially, it collected the cases already known to study centers in the period 1985-1993; then, it collected the new cases, until December 31, 2002. The registry envisaged the presence of a data coordinating center (Dermatology Clinic in Bergamo), 3 regional centers (CMCE in Milan, IDI in Rome and Bari Hospital) that collected patients from North, Center and South Italy respectively, and DEBRA Italy. Results. In total, 697 cases have been notified (9 not yet classified), with 28% epidermolytic EB, 10% junctional EB and 62% dermolytic EB. EB incidence at December 31, 2002 was 0.1 new cases per million live births; prevalence at December 31, 2002 was 10.1 affected patients per million Italians. Conclusion. This epidemiological evaluation is representative of the Italian situation; from these data a geographic distribution of the disease in our country can be traced, with significant effects on prevention strategy. KEY WORDS: Hereditary epidermolysis bullosa - Registry - Incidence - Prevalence. Paper presented at the GISED National Congress, October 3-6, 2001, Genoa, Italy. Fundings. Grant offered by Lions Club Multidistretto Italy - Leo Club Telethon. Address reprint requests to: Dr. G. Tadini, Institute of Dermatological Sciences, Center for Inherited Cutaneous Diseases, University of Milan, IRCCS, Ospedale Maggiore Mangiagalli e Regina Elena, Via Pace 9, Milan, Italy. Vol. 140 - N. 4 1Institute of Dermatological Sciences, Center for Inherited Diseases University of Milan, IRCCS, Ospedale Maggiore Policlinico Mangiagalli e Regina Elene, Milan, Italy Department of Dermatology I, IRCCS, Milan, Italy 2Department of Dermatology IV S. Paolo Hospital, Milan, Italy 3Unit of Biogenetics Department of Biomedical and Biotechnological Sciences University of Brescia, Brescia, Italy 4Istituto Dermopatico dell’Immacolata, Rome, Italy 5Associazione Pugliese Epidermolisi Bollose, Bari, Italy 6Department of Pediatric Dermatology Bambin Gesù Pediatric Hospital, Rome, Italy 7Department of Pediatric Dermatology V University of Milan, City Hospital, Bergamo, Italy 8Department of Pediatric Dermatology University of Bari, Bari, Italy 9DEBRA Italy, Catania, Italy 10Department of Dermatology S. Orsola Hospital, Bologna, Italy 11Department of Dermatology University of Naples, Naples, Italy H ereditary epidermolysis bullosa (EB) represents a heterogeneous group of genetically determined mechano-bullous dermatosis. Though these diseases are rare in the general population, they have great clinical relevance because of the possible reduction in life quality and life expectancy they could cause. The common clinical features of all EB is the marked cutaneous and mucous fragility, which lead to blister GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 359 TADINI HEREDITARY EPIDERMOLYSIS BULLOSA ITALIAN REGISTRY DERMO-EPIDERMICAL JUNCTION AND FORMS OF EB BASAL TONO-FILAMENTS KERATINOCYTE EMI-DESMOSOME Lamina lucida Keratin K5 K14 EBS Plactin Iam 5, α6β4, BP180 ANCHORAGE FILAMENTS EBG Lamina densa Sub Lamina densa ANCHORAGE FIBERS VII coll. EBD Figure 1.—Pattern of dermo-epidermal junction and proteins implicated in the pathogenesis of inherited epidermolysis bullosa. formation after minimal traumatism. The adnexial structures could be involved, resulting sometimes in a partial or complete absence. Depending on the ultrastructural level within which the cleavage plane of the blister occurs, we can classify epidermolytic, junctional and dermolytic EB. This is possible with immunohistochemical assays and with electronic microscopy. In epidermolytic EB, the molecular defect involves keratins K5 and K14 and plectin, a protein which is also present in the neuromuscular plaque, inducing a cleavage at the basal level of the epidermis. In junctional EB the altered molecules are laminin 5 (chain alfa 3, beta 3, gamma 2), collagen XVII and the integrins alfa 6 and beta 4; the cleavage is at the level of lamina lucida. In dermolytic EB the molecular defect involves collagen VII and the cleavage occurs below lamina densa 1 (Figure 1). 360 From a clinical point of view, the bullous manifestations of epidermolytic EB tend to resolve without any scars or milia, and generally don’t affect extracutaneous areas with the exception of the oral mucosa (Figure 2-4). Epidermolytic EB is divided into 4 major subgroups (Table I), each of them with characteristic aspects (Table II). In junctional EB blisters resolve with difficulty and tend to result in atrophic lesions without retractions or milia; in some subtypes the extracutaneous involvement (mucosa of the gastroenteric, respiratory and genitourinary tract) could be impressive and give rise to severe complications which could also provoke death. Dermolytic EB types are characterized by scarring, which in time could cause retractions and contractures of limbs and sometimes also esophageal strictures.2 Until now epidemiological studies about EB have been mostly incomplete or regard only small samples GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 HEREDITARY EPIDERMOLYSIS BULLOSA ITALIAN REGISTRY Figure 2.—Image showing epidermolysis bullosa of the back. TADINI Figure 3.—Infant affected by epidermolysis bullosa. or some subgroups of the disease, thus giving only information which is not very indicative.3 The purpose of the Italian EB Registry is to fill this gap, promoting the development of clinical knowledge on EB by solving the problems related to the extreme rarity of this disease. Therefore, the purpose of the register is to: — centralize important clinical information; — develop a uniform clinical and pathological approach to the disease (creation of reference institutes); — promote systematic genetical studies (through a network of laboratories collaborating with the Registry); — assess periodically the cases included in the Reg- Figure 4.—Epidermolysis bullosa of the feet. istry, using standardized methods; — collaborate with the “rare diseases” study group of the Superior Institute of Health in order to promote was essential for the realization of this project, which knowledge on EB and its social and medical impact. made it possible to calculate the incidence and the The creation of a register for EB was suggested prevalence of EB in the Italian population. In order to about 10 years ago by the Center for hereditary cuta- get an epidemiologically valid result, in 1992 the Genneous diseases of Milan, because of the need for an odermatoses Project was set up. This project involved more than 500 dermatologists who were not members exact estimation of Italian EB patients. The collaboration of the Italian Group for Epi- of the National Health service in order to reach as demiologic Studies in Dermatology, of many univer- many EB patients as possible. Part of this project was sity clinics and hospitals and of some dermatologists the distribution of educational material on genoder- Vol. 140 - N. 4 GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 361 TADINI HEREDITARY EPIDERMOLYSIS BULLOSA ITALIAN REGISTRY TABLE I.—Classification of the main inherited EB. Major type of EB Major subtype of EB EB epidermolytic (EB simplex, EBS) Protein/gene system EBS, Weber-Cockayne (EBS-WC) EBS, Koebner (EBS-K) EBS, Dowling-Meara (EBS-DM) EBS with associated muscular dystrophy (EBS-MD) EB type “Ogna”(without muscular dystrophy) JEB, Herlitz (JEB-H) JEB, non-Herlitz (JEB-nH) JEB with associated pyloric atresia (JEB -PA) DDEB RDEB, Hallopeau-Siemens (RDEB-HS) RDEB, non-Hallopeau -Siemens (RDEB-nHS) EB junctional (JEB) EB dermolytic (EB dystrophica, DEB) K5, K14 K5, K14 K5, K14 Plectin Plectin Laminin-5 Laminin-5; collagen XVII α6β4 integrin+ Collagen VII Collagen VII Collagen VII TABLE II.—Classification of rare inherited EB. Major EB type Major subtype EB Protein/gene system EB epidermolytic (EB simplex, EBS) EBS “mottled pigmentation” (EBS-MP) EBS autosomal recessive without muscular dystrophy (EBS-AR) EBS superficialis (EBSS) JEB, inversa (JEB-H) JEB, delayed outbreak (JEB-nH) DDEB, pretibial (DDEB-Pt) DEB, transient dermolysis bullosa of newborn (DEB-TBDN) DDEB or RDEB, “pruriginosa” (DDEB-Pr ; RDEB-Pr) RDEB, inversa (RDEB-I) RDEB, centripetalis (RDEB-Ce) DEB, AD/AR heterozygote K5 K14 Unknown Laminin-5 Unknown Collagen VII Collagen VII Collagen VII Collagen VII Unknown Collagen VII EB junctional (JEB) EB dermolytic (EB dystrophica, DEB) matoses and the explanation of how to fill in the recruitment cards. Prospects The register will give the opportunity to integrate different competencies in the study of EB and, on the other hand, the possibility to build a representative cohort of EB cases. The advantages will be as follows: — estimation of genetic heterogeneity within homogeneous diagnostic groups, and study of the relationship between specific mutations, phenotypes and clinical evolution in a specific diagnostic group. The study of genetic heterogeneity is important for the development of prenatal diagnosis; — possibility to organize studies and therapeutic trials with a large group of patients, which make these studies and trials statistically significant; — possibility to estimate the incidence of some clinical subgroup in certain geographic areas, studying the possible differences between these areas and organizing studies of genetic epidemiology; 362 — estimate of the incidence of most severe complications and study of their management; — assessment of the causes of deaths. The register will help to address the important challenges offered by EB in all medical fields; from a methodological point of view it would be possible to develop new research instruments or to evaluate old ones (for example, it would be possible to evaluate the validity of single patient trials in therapeutic questions). The register could also promote, together with patient associations, information campaigns about EB. Materials and methods Definition of register In biomedical research “register” means: systematic and continuous collection of information regarding all reported cases of a given disease. There are 2 kinds of registers: population registers and hospital GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 HEREDITARY EPIDERMOLYSIS BULLOSA ITALIAN REGISTRY registers. In population registers the main interests are descriptive epidemiology (assessment of incidence) and the organization of medical resources available for a specific disease. Hospital registers (like that of EB), on the other hand, assess clinical problems such as the description of the medical evolution of a disease or the definition of its prognosis. These kinds of registers also allow the creation of a patient group, which is available for etiological, biological and therapeutic studies. The validity of data obtained from a register depends on the completeness of the information contained: so the validity of a hospital register depends on the possibility of containing all the cases observed in the structure. If there are registered cases of disease which are of good quality and with complete information and if they regard all the cases seen in the past, it is possible to use them for the register although the information was collected before the setting up of the registry (retrospective registry). This information is very useful if someone wants to reconstruct the natural history of a disease over a long period. Structure of the registry The Italian registry of EB, a hospital register, has been proposed to all Italian centers interested. During the first stage of the project, all the cases known to the participant centers until 1993 were collected. During the second stage, the register continued its activity by registering all new cases diagnosed. The structure of the registry envisages a center for the coordination of all data. In this center the Dermatological Clinic of Bergamo is responsible for the epidemiological aspects while the CMCE of Milan controls the clinical aspects and manages the whole project, collecting the data coming from all members taking part in the registry. Four supra-regional centers refer to this coordination center: CMCE of Milan, Istituto Dermopatico dell’Immacoloata (IDI) of Rome, Pediatric Dermatology Unit of the Hospital of Bari and the Dystrophic Epidermolysis Bullosa Recessiva Association (DEBRA) of Catania. Their first task has been to collect all patients present in the territories of pertinence, which are respectively the North of Italy, the Center, the South and the Islands. These centers also had to undertake laboratory assessments if necessary to confirm the diagnosis of the different EB sub-types. Vol. 140 - N. 4 TADINI Regarding this, every center had its own laboratory or collaborated with a laboratory which could perform histological, immunohistochemical, ultrastructural and molecular assays. Other hospital divisions and clinics joined these first reference centers in time, and so the collection web over the territory got bigger and wider. In the centers a new figure was created: the monitor (who already exists in the GISED). This figure is a dermatologist with experience in EB, making it possible to carry out a widespread targeted survey. The 80 monitors, who took part in the registry gave easily available and prompt information about the disease to all ambulatorial dermatologists, called observers, distributed all over Italy. These observers, who numbered between 350 and 500 per year, represented a grassroots web of information over the whole territory, which has identified and reported to the nearest monitor all cases compatible with a diagnosis of EB. Patient recruitment Previous to patient recruitment an informative campaign for dermatologists was carried out: training courses in genodermatoses, with an average of 400 participants per year, were organized and informative material about these rare diseases was distributed (“Project Genodermatoses”). Moreover, a promotional campaign for the general population was organized: gadgets were distributed in the streets of many cities and articles about this dermatosis were published in different magazines. All this was done with the help of the Italian Lions Club organization. Essential both for financial help and for marketing was the contribution of the TV program “Telethon”, which has made it possible to collect a conspicuous amount of money for the creation of many scholarships. Instruments for data collection Report cards were developed where physicians have to enter the personal data of the patient, the familiarity of the disease, the clinical history (symptoms, diagnostic procedures and therapies carried out) and, if possible, the supposed type of EB affecting the patient. These report cards were distributed to all members of the project during the meetings or by mail. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 363 TADINI HEREDITARY EPIDERMOLYSIS BULLOSA ITALIAN REGISTRY TABLE III.—Distribution of Registry cases by type and subtype of EB. Type/Subtype Epidermolytic EB — Koebner — Weber-Cockaine — Dowling-Meara — With muscular dystrophy Junctional EB — Non-Herlitz — Herlitz — With pyloric atresia Dermolytic EB — Cockaine-Touraine — Hallopeau-Siemens — Non Hallopeau-Siemens EB unclassifiable Frequency % 192 75 67 50 1 67 46 12 9 438 159 182 91 9 28 10.8 8.5 8.2 0.2 10 5.8 2.3 1.6 62 24.5 29 8.8 — Epidermol. EB (192, 28%) Junctional EB (67, 10%) N.B.: Unclassified EB are not calculated for incidence and prevalence. Dermol. EB (438, 62%) TABLE IV.—List of main Clinical Centers involved in the study. Clinical Centers — Center for Inherited Cutaneous Diseases Milan — Istitute Dermopatico dell’Immacolata (IDI) Rome — DEBRA Italy, Catania — Pediatric Dermatology Hospital, Bari — Department of Genetics, University of Brescia — Observers GISED — Gaslini Hospital, Genova — Dermatologic Department, Bologna — Federico II Hospital, Napoli Data used for the analysis To estimate EB prevalence and incidence in our country, all data contained in the registry regarding the period 1991-2002 were used. Diagnosis was based on the concordance with clinical and laboratory criteria described in the literature and validated in practice all over the world. Figure 5.—Total case of inherited epidermolysis bullosa. sification of EB presented by the Consensus Conference of Chicago in 1999: — epidermolytic EB: Koebner, Weber-Cockaine, Dowling Meara, muscular dystrophic-EB; — junctional EB: Herlitz, not-Herlitz, junctional with pyloric atresia — dermolytic EB: Cockaine-Touraine, HallopeauSiemens, not Hallopeau-Siemens. The incidence is calculated considering the number of live-born EB patients per 1 million births in the two-year period 1997-98. The prevalence is based on all living patients affected by EB in the year, the last year of the survey. Results Approach to analysis Total number of cases and contribution of every supraregional center All EB patients of the registry having adequate data, entered the survey. The diagnosis of every type of EB was confirmed with the help of immunofluorescence assays or electronic microscopy, which shows the cleavage plane of the blister, with the study of genetic transmission of the disease and, if possible, with bio-molecular assays. The patients were divided into the principal types and subtypes of EB which are described in the new clas- Table III and Table IV show all the cases observed in 10 years of registry activity and the contribution of the main supra-regional centers in data collection. At the end of 2002 the patients affected by EB were about 697 clearly defined cases and 9 non-classifiable cases (Figure 5). This last number depends on the fact that in the study some case reports were considered which were taken before the project had been found and which were therefore incomplete. 364 GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 HEREDITARY EPIDERMOLYSIS BULLOSA ITALIAN REGISTRY TADINI Muscl. dystr. (1%) Herlitz (22%) Pyloric atrophy (16%) Dow-Meara (30%) Koebner (38%) Web-Cock (31%) Figure 6.—Table of data concerning epidermolytic EB. These percentages refer to total number of epidermolytic EB and not to the total of cases picked up. Non Herlitz (62%) Figure 7.—Table of data concerning the junctional EB. These percentages refer to the total number of junctional EB and not to total cases collected. The Italian registry has operated constantly up to Non Hallopeau (15%) now.4 The distribution of cases between the different types and subtypes of EB, according to the up to date classification,5 is the following: — 192 cases of epidermolytic EB (28% of the total); within this group the ratio of different subgroups fluctuates from 8.2% for Dowling Meara to 10.8% for Cockayne Hallopeau Koebner (Figure 6); Touraine (46%) — 67 cases of junctional EB (10%) of the total (Fig(39%) ure 7); the non-Herlitz cases represent 5.8% of cases. It is important to point out that in all 9 cases of junctional EB with pyloric atresia (1.6%) the genetic mutation was identified. The dermolytic forms represent most of the cases in the Italian Registry: 438 patients (62%). Among these the recessive form named Hallopeau- Figure 8.—Table of the data concerning dermolytic EB. These percentaSiemens, which represents 29% of all cases, together ges refer to total number of dermolytic EB and not to total cases collected. with the dominant form of Cockaine-Touraines, which represents 24.5% of all cases, account for 53.5% of all is calculated as 20.1 per million live newborns. If we patients with dermolytic EB (Figure 8). consider the main groups of EB, we find that the incidence of dermolytic EB is of 12.4 per million. The Incidence and prevalence incidence of junctional and epidermolytic EB is simIn the period 1997-98, taken as a sample recruit- ilar: 3.8 per million. In the period 1997-1998, taken as a sample period, ment period, 21 newborns affected by EB were observed. At the same time 1 044 340 healthy new- the prevalence was of 10.1 EB patients per million (in borns were registered in Italy: so the incidence of EB a population of 57 679 855 people). Vol. 140 - N. 4 GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 365 TADINI HEREDITARY EPIDERMOLYSIS BULLOSA ITALIAN REGISTRY Table III shows epidemiologic data using the new classification. Discussion Knowledge of the epidemiology of hereditary EB is approximate for several reasons: 1) Only few countries have worked on this kind of studies and among these are Japan,3 Finland,6 Northern Ireland,3 South Africa,7 Sweden and Norway,8 Scotland and Great Britain 9 and recently the USA.3 2) The epidemiologic studies considered parts of the population or only some regions of a country. Some studies also considered only one subset of EB 3) In general, data are old and/or not recently revised. All these factors don’t allow us to know the situation of all the patients with EB in a country accurately. For example, in Croatia only 56 patients of 50 families were considered suggesting an incidence for EBDr H-S of 19.2/1milion newborns.10 In Norway, in 1995, the incidence of Epidermolytic EB was estimated to be 42 cases/1million without considering the different clinical subtypes of Epidermolytic EB. In the USA the prevalence was obtained considering the data from the American DEBRA: 327 newborns with EB from 1986 to 1991 with an incidence of 16.62/1milion newborns,3 these results are not complete because they consider only the severe forms of EB, excluding milder subtypes. Later, in the USA, methods for recruitment were reinforced and an incidence of 19.60/1milion newborns was obtained: 10.75 with EBE, 2.86 with EBDd, EBDr and EBJ 2.04 each. We note that in Croatia the incidence of EBDr H-S was 47 times more than in the USA (19.23 vs 0.43) because of different methods for epidemiologic studies in different countries. In the USA in 1990 the prevalence was 2 044 patients (8.22/1milion). It is very important to consider that our prevalence data (10.1) are very similar to the data reported in the USA Registry and underline the relevance of the 2 Registries, even in the presence of a different socio-sanitary and geographical environment. Nevertheless, the recruitment of patients in the foreign Registries does not follow an adequate sampling of the population given that the recruitment itself does not refer to peripheral Centers distributed in the whole territory. 366 In Italy, before the present collection, incidence and prevalence data were not available and were estimated on the basis of foreign data. The Italian Registry enables a widespread sampling of patients with EB to be carried out, recruiting also the highest possible number of milder and paucisymptomatic cases (i.e. familiar mild forms of palmo-plantar Epidermolytic EB recruited by peripheral- dermatologists or by the Centers of Military Medicine) that often in this kind of investigation are underestimated because of the relative paucity of their symptoms. Our information reflects the real situation of patients with EB in Italy and this is very important for social, clinical and scientific purposes. Knowing the clinical features of EB and how they are spread all over Italy, we are able to organize medical centers for the management of all these patients with a chronic disease; that’s why the General Institute of Health promoted this study and now the Italian Registry of EB is part of the Register of Rare Diseases of the Ministry of Health. From a scientific point of view all the data are a rich store of knowledge about all the features of this pathology: — clinical features and all their variants in every group or subgroup of EB; — traditional and molecular diagnostic trials; — study of recurrent or ancestral mutations in the Italian population; — examination of recurrent pathologies, especially cancer, and management of their prevention, therapy and follow-up. Finally, in perspective, we can hypothesize a significant impact in the strategy of genetic counseling which should classify EB patients following the molecular classification in order to find all carriers in the families of EB affected patients and to perform molecular prenatal diagnosis when requested in lethal or very severe forms of EB. At this point we remember that 70 mutations were discovered in Italian patients with EBJ and EBD in the 2 main laboratories in Rome (Istituto Dermopatico dell’Immacolata) and Brescia (Institute of Genetics). Mortality rates Until today it was possible to make a rough calculation of the mortality rate only in patients of the Center of Inherited Diseases of Milan representing 44.7% of all the patients considered in Italy: 18 patients died in GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 HEREDITARY EPIDERMOLYSIS BULLOSA ITALIAN REGISTRY TABELLA V—Mortality of patients at Inherited Cutaneous Disease Center (Milan). Number of deaths (period 1992-2002) on the basis of epidermolysis bullosa subtypes Epidermolytic Junctional Dermolytic — 8 10 Total 18 the 13 years of the survey in a group of 263 patients (mortality rate = 6.8%). Of these, 8 had an EBJ and 10 an EBD (Table V). Of the patients with EBJ, 2 had EBJ with pyloric atresia and 6 had the variant of Herlitz, of these 2 died when they were 6 and11 year-old, and 6 did not pass the first year of life. The most frequent causes of death were infections, especially in EBJ, and metastasis from squamous cell carcinomas of the skin ( 8-10 patients with EBD died in adult age). The last 2 EBD patients died soon after birth due to sepsis. Complications A lot of complications have been described in patients with EB; our data refer only to 2 of the most frequent and severe of them, squamous cell carcinomas and esophageal stenosis. Of the 263 patients of the Center of Milan 7 patients (2.7%) presented squamous cell carcinomas and 2 of them died due to metastases. Of these, 6 patients had a squamous cell carcinoma and only 1 had a basal cell carcinoma. Twenty-five patients (9.5%) presented an esophageal stenosis. Conclusions The incidence and prevalence data are representative of the Italian situation given that all the severe cases have been collected and we think that almost every case with milder forms of EB have also been identified using the widespread network of dermatologists throughout Italy. In fact, no other registry or collection of patients shows such an accurate or complete sampling as ours. The methods of recruitment of the Italian Registry allow us to state that just a very small percentage of cases, of little consequence for the prevalence and incidence data, escaped recruitment. Vol. 140 - N. 4 TADINI The rarity of these genodermatoses and their severity, considered from the human, medical or social point of view, forced us to create a structure devoted to the collection of all data regarding EB. From the epidemiological data, we can derive the geographical distribution of the patients nationally and its relationship with medical and social organizations. EBs are genetically determined diseases with genetic variability much greater than clinical and the respective data are available for anyone interested in these fields at any level. We can hypothesize a significant impact in the strategy of prevention, especially for the identification of the mutations in all EB families in order to find carriers and avoid consanguineous marriages. Comparison with the epidemiological data of other Registries may be of great utility. Finally, the medical staff of the Italian Registry of EB promotes a European Registry of Epidermolyss Bullosa. Acknowledgments.—The compilation of the Italian Registry of Hereditary Epidermolysis has been made possible by the contribution of about 600 Italian dermatologists that worked together giving their time and experience to this project. We thank all contributors, wishing to have them again as companions in the future projects of other epidemiological studies on genodermatosis. References 1. Lin AN, Carter DM. Epidermolysis bullosa: basic and clinical aspects. New York: Springer; 1992. 2. Tadini G, Brusasco A, Cambiaghi S, Camozzi S, Cavalli R, Restano L. Epidermolisi bollose ereditarie - Ittiosi. Milano: EdiSES; 1995. 3. Fine J-D, Bauer EA, Mc Guire J, Moshell A. Epidermolysis bullosa: clinical, epidemiologic and laboratory advances, and the findings of the National Epidermolysis Bullosa Registry. Baltimore: John Hopkins University Press; 1999:101-13. 4. Tadini G, Naldi L, Locati L, Cainelli T. Epidemiological survey on epidermolysis bullosa in Italy. J Invest Dermatol 1994;103:853. 5. Fine J-D, Eady RAY, Bauer EA, Briggman RA, Bruckner-Tuderman L, Christiano A et al. Revised classification system for inherited epidermolysis bullosa Report of the Second International Consensus Meeting on Diagnosis and Classification of Epidermolysis Bullosa. Special report. J Am Acad Dermatol June 2000;42: 1051-66. 6. Kero M. Occurrence of epidermolysis bullosa in Finland. Acta Derm Venereol (Stockh) 1984;64:57-62. 7. Winship IM. Epidermolysis bullosa in South Africa: formation of a National Registry. Epidermolysis Bullosa. A comprehensive review of classification management and laboratory studies.Crowthorne, Berkshire, UK: DEBRA; 1990. p.134-6. 8. Gedde-Dahl Jr T. Epidermolysis bullosa. A clinical, genetic and epidemiological study. Universitets Forlaget (Oslo). Baltimore: The John Hopkin Press; 1971. 9. Davison BCC. Epidermolysis bullosa. J Med Genet 1965;2:233-42. 10. Pavicic Z, Kmet-Vizintin P, Kansky A, Dobric I. Occurrence of hereditary bullous epidermolysis in Croatia. Pediatric Dermatol 1990; 7:108-10. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 367 TADINI HEREDITARY EPIDERMOLYSIS BULLOSA ITALIAN REGISTRY Il registro italiano delle epidermolisi bollose ereditarie L e epidermolisi bollose (EB) ereditarie costituiscono un gruppo eterogeneo di patologie meccanobollose geneticamente determinate. Sono dermatosi rare nella popolazione generale, ma di grande rilevanza clinica, in quanto possono determinare una notevole riduzione della qualità di vita e dell’aspettativa di vita dei pazienti affetti. Le peculiarità cliniche comuni alle EB sono rappresentate dalla marcata fragilità cutanea e mucosa, per cui anche il minimo trauma determina l’insorgenza di lesioni bollose. Gli annessi sono coinvolti in modo più o meno marcato, risultando in alcuni casi mancanti. Si distinguono EB epidermolitiche, giunzionali e dermolitiche in base al sito di clivaggio nella regione della giunzione dermo-epidermica, evidenziabile con tecniche immunoistochimiche (immunofluorescenza diretta) e con la microscopia elettronica. Nelle EB epidermolitiche il difetto molecolare interessa le citocheratine K5 e K14 e la pectina, una proteina non collagenica presente anche nelle giunzioni neuromuscolari, determinando il clivaggio a livello della membrana basale dei cheratinociti; le EB giunzionali conseguono all’alterazione genetica della laminina 5 (catene α3, β3 e γ2), del gene del collagene XVII e delle integrine α6 e β4, e le dermolitiche del collagene VII, con conseguente separazione a livello della lamina lucida e al di sotto della lamina densa, rispettivamente 1 (Figura 1). Dal punto di vista clinico le lesioni bollose di tipo epidermolitico tendono a guarire rapidamente senza esiti cicatriziali o grani di «milium», generalmente senza coinvolgere sedi extracutanee, se non la mucosa orale (Figure 2-4): all’interno di questo gruppo esistono comunque 4 varianti principali (Tabella I), ciascuna con aspetti peculiari (Tabella II). Nelle EB giunzionali le bolle riepitelizzano con difficoltà, ed esitano in lesioni atrofiche, ma senza cicatrici retraenti o grani di milium; in alcune varianti il coinvolgimento extracutaneo (mucose degli apparati gastroenterico, respiratorio, genitourinario) può essere imponente, con complicanze sistemiche anche molto gravi o letali. Tipici delle forme dermolitiche sono gli esiti cicatriziali, la cui continua formazione può provocare nel tempo retrazioni e contratture degli arti interessati o restringimenti esofagei 2. Le stime epidemiologiche sulle EB condotte fino a questo momento sono scarse e incomplete, o comunque relative solo a piccoli campioni o a poche varianti della malattia, fornendo perciò dati parziali e poco significativi 3. Obiettivo generale della costituzione di un Registro italiano delle EB è quello di favorire lo sviluppo delle conoscenze cliniche sulle EB superando i problemi connessi con la rarità di tali patologie. A tal fine il Registro prevede: — la centralizzazione delle informazioni cliniche rilevanti; — lo sviluppo di un inquadramento clinico-patologico uniforme e riproducibile (revisione esperta di casi, istituzione di Centri di riferimento clinico all’interno del Registro); 368 — la conduzione di indagini genetiche sistematiche (attraverso una rete di laboratori di biologia molecolare che collaborano al registro); — la valutazione clinica periodica dei casi inseriti nel Registro, secondo modalità il più possibile standardizzate; — l’adesione al gruppo di studio dell’Istituto Superiore di Sanità sulle malattie rare per portare a conoscenza delle Istituzioni preposte la realtà delle epidermolisi Bollose e il loro impatto socio-sanitario. La proposta di creazione di un Registro delle Epidermolisi Bollose Ereditarie è stata fatta circa 10 anni fa dal Centro Malattie Cutanee Ereditarie di Milano, in seguito alla necessità di avere una stima quanto più vicina al vero della casistica italiana dei pazienti affetti da EB. La collaborazione con il Gruppo Italiano Studi Epidemiologici in Dermatologia (GISED), con numerosi centri universitari e ospedalieri, e con dermatologi ambulatoriali ha consentito la realizzazione di tale progetto, rendendo disponibili i valori di incidenza e prevalenza nella popolazione italiana. Per ottenere un risultato epidemiologicamente valido nel 1992 è stato organizzato il «Progetto Genodermatosi» che ha consentito di programmare il coinvolgimento di più di 500 dermatologi ambulatoriali esterni del Sistema Sanitario Nazionale, nell’intento di allargare la base di reclutamento dei pazienti con EB. Il Progetto veniva completato con una parte didattica sulle Genodermatosi in generale, compresa la fornitura di materiale didattico e l’informazione sulla compilazione delle schede di arruolamento dei pazienti affetti da EB. Prospettive Il Registro offrirà la possibilità di integrare differenti competenze nello studio delle EB e permetterà di costruire una coorte rappresentativa dei casi di EB. I vantaggi sono evidenti; sarà infatti possibile: — valutare la presenza di un’eterogeneità genetica all’interno di categorie diagnostiche omogenee, studiando la relazione tra specifiche mutazioni, fenotipo e storia evolutiva delle singole entità diagnostiche (prognosi). La documentazione di un’eterogeneità genetica ha particolare importanza per lo sviluppo di strumenti di diagnosi prenatale; — avviare studi e trials terapeutici su una larga base di pazienti, con disegni formali che garantiscano validità e riproducibilità dei risultati e potere statistico sufficiente; — stimare in aree campione l’incidenza delle varietà clinico-patologiche più frequenti, valutando eventuali differenze tra aree geografiche ed avviando studi di epidemiologia genetica; — analizzare l’incidenza delle complicazioni più severe e il loro conseguente trattamento; — studiare le cause dei decessi. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 HEREDITARY EPIDERMOLYSIS BULLOSA ITALIAN REGISTRY Il Registro potrà fungere da catalizzatore per affrontare problemi complessi all’interfaccia tra differenti discipline; da un punto di vista metodologico potranno essere sviluppati strumenti di ricerca originali o potranno essere valutati strumenti esistenti (ad esempio, valutazione della validità di disegni del tipo «single patient trial» in ambito terapeutico). Il Registro potrà inoltre promuovere, in collaborazione con le Associazioni dei pazienti, un’attività di informazione e documentazione sulle EB. Materiali e metodi Definizione di registro Si definisce «registro», nella ricerca biomedica, la raccolta sistematica e continua di alcune informazioni su tutti i casi notificabili di una malattia. Si possono distinguere registri di popolazione e registri ospedalieri. Nei registri di popolazione prevalgono gli interessi di epidemiologia descrittiva (stime di incidenza nel tempo) e di pianificazione delle risorse sanitarie disponibili per la patologia in esame. I registri ospedalieri, come è quello delle EB, sono invece maggiormente orientati alla valutazione di problemi clinici come la descrizione della storia evolutiva della malattia e la definizione della prognosi, permettendo inoltre la costituzione di una base di pazienti per studi eziologici, biologici (banche biologiche) o di terapia. L’interesse e la riproducibilità delle informazioni ottenute in un registro dipendono dalla completezza della registrazione, per cui la validità di un registro ospedaliero dipende dalla possibilità di segnalare tutti i casi osservati presso l’istituzione. Tuttavia, quando sia garantita una buona qualità e completezza delle informazioni retrospettive e sia possibile recuperare informazioni su tutti i casi di malattia, è possibile utilizzare anche i casi diagnosticati in un definito periodo precedente l’avvio del Registro (registro retrospettivo). L’utilizzo di tali informazioni da tali casi è particolarmente utile quando si voglia ricostruire la storia naturale della patologia in esame su di un lungo periodo di tempo (studi di coorte in parte retrospettivi). Struttura del Registro Il Registro Italiano delle EB è stato proposto come registro su base ospedaliera a tutti i centri italiani interessati. In una prima fase sono stati raccolti tutti i casi noti ai Centri partecipanti fino al 1993. Successivamente il Registro ha proseguito la sua attività attraverso la segnalazione di tutti i nuovi casi diagnosticati (casi incidenti). La struttura del Registro prevede la presenza di un centro di coordinamento dei dati, gestito dalla Clinica Dermatologica di Bergamo per la parte epidemiologica e dal CMCE di Milano per la parte clinica, col compito di gestire e controllare l’intero progetto e raccogliere tutti i dati forniti dai diversi componenti del progetto stesso. A questo fanno riferimento 4 centri sovraregionali: il Centro Malattie Cutanee Vol. 140 - N. 4 TADINI Ereditarie di Milano, l’Istituto Dermopatico dell’Immacolata (IDI) di Roma, la Cattedra di Dermatologia Pediatrica dell’Ospedale di Bari e la Dystrophic Epidermolysis Bullosa Recessiva Association (DEBRA) Italia con sede a Catania. La loro funzione primaria è stata quella di individuare e raccogliere i pazienti provenienti dalle aree del territorio italiano di loro competenza, rispettivamente il Nord, il Centro, e il Sud peninsulare e insulare. Questi centri dovevano inoltre ricorrere, quando indicato, ad appropriate indagini di laboratorio per la conferma diagnostica del tipo di EB incontrata. A questo proposito, si sottolinea che ogni centro di riferimento era in contatto o disponeva di un laboratorio di analisi per l’esecuzione di indagini istologiche, immunoistochimiche, ultrastrutturali e molecolari. A queste strutture di riferimento si sono poi affiancati divisioni ospedaliere e cliniche universitarie, ampliando così la rete di arruolamento sul territorio. Nell’ambito di questi centri è stata istituita la figura del «monitor» (già operanti nella rete GISED), cioè quella di uno specialista dermatologo, che, dimostrando una conoscenza specifica di queste patologie, ha permesso di raccogliere una casistica ampia e selezionata. Gli 80 monitor che hanno preso parte al registro hanno inoltre fornito una consulenza pronta e facilmente raggiungibile ai colleghi dermatologi ambulatoriali, denominati «observers» e distribuiti sul territorio italiano. Questi, in numero variabile da 350 a 500 per anno, hanno costituito una rete informativa capillare su tutto il territorio, identificando e segnalando al più vicino «monitor» o centro clinico regionale tutti i casi certi o compatibili con diagnosi di EB. Reclutamento dei pazienti Il reclutamento dei pazienti è avvenuto mediante una campagna informativa rivolta ai dermatologi, nell’ambito della quale sono state organizzate riunioni di aggiornamento clinico, che hanno raccolto in media 400 partecipanti per anno, ed è stato stampato materiale didattico in cui erano descritte le più importanti caratteristiche di queste rare malattie (Progetto Genodermatosi). Inoltre è stata avviata una campagna promozionale anche nei confronti della popolazione generale, mediante la distribuzione di gadgets nelle piazze e la pubblicazione di articoli informativi sui diversi quotidiani, organizzata in cooperazione con il Multidistretto Lions Clubs Italia. Fondamentale, sia in termini economici che di risonanza pubblica, è stato anche il contributo derivato dalla trasmissione televisiva «Telethon», che ha permesso di raccogliere una cospicua quota di fondi utilizzati per il finanziamento di borse di ricerca. Strumenti per la raccolta dei dati Sono state sviluppate delle schede informative in cui venivano richiesti i dati anagrafici del paziente, la familiarità per tali malattie, la storia clinica (sintomi riferiti, indagini diagnostiche eseguite e terapie intraprese) e, ove possibile, una diagnosi orientativa per il tipo di EB espressa dal probando. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 369 TADINI HEREDITARY EPIDERMOLYSIS BULLOSA ITALIAN REGISTRY Le schede sono state distribuite a tutti i collaboratori del progetto, o durante gli incontri di aggiornamento o mediante mezzo postale a chi ne facesse richiesta. Dati utilizzati per l’analisi Per stimare la prevalenza e l’incidenza delle EB nel nostro paese, le analisi sono state eseguite su tutti i dati disponibili del Registro italiano dal 1991 al 2002. Le diagnosi si sono basate sulla concordanza con specifici criteri clinici e di laboratori descritte in letterature e ampiamente validate dall’utilizzo in tutto il mondo. Approccio all’analisi Lo studio di popolazione si è basato su tutti i pazienti affetti da EB facenti parte del Registro, sui quali fossero disponibili dati informativi adeguati. La diagnosi di ciascuna categoria principale di EB è stata confermata sulla base della sede di formazione del distacco dermo-epidermico identificabile a livello ultrastrutturale con l’immunofluorescenza e la microscopia elettronica, e sulla trasmissione genetica della malattia e, dove effettuata, anche sulla diagnosi di biologia molecolare (mutazione). I pazienti sono stati progressivamente suddivisi nei principali tipi e sottotipi di EB di seguito riportati seguendo la nuova classificazione per le EB redatta dalla Consensus Conference di Chicago del 1999: — EB epidermolitiche: Koebner, Weber-Cockaine, Dowling-Meara, epidermolitiche con distrofia muscolare associata. — EB giunzionali: non-Herlitz, Herlitz, giunzionale con atresia pilorica. — EB dermolitiche: Cockaine-Touraine, Hallopeau-Siemens, Non-Hallopeau-Siemens. L’incidenza viene calcolata in base al numero dei nati vivi affetti da EB per 1 000 000 nati in Italia nel biennio 1997-98, presi come biennio campione. Il calcolo della prevalenza si è basato sulla determinazione di tutti pazienti vivi affetti da EB nel corso dell’ ultimo anno dello studio, preso come anno campione. Risultati Casistica totale raccolta e contributo di ciascun centro sovraregionale Nelle Tabelle III-IV viene segnalato il totale dei casi accertati nei 10 anni di attività del Registro e il contributo dei principali Centri Clinici Sovraregionali nella raccolta della casistica. Alla fine del 2002 la stima dei pazienti colpiti da EB ammontava a 697 casi accertati e 9 casi di EB non ulteriormente classificabili (Figura 5). Quest’ultimo valore è legato al fatto che sono state esaminate anche segnalazioni antecedenti al progetto, le quali presentavano solo informazioni parziali, tali da non poter meglio inquadrare questi pazienti. 370 Il registro italiano rimane comunque, costantemente operativo 4. La distribuzione per tipo e sottotipo di EB, basata sulla più recente classificazione di tali genodermatosi 5, evidenzia per le forme epidermolitiche 192 casi, circa il 28% del totale; all’interno di questo raggruppamento i valori percentuali dei diversi sottotipi di EB oscillano tra 8,2% per la Dowling-Meara e 10,8% per la forma generalizzata di Koebner (Figura 6). Le EB giunzionali hanno formato una casistica di 67 pazienti, pari al 10% del totale (Figura 7); la variante non-Herlitz costituisce il 5,8% dei casi. È importante segnalare che nei 9 casi (1,6%) di EB giunzionale con atresia pilorica, è stata individuata la mutazione corrispondente. Le forme dermolitiche costituiscono la parte preponderante della casistica del registro italiano con 438 casi, pari al 62% del totale. In quest’ambito la variante recessiva grave di Hallopeau-Siemens con il 29% dei casi sommata a quella dominante di Cockaine-Touraine con il 24,5%, costituiscono ben il 53,5% di tutti i casi (Figura 8). Incidenza e prevalenza Nel corso del biennio 1997-98, preso come biennio campione, sono stati segnalati 21 nuovi casi di bambini nati vivi e affetti da EB. Nello stesso periodo i nati vivi e sani ammontavano nel nostro paese a 1 044 340. Il valore di incidenza che ne è derivato è di 20,1 nati affetti per milione di nati vivi. Se si considerano i principali gruppi di EB, troviamo che le forme dermolitiche in questi due anni mostravano un’incidenza pari a 12,4 nati affetti per 1 000 000 nuovi nati. Per le giunzionali e le epidermolitiche il valore di incidenza è lo stesso ed è pari a 3,8. La prevalenza, calcolata al 31 dicembre 1998, era pari a 10,1 pazienti affetti per milione di abitanti (calcolata su una popolazione di 57 679 855). Applicando la nuova classificazione, l’indagine epidemiologica definitiva fornisce i dati raccolti nella Tabella III. Discussione Le conoscenze sugli aspetti epidemiologici delle epidermolisi bollose ereditarie sono piuttosto approssimative per una serie di differenti ragioni. La prima risiede nel fatto che solo pochi paesi si sono impegnati in questi tipi di ricerca: tra questi, Giappone 3, Finlandia 6, Nord Irlanda 3, Sud Africa 7, Svezia e Norvegia 8, Scozia e Gran Bretagna 9 e recentemente anche gli Stati Uniti 3. La seconda ragione è legata al fatto che tali indagini epidemiologiche sono state fatte su campioni parziali di popolazione o prendendo in considerazione solo alcune regioni di uno stato. In alcuni inoltre, è stato considerato solo un determinato sottogruppo di epidermolisi bollose ereditarie, escludendo tutte le altre varianti. La terza e ultima ragione è che si tratta di stime non recenti o comunque non più aggiornate. Questi fattori, combinati fra loro, non consentono di stabilire una stima rappresentativa della reale situazione dei GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 HEREDITARY EPIDERMOLYSIS BULLOSA ITALIAN REGISTRY pazienti affetti da EB in quel determinato paese. Per esempio, in Croazia sono stati esaminati solo 58 pazienti di 50 famiglie, suggerendo un’incidenza per la forma dermolitica recessiva di Hallopeau-Siemens di 19,2 affetti per milione di nati vivi 10. In Norvegia, nel 1995, è stata calcolata un’incidenza delle EB epidermolitiche pari a circa 42 casi per milione, non considerando però i diversi sottotipi della forma stessa. Negli Stati Uniti la prevalenza è stata calcolata basandosi su dati forniti dalla DEBRA americana: è stato infatti riportato un totale di 327 nati affetti da EB nel periodo 19861990, con un’incidenza pari a 16,62 nati per milione di nascite 3. In realtà questi dati sono relativi a una maggioranza di casi di pazienti affetti dalle forme più severe, mentre gran parte dei casi meno severi non erano noti al progetto. I redattori del Registro USA, nel tentativo di fornire una stima più accurata, hanno perciò migliorato negli anni successivi le metodiche di reclutamento e di sviluppo di centri regionali di riferimento. Usando questo approccio, l’incidenza complessiva di EB è risultata pari a 19,60 nuovi casi per milione di nascite, con una quota maggiore di EB epidermolitiche (10,75), seguite dalla EB dermolitica dominante (2,86), dalla recessiva e dalla EB giunzionale (2,04 ciascuna). È interessante notare che l’incidenza stimata per la EB dermolitica tipo Hallopeau-Siemens in Croazia sia all’incirca 47 volte (19,23 vs 0,43) più alta di quella osservata negli Stati Uniti e ciò è spiegato proprio dai differenti sistemi di arruolamento dei pazienti e della precisione di tale raccolta. La prevalenza complessiva di EB ereditaria negli Stati Uniti nel 1990 è stata stimata essere 2 044 pazienti, corrispondenti ad un tasso di prevalenza di 8,22 casi di EB per milione di abitanti. È importante sottolineare che la nostra casistica e il nostro dato assoluto di prevalenza (10,1) sono molto simili a quelli riferiti dal Registro statunitense e ribadisce la validità di entrambi gli approcci fatte salve le differenze sociali socio-sanitarie e geografiche. Si fa notare che comunque la raccolta della casistica finora utilizzata all’estero non segue un campionamento adeguato della popolazione, in quanto la raccolta dei casi non è stata affidata a centri di riferimento periferici ben inseriti in ogni regione del territorio. Nel nostro Paese, finora, i dati relativi all’incidenza o prevalenza di queste genodermatosi non erano disponibili, ma erano desunti in modo proporzionale da quelli degli altri Paesi. Il Registro Italiano con la sua organizzazione ha permesso un campionamento sul territorio quasi in modo capillare dei pazienti affetti da EB, identificando anche un numero significativo di casi molto modesti di EB epidermolitiche (forme familiari fruste di EB epidermolitiche palmo-plantari individuate dai dermatologi del SSN a cui i pazienti si riferivano per altre patologie oppure diagnosticate nei Centri di Medicina Militare) che spesso, in questo tipo di indagini, non riescono a essere individuati proprio per la esiguità delle manifestazioni cliniche. La disponibilitá di informazioni sufficientemente rappresentative della reale situazione dei pazienti affetti da EB nel nostro paese è estremamente importante sia da un punto di Vol. 140 - N. 4 TADINI vista medico-sanitario sia per gli aspetti prettamente scientifici. Nel primo caso, conoscendo le caratteristiche cliniche delle EB, e la loro distribuzione nel territorio si possono organizzare centri sanitari con strutture congrue alla gestione di questi malati che sono portatori di una patologia cronica. Proprio per questa necessitá l’Istituto Superiore di Sanitá, ha stimolato lo sviluppo di tale indagine epidemiologica e a oggi i dati del Registro Italiano delle EB fanno parte del Registro Italiano delle Malattie Rare del Ministero della Salute. Da un punto di vista scientifico la casistica raccolta rappresenta un grosso bagaglio informazionale su tutti gli aspetti della patologia: — Aspetti clinici e loro varianti all’interno dello stesso gruppo o sottotipo. — Aspetti diagnostici tradizionali e molecolari. — Studio delle mutazioni ricorrenti ed ancestrali della popolazione italiana. — Valutazione delle patologie ricorrenti nei pazienti con particolare riguardo ai tumori ed alla loro prevenzione e terapia. Si prospetta infine un impatto significativo nella strategia della consulenza genetica la quale prevede di disporre della classificazione molecolare dei soggetti affetti per individuare i portatori fra loro consanguinei e gli incroci a rischio, e infine per rendere possibile la diagnosi prenatale molecolare per ciascuna delle famiglie a rischio per EB letali o gravemente invalidanti A questo proposito ricordiamo che sono state individuate circa 70 mutazioni nei pazienti italiani affetti da epidermolisi bollosa giunzionale e dermolitica, nei 2 laboratori di riferimento italiani di Roma (Istituto Dermopatico dell’Immacolata e di Brescia, Dipartimento di Genetica Medica dell’Università di Brescia). Dati di mortalità Fino ad oggi è stato possibile effettuare una stima dei dati di mortalità esclusivamente sui pazienti afferenti al Centro Malattie Cutanee Ereditarie di Milano, che costituiscono il 44,7% della casistica complessiva: su 263 pazienti, sono stati registrati 18 decessi (tasso di mortalità = 6,8%), di cui 8 pazienti erano affetti da EB giunzionale e 10 da EB dermolitica (Tabella V); non si sono verificati decessi di pazienti con EB epidermolitica. Dei pazienti con EB giunzionale, 2 erano affetti dalla variante associata ad atresia del piloro e 6 dalla variante di Herlitz: tra questi ultimi, 2 pazienti sono deceduti tra i 6 e gli 11 anni, mentre 6 non hanno superato l’anno di vita. Tra le più frequenti cause di morte si segnalano le complicanze infettive (soprattutto nelle varianti giunzionali) e le metastasi a partenza da carcinomi spinocellulari (presenti in 8 dei 10 soggetti con EB dermolitica, variante Hallopeau-Siemens, deceduti entrambi in età adulta; gli altri 2 pazienti affetti da EB dermolitica sono deceduti poco dopo la nascita per sepsi). I dati che si riferiscono ai decessi riferiti dagli altri Centri di reclutamento saranno elaborati ed GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 371 TADINI HEREDITARY EPIDERMOLYSIS BULLOSA ITALIAN REGISTRY esposti in successive comunicazioni di aggiornamento del Registro. Complicanze Sono state descritte numerosissime complicanze nei pazienti affetti da EB ereditarie: i dati disponibili sono relativi a due delle più frequenti e severe (sviluppo di carcinomi, stenosi esofagea) e rappresentano quindi solo una stima parziale rispetto alla loro reale incidenza. Nella casistica dei pazienti del Centro Malattie Cutanee Ereditarie di Milano, 7 pazienti su 263 (pari al 2,7%) hanno sviluppato carcinomi cutanei: tra questi, 2 sono deceduti in seguito allo sviluppo di metastasi. In 6 casi si trattava di carcinomi spinocellulari; in un solo paziente era presente un carcinoma basocellulare. Inoltre, 25 pazienti tra quelli afferenti al CMCE di Milano (N=263) erano affetti da stenosi esofagea, ovvero una percentuale pari al 9,5%. Anche per le complicanze, i dati di riferimento degli altri Centri sono in via di rielaborazione. Conclusioni Le stime di in incidenza e prevalenza sopra riportate sono rappresentative della reale situazione italiana per una serie di motivi. In primo luogo, si ritiene che tutti i casi più gravi siano stati raccolti e conteggiati e, in secondo luogo, anche i casi relativi alle forme di EB meno gravi sono stati quasi completamente individuati grazie alla rete informativa capillare creata sul territorio. Infatti, nessun altro registro ha potuto disporre di un campionamento così completo; in questo modo solo una percentuale minima, non in grado di alterare significativamente il valore di incidenza, sarebbe sfuggita alla raccolta. Comunque le cause di questo sia pure modesto «bias» risiedono nel livello socio-culturale dei soggetti affetti e dei familiari, dalla esiguità del quadro clinico, dalla inconsapevolezza di essere affetto da EB e da ultimo dalla mancata diagnosi. La rarità di tali Genodermatosi e la loro gravità, intesa sia da un punto di vista umano sia sanitario ed economico, rendeva obbligatoria l’istituzione di una struttura atta a raccogliere tutte le informazioni possibili su queste materie. Dalla disponibilità dei dati epidemiologici sopra esposti si può evidentemente derivare la distribuzione geografica della malattia sul territorio nazionale e la sua interferenza nella organizzazione degli interventi clinici assistenziali. Le EB sono malattie geneticamente determinate e con 372 una variabilità genetica ben più ampia di quella clinica; dati di qualunque natura così ottenuti, sono disponibili a chiunque ne sia a qualsiasi titolo interessato. Si prospetta inoltre un impatto significativo nella strategia della prevenzione, la quale prevede di disporre della classificazione molecolare dei soggetti affetti per individuare i portatori fra loro consanguinei, per individuare i matrimoni a rischio e, infine, per rendere possibile la diagnosi molecolare di tutte le famiglie italiane affette. In virtù di questa prerogativa, i dati ottenuti potranno essere confrontati e scambiati con quelli di altri Paesi. Lo staff medico del Registro italiano ha proposto infatti in numerosi sedi congressuali e istituzionali la creazione di un Registro Europeo delle Epidermolisi Bollose Ereditarie Riassunto Obiettivo. Attualmente in Italia non esiste uno studio epidemilogico completo sulle Epidermolisi Bollose (EB) ereditarie: la necessità di avere una stima accurata della casistica italiana ha favorito la costituzione di un Registro nazionale, che ha permesso di raccogliere tutti i casi notificabili di malattia, con importanti implicazioni nell’ambito delle conoscenze cliniche dell’EB, per lo sviluppo di strumenti di diagnosi prenatale e per l’avvio di studi di epidemiologia genetica. Metodi. Abbiamo costituito un registro su base ospedaliera che, in una prima fase, ha raccolto i casi già noti ai centri partecipanti dal 1985 al 1993, e, successivamente, ha proseguito con la segnalazione dei nuovi casi incidenti, fino al 31 Dicembre 2002. Il Registro prevedeva la presenza di un centro di coordinamento dati (Clinica Dermatologica di Bergamo), di 3 centri sovraregionali (CMCE di Milano, IDI di Roma, Ospedale di Bari) che hanno raccolto i pazienti provenienti rispettivamente dal Nord, Centro e Sud del Paese e della DEBRA Italia. Risultati. Sono stati notificati in tutto 697 casi e 9 casi non ulteriormente classificabili, costituiti da un 28% di EB epidermolitiche, 10% di EB giunzionali e 62% di EB dermolitiche. L’incidenza delle EB al 31 Dicembre 2002 era di 0,1 pazienti affetti per milione di abitanti; la prevalenza, calcolata al 31 dicembre 2002, era pari a 10,1 pazienti affetti per milione di abitanti. Conclusioni. Tali stime epidemiologiche sono rappresentative della situazione italiana e tracciano una distribuzione geografica della malattia sul nostro territorio, con un impatto significativo nella strategia della prevenzione. PAROLE CHIAVE: Epidermolisi Bollosa - Registro - Incidenza - Relevanza. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 G ITAL DERMATOL VENEREOL 2005;140:373-9 Methodological procedure for evaluation of risk factors for cutaneous malignant melanoma in a representative sample of the Tuscan population P. RUBEGNI 1, P. SBANO 1, G. CEVENINI 2, M. RISULO 1, E. STANGHELLINI 1, P. BARBINI 2 M. R. MASSAI 2, L. ANDREASSI 1, M. FIMIANI 1 Aim. The incidence of melanoma is rising steadily in countries with white populations and, despite all attempts at treatment, a considerable proportion of patients with malignant melanoma die of the disease. Primary prevention is, therefore, important to reduce mortality at present. Here we report the results of a case-control study of melanoma risk factors conducted in Tuscany (Italy) in 2002-2003. Methods. One-hundred-forty Italian subjects who underwent surgical exeresis of non familial cutaneous malignant melanoma and 280 age- and gender-matched controls filled in a standardized questionnaire about occupational and recreational sun exposure, underwent complete skin examination by a dermatologist to assess the number of nevi and presence of clinically atypical nevi, eye color, hair color and Fitzpatrick phototype. Moreover skin color was measured with a Minolta CR300 colorimeter. Univariate and stepwise logistic regression statistical analysis were performed to analyze differences in variables between melanoma patients and control subjects. Results. We demonstrated a highly significant difference between controls and melanoma patients in our study: in nevi number and presence of atypical nevi, constitutional skin color (Fitzpatrick phototype was completely explained by colorimetric variables of skin color) and eye color. Conclusion. We agree with what was recently proposed by others that objective skin color measurements must be combined with phenotype parameters and sun exposure history for exact assessment of individual risk KEY WORDS: Cutaneous melanoma - Risk factors - Colorimeter Skin color. Received: May 10, 2005 . Accepted for publication: July 1, 2005. Address reprint requests to: P. Rubegni, MD, Istituto di Scienze Dermatologiche, Università degli Studi di Siena, Policlinico Le Scotte, 53100 Siena, Italy. E-mail: [email protected]. Vol. 140 - N. 4 1Unit of Dermatology, Department of Clinical Medicine and Immunological Science University of Siena, Siena, Italy 2Department of Surgery and Bioengineering University of Siena, Siena, Italy T he incidence of melanoma is rising steadily in countries with white populations and, despite all attempts at treatment, a considerable proportion of patients with malignant melanoma (MM) die of the disease.1-4 Early diagnosis and primary prevention are, therefore, the only way to reduce mortality at present. Many advances in early diagnosis have been made through innovative non invasive techniques, such as digital dermoscopy analysis (DDA), which have shown the importance of objective numerical parameter measurements.5 Less progress seems to have been made with primary prevention, despite the fact that instruments for objective numerical evaluation of important phenotypic characteristics are now available. Features known to be correlated with melanoma risk include eye color, hair color, tanning ability, freckling, nevus number, skin phototype and skin colour.6, 7 Skin color and phototype characterise skin biotype.8 In an extensive review of case-control studies, Evans et al. noted that 6 out of 9 studies demonstrated that the relative risk of melanoma was 2-18 fold higher for fair or pale complexions than for non fair complexions.9 However, in almost all these studies, skin color was evaluated by a visual score and was therefore sub- GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 373 RUBEGNI METHODOLOGICAL PROCEDURE FOR EVALUATION OPF RISK FACTORS FOR CUTANEOUS MALIGNANT MELANOMA jective, non reproducible and non quantifiable. The increasing availability of colorimeters now makes it possible to objectively evaluate skin color in an easy and largely reproducible manner.10, 11 Here we report the results of a case-control study of melanoma risk factors conducted in Tuscany (Italy) in 2002-2003. The aim of the study was to assess the significance of risk factors commonly associated with MM in a representative sample of the Tuscan population, combining objective measurement of skin color with other known data acquisition methods (questionnaire and dermatological examination). Materials and methods Selection of patients and controls From October 2002 to May 2003 we studied all subjects (49 men, 91 women, total 140) of native Italian origin from Tuscany who underwent surgical exeresis of non familial cutaneous MM and non acral lentiginous melanoma. Twenty four patients had nodular melanoma and 84 had superficial spreading melanoma. Their age and gender distributions were similar to those of the Tuscan Cancer Registry from 1985 to 1987. A group of 280 age- and gender-matched controls of native Italian origin living in Tuscany was evaluated in the same period. The control group consisted of subjects chosen by random sampling from the computerized demographic files of Siena, Arezzo and Grosseto hospitals. These files contained all the data required for the present study of subjects attending the dermatology clinics. Subjects with a history of phototherapy or skin tumors were excluded from the control group. The control group may, of course, have contained subjects who will develop melanoma, however, since the recent mean prevalence of melanoma in Tuscany is very low (68 cases per million), we regard them as normal, though low-risk would be a more appropriate term. Qualitative risk factors All subjects gave their informed consent and filled in a standardized questionnaire about occupational and recreational sun exposure. Occupational exposure was scored in 5 categories (minor, a few years, parttime, most of the time and full-time) and recreational exposure in 4 categories (none, minor, medium and 374 TABLE I.—Frequency counts of categorical risk factors together with statistical significances (HS=highly significant, P<0.01; S=significant, P<0.05; NS=not significant, P>0.05) of χ2 test, Fisher exact test and Spearman correlation analysis applied to contingency tables. Risk factor Categories Occupational exposure Minor χ2 (NS) A few years Spearman (NS) Part-time Most of time Full-time Recreational exposure None χ2 (NS) Minor Spearman (NS) Medium Strong Number of nevi and High-risk presence of atypical nevi Medium-risk χ2 (NS) Low-risk Spearman (NS)c Fitzpatrick phototype I χ2 (NS) II Spearman (NS) III IV Eye color Fair (green, blue) Fisher exact (S) Dark (brown, black) Spearman (S) Hair color Fair (red, blond) Fisher exact (NS) Dark (brown, black) Spearman (NS) Freckles No Fisher exact (NS) Yes Spearman (NS) Sunburn No Fisher exact (NS) Yes Spearman (NS) Controls Melanomas Totals 20 16 20 8 216 40 152 76 12 76 164 40 6 8 8 10 108 16 54 70 0 32 44 64 26 24 28 18 324 56 206 146 12 108 208 104 16 88 120 56 40 240 4 60 74 2 46 94 20 148 194 58 94 326 48 232 26 114 74 346 192 88 80 60 272 148 180 100 74 66 254 166 strong). Patients were also asked to recall episodes of sunburn in infancy or adolescence (yes/no). Complete skin examination was performed by a dermatologist to assess the number of nevi, distinguishing 3 categories: less than 10, between 10 and 30, and more than 30. Eye color was recorded as fair (green-blue) or dark (brownblack), hair color as fair (red-blond) or dark (brownblack), Fitzpatrick phototype as I, II, III or IV and freckling (as yes/no). The exact description of categories is shown in Table I. Semi-quantitative risk factors Complete skin examination was performed by the same dermatologist to assess the number and type of nevi, as recently suggested by Carli et al.,12 distinguishing subjects into 3 categories: high-risk when there were 30 or more common acquired nevi and 3 or more atypical nevi; medium-risk when there were less than 30 common acquired nevi and 3 atypical nevi but more than 15 acquired nevi; low-risk when there were GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 METHODOLOGICAL PROCEDURE FOR EVALUATION OPF RISK FACTORS FOR CUTANEOUS MALIGNANT MELANOMA less than 15 acquired nevi. Clinically atypical nevi were defined as acquired macular or slightly palpable nevi with a minimum of 3 of the following 5 criteria: diameter 5 mm or more, asymmetrical shape, ill-defined border, irregular brown pigmentation, and erythema. Nevi showing only 1 or 2 criteria were counted as normal nevi, although histological features of dysplasia can sometimes be found in these nevi. Quantitative risk factors Six quantitative variables representing objective skin color were also considered. Skin color was measured with a Minolta CR-300 colorimeter consisting of a detector and a microcomputer. The detector contains a pulsed xenon arc lamp in a mixing chamber and provides diffuse illumination over the sample to be analyzed. Six high-sensitivity silicon photocells, filtered to match the Commission International d’Eclairage standard observer response in a double-beam feedback system, measure incident and reflected light. The CR-300 detects any slight deviation in the spectral power distribution of the pulsed radiation and corrects it automatically. The opening of the detector is fitted with an applicator so that dermal vessels are not compressed. The CR-300 expresses the results in 5 different color systems. We chose the Yxy system because it gives parametric color measurements and is widely used in dermatology. After 15 min acclimatization in a room with air conditioning at 18°C, skin color was measured on the upper medial quarter of the buttock (constitutional color) and on the cheek (facultative color), taking care not to press the detector heavily onto the surface, which could cause ischemia. The chromameter was calibrated between individuals. The color measurement was read 3 times and averaged. Two terns of variables Yxy were, therefore, measured for each subject, specifically 3 colorimetric values, Yc, xc and yc, for constitutional skin color and 3 more colorimetric values, Yf, xf and yf, for facultative sunexposed skin color. Statistical analysis Fourteen variables were analyzed for involvement with melanoma risk. Frequency count and contingency tables were calculated for each categorical variable and the χ2 test was used to analyze differences in variables between melanoma patients and control subjects. For 2×2 tables, the more powerful Fisher exact test was Vol. 140 - N. 4 RUBEGNI used instead of the χ2 test. The Spearman correlation coefficient was also computed to check statistically significant associations between melanoma and the categories of ordinal variables used. Colorimetric variables were described statistically as mean, standard deviation (SD) and range for all cases and separately for melanoma patients and controls. Univariate differences between groups were tested by F statistics. Stepwise logistic regression analysis was then carried out with all 14 variables (covariates) to identify a statistically significant minimum subset of variables with the highest possible power in discriminating melanoma patients from controls and quantifying risk. Logistic discrimination is generally preferable to linear discrimination in small samples, especially when distributions are suspected to be non-Gaussian. In logistic regression the dependent variable is binary, i.e. with only 2 possible values, in our study melanoma/control. The method assumes that the posterior probabilities P1 and P2 of group membership follow the logistic model: eV P1 = 1 + eV 1 + eV 1 P2 = 1 - P1 = 1 + eVV 1+e where V is a linear function of one or more independent variables, that is: V = b0 + b1x1 + b2x2 + ... + +bnxn where x1, x2, ..., xn are the n independent variables and b0, b1, b2, ..., bn the corresponding model parameters to be estimated from experimental data. The ratio of probabilities P1 = eV P1 = eV P2 = eV expresses the risk of group 1 (melanoma patients) with respect to group 2 (healthy subjects). The exponential quantities ebi (i=1,2, ..., n) are known as odds ratios. Supposing the variables (risk factors) to be linearly uncorrelated, the odds ratio represents the contribution of unit variable to relative risk. Therefore, for dichotomous variables binary coded 0 or 1, the odds ratio is simply the relative risk of the category coded 1 with respect to the category coded 0. For qualitative variables with n categories, we have n-1 odds ratios related to the remaining category taken as reference. To interpret odds ratios of quantitative variables it is convenient to standardize them, that is to transform them GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 375 RUBEGNI METHODOLOGICAL PROCEDURE FOR EVALUATION OPF RISK FACTORS FOR CUTANEOUS MALIGNANT MELANOMA into z-scores with 0 mean and unit SD. Odds ratios can then be interpreted as representing an increase of one SD from the mean value of the variable. In the presence of confusing variables which may affect the relative risk due to causes not related to the study (for example sample distortions in age or gender), estimates of odds ratios related to real risk factors can be corrected by including these variables in the analysis. Their inclusion should be decided on the basis of a preliminary univariate analysis for testing their effective statistical influence on relative risk. In the stepwise process, an independent variable is added to or removed from the model at each step on the basis of a statistical criterion. Many statistical criteria for inclusion or exclusion of variables are available in the following 2 main configurations: — forward, starting (step 0) with all the variables out of the model and adding them stepwise; the removal process can only begin when at least 2 variables have been entered in the model; — backward, starting with all the variables in the model and removing them stepwise. The process stops when addition or removal of variables no longer improves statistical significance. The subset of variables entered in the model is then considered for interpretation of the multivariate model. We used the criterion of maximum likelihood ratio in the forward configuration. The Hosmer-Lemeshow test was used to evaluate the fit of the logistic multivariate model step by step. An associated value of P=1 indicated perfect fit and P < 0.05 indicated 95% statistical significance of disagreement between model and experimental data. Values of P between 0.05 and 1, therefore, demonstrate a statistically significant fit. A classification matrix was formed by assigning each case to the group having a probability greater than 0.5. The percentage of correct classification was calculated. Univariate logistic regression was also obtained from stepwise results at step 0. Any undesired confusing effect of age and gender on melanoma risk was also examined by univariate logistic regression. Odds ratios and 95% confidence intervals (CI) were evaluated to establish the relative risk of melanoma by uni- and multivariate models, for statistically significant variables. For this purpose, quantitative colorimetric variables were standardized, so that odds ratios represented the relative risk associated with an increase of one SD from their mean value. Statistical analysis was performed using SPSS statistical software. 376 TABLE II.—Descriptive statistics for colorimetric variables and Fisher statistics significance of differences between control and melanoma cases: HS=highly significant, P<0.01; S=significant, P<0.05; NS=not significant, P>0.05. Colorimetric variables Yc Fisher (S) Xc Fisher (HS) Yc Fisher (HS) YF Fisher (S) XF Fisher (NS) YF Fisher (NS) Group Mean Standard deviation Min Total Melanoma Control Total Melanoma Control Total Melanoma Control Total Melanoma Control Total Melanoma Control Total Melanoma Control 38.27 39.09 37.46 0.3591 0.3557 0.3625 0.3424 0.3398 0.3449 27.53 26.65 28.40 0.3826 0.3830 0.3823 0.3440 0.3432 0.3449 4.3 3.3 5 0.0096 0.0075 0.010 0.0066 0.0059 0.0063 4.5 4.3 4.5 0.011 0.011 0.0095 0.0074 0.0061 0.0071 22.62 30.86 22.62 0.3354 0.3354 0.3456 0.3300 0.3300 0.3334 19.08 19.42 19.08 0.3539 0.3539 0.3619 0.3230 0.3277 0.3230 Max 46.93 46.93 46.55 0.3885 0.3822 0.3885 0.3648 0.3576 0.3648 38.51 38.27 38.51 0.4076 0.4045 0.4076 0.3643 0.3616 0.3643 Results Frequency counts of categorical variables are shown in Table I. Univariate statistically significant differences between control and melanoma subjects are also reported for each categorical risk factor (P-value of χ2 or Fisher-exact test). Spearman correlation analysis assessed statistically significant associations between risk factors and melanoma. Highly significant differences between controls and cases were found for the 3 classes of number of nevi and presence of atypical nevi and for Fitzpatrick phototype (χ2 test, P<0.01). Recreational sun exposure and eye color gave less significant differences (χ2 or Fisher exact test, P<0.05). Hair color type, freckles, sunburn and occupational sun exposure did not show significant differences (χ2 or Fisher exact test, P>0.05). A statistically significant ordinal association with melanoma was only found for a number of nevi and the presence of atypical nevi (Spearman, P<0.01) and eye color (Spearman, P<0.01). Table II shows descriptive statistics for colorimetric variables and Fisher test for statistical comparison of melanoma and control cases. Significant differences were found in constitutional colorimetric variables xc and yc (P<0.01) and in constitutional, Yc, and facultative, Yf, reflectances (P<0.05). No statistically significant differences were found in facultative colori- GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 METHODOLOGICAL PROCEDURE FOR EVALUATION OPF RISK FACTORS FOR CUTANEOUS MALIGNANT MELANOMA metric variables xf and yf (P>0.05). Univariate analysis showed that the major colorimetric factor related to probability of developing melanoma was chromaticity of unexposed skin, followed by reflectance of unexposed and chronically exposed skin. Univariate logistic regression of age and gender failed to reveal statistical significance, indicating that it is not necessary to correct for confusing effects of these variables on melanoma risk. The results of univariate logistic regression applied to the 14 variables investigated are shown in Table III. Only risk factors giving a statistically significant odds ratio (P>0.05) are reported. It was found that fair eyes are associated with a significantly greater risk of melanoma than dark eyes (odds ratio 2.4, 95% CI 1.1-5.3). Referring to the class number of nevi and the presence of atypical nevi, an increased risk difference was demonstrated for high-risk class in comparison with a low-risk class (odds ratio 3.8, 95% CI 1.4-10), whereas medium-risk class does not supply appreciable risk differences. Although odds ratios of Fitzpatrick phototypes had large CI indicating poor accuracy in phototype estimation, the risk associated with types I-III was much greater than that associated with type IV. The fairest constitutional skin color is also associated with increased risk of melanoma: an increase of one SD in reflectance Yc leads to a significant increase in risk (odds ratio 1.5, 95% CI 1-2.1) and the same increase (one SD) in chromaticity xc and yc is associated with a significant decrease in risk (odds ratios: xc=0.44, 95% CI 0.290.66; yc=0.39, 95% CI 0.25-0.60). With regard to facultative skin color, an increase in reflectance Yf led to a decrease in risk (odds ratio 0.67, 95% CI 0.470.94). This is in line with the fact that subjects who expose their skin less to sunlight (and therefore have paler skin color on the cheek) are at lower risk for MM. No significant differences were found between cases and controls with regard to type of exposure (occupational or facultative), hair color, freckling or early history of sunburn. The multivariate logistic model obtained by stepwise regression is shown in Table IV. Some of the results are interesting. When the constitutional skin color component yc was entered, at the first step, the other constitutional skin color components Yc and xc lose significance, indicating substantial correlation among the 3 colorimetric components. One colorimetric component is, therefore, sufficient to account for relative melanoma risk due to constitutional skin col- Vol. 140 - N. 4 RUBEGNI TABLE III.—Univariate logistic regression. Odds ratios and 95% confidence intervals (CI) are only reported for statistically significant (P<0.05) variables. Risk factors Catogories Reported category 95% CI Odds ratios Lower Upper Eye color Fair Dark 2.4 Number of nevi and Medium risk Low risk 0.64 presence of atypical nevi High risk 3.8 Fitzpatrick phototype I IV 7 II 19.1 III 17.3 Yc * 1.5 xc * 0.44 yc * 0.39 Yf * 0.67 1.1 5.3 0.27 1.5 1.4 10 0.50 98 2.3 156 2.1 139 1 2.1 0.29 0.66 0.25 0.60 0.47 0.94 *Odds ratios of quantitative colorimetric variables represent an increase of one standard deviation with respect to the mean TABELLA IV.—Multivariate stepwise logistic regression. Odds ratios and 95% confidence intervals (CI) are only reported for statistically significant (P<0.05) variables. Step N. 1 2 3 4 5 6 Risk factors Categories 95% CI Reported Odds category ratios Lower Upper yc * 0.20 Number of nevi Medium risk Low risk 0.83 and presence of High risk 6.74 atypical nevi Yf * 0.33 Fitzpatrick I IV 11.3 phototype II 12.1 III 0.65 Recreational Minor Null 1.35 exposure Medium 7.62 Strong 0.073 yf * 2.05 0.088 0.29 1.85 0.44 2.40 24.60 0.18 0.60 1.36 92.90 1.38 105.00 0.027 15.70 0.35 5.20 1.65 35.20 0 1.4×1015 1.11 3.81 *Odds ratios of quantitative colorimetric variables represent an increase of one standard deviation with respect to the mean or. Once quantitative constitutional and facultative skin color was entered at step 3, the model already has a satisfactory fit and power of discrimination between melanoma and healthy subjects. The Hosmer-Lemeshow test gave a P-value of 0.25 indicating a significantly good model fit. Model sensitivity and specificity were 70% and 75.7%, respectively. The Fitzpatrick phototype was entered at step 4. Estimated odds ratios indicated a risk of melanoma about 11 and 12 times greater for phototypes I and II, respectively, than for phototype IV, though phototypes had poor accuracy (large CIs). Recreational exposure entered at step 5. This furnishes less accurate esti- GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 377 RUBEGNI METHODOLOGICAL PROCEDURE FOR EVALUATION OPF RISK FACTORS FOR CUTANEOUS MALIGNANT MELANOMA mates of odds ratio but towards an increase of risk for greater exposures. At step 6 yf entered, with a well-estimated odds ratio of about 2 which confirms that higher facultative pigmentation significantly increases the risk of melanoma. Finally, the multivariate logistic model gives a good fit (Hosmer-Lemeshow test, P=0.42) with appreciable sensitivity, specificity and accuracy: 78.6%, 75.7% and 77.1% respectively. Discussion and conclusions Many environmental and constitutional risk factors have been associated with MM.1 Among constitutional factors, numerous melanocytic nevi or the presence of atypical nevi is the major known risk factor.1, 13, 14 The former has also emerged as a risk factor in studies on Caucasian and Mediterranean peoples,12, 15 and in Celtic people with fair phototype (northern Europe and Australia). 16 We also encountered a highly significant difference in nevus number and type between controls and melanoma patients in our study. This finding emerged both with univariate and multivariate statistical analysis. We found that subjects we defined as high risk on the basis of these 2 factors had a much higher risk of melanoma than those defined as low risk. An interesting new finding, different from those of other studies, was that subjects defined by us as medium risk actually had a risk of melanoma similar to low risk subjects. We could, therefore, have divided the population into 2 (high and medium-low risk) instead of 3 groups on the basis of nevus number. A variable we found highly significant despite an odds ratio indicative of high CIs was the Fitzpatrick phototype, and specifically, the risk associated with types I-III was much greater than that associated with type IV. However, this variable turned out to be correlated and completely explained by colorimetric variables of skin color. This means that we could have omitted phototype and studied only skin color. The major skin color parameters correlated with risk of MM were chromaticity of unexposed skin (buttock y) and reflectance (fairness) of unexposed and chronically exposed skin (buttock and cheek Y). It is well known that fair subjects are more susceptible to skin cancer because they are more vulnerable to UV light, the major environmental risk factor for skin cancer.17 378 With regard to facultative skin color, an increase in reflectance (Yf), i.e. fair skin on the cheek, is associated with a decrease in melanoma risk. This finding is in line with the fact that subjects with lower exposure to sunlight (and hence fairer exposed skin on the cheek) have a lower risk of MM.17 As indicated by all the international literature,1, 12, 15, 16 univariate and multivariate analysis identified fair eyes as a significantly greater risk factor than dark eyes, in our study. On the other hand, no significant differences in type of exposure (occupational or facultative), hair color, freckling or early sunburn history were found between cases and controls by univariate analysis. Some of these findings disagree with the results of epidemiological studies from other geographical areas,1, 16-18 suggesting that risk factors on which to base prevention campaigns have to come directly from population subgroups and cannot be based on generalizations from studies on climatically, geographically and racially different peoples. The absence of correlations between risk of melanoma, hair color and freckling, for example, may be due to the fact that few Tuscans have red hair and develop freckles. With regard to early history of sunburn, the lack of significance of this factor had already been reported in a population geographically and culturally similar to ours 12 and this could be due to the large percentage of subjects with phototype III or IV, who are unlikely to burn, and to nearness to the sea and temperate climate which permit continuous exposure, so that skin is protected against acute sunburn. Indeed, in our study population, only 6% of melanoma patients and 3.8% of controls had a history of sunburn. As far as we know, 7 case-control studies have been conducted on melanoma risk factors in the Italian population.12, 15, 19-24 Because these studies differed in aim, method (qualitative and/or quantitative) and study population, their results cannot readily be compared. In particular, northern and southern Italy are characterized by prevalences of fair and Mediterranean phototypes, respectively, and by quite different climates. Moreover, most of these case-control studies were based on retrospective assessment of sun exposure and self-reported information on individual sensitivity to UV radiation. In conclusion, we agree with what was recently proposed by Brenner et al.23 that objective skin color measurements must be combined with phenotype parameters and sun exposure history for exact assessment of individual risk. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 METHODOLOGICAL PROCEDURE FOR EVALUATION OPF RISK FACTORS FOR CUTANEOUS MALIGNANT MELANOMA Riassunto Procedura metodologica per la valutazione dei fattori di rischio per melanoma cutaneo maligno in un campione rappresentativo della popolazione toscana Obiettivo. Numerosi fattori di rischio costituzionali e ambientali sono stati messi in relazione con l’insorgenza del melanoma cutaneo. I risultati degli studi epidemiologici effettuati sino ad oggi sono, tuttavia, spesso non confrontabili tra loro a causa della carenza di parametri oggettivi (numerici) quantificabili e della diversità razziale esistente tra le varie popolazioni esaminate. Lo scopo di questo lavoro è stato valutare la significatività dei fattori di rischio comunemente associati al melanoma, in un campione rappresentativo della popolazione della Toscana, associando ai già utilizzati strumenti di raccolta dati (questionario e visita dermatologica), la misurazione oggettiva del colore della pelle. Metodi. Centoquaranta soggetti di origine italiana, nati e residenti in Toscana, sottoposti ad asportazione chirurgica di melanoma cutaneo (non familiare e non acrale-lentiginoso) e 280 soggetti controllo, simili ai primi per età, sesso e origine hanno compilato un questionario e sono stati sottoposti a visita dermatologica e valutazione strumentale del colore della pelle. Risultati. L’analisi statistica univariata e multivariata ha dimostrato differenze statisticamente significative tra il gruppo dei pazienti e quello dei controlli per le 3 classi “numero di nevi e presenza di nevi atipici” e per il fototipo secondo Fitzpatrick. Inoltre differenze significative sono state apprezzate per il colore degli occhi e il colore costituzionale della cute. L’analisi univariata ha mostrato che la variabile maggiormente correlata al rischio di ammalarsi di melanoma è la cromaticità della cute non esposta al sole, seguita dalla reflettanza della cute non esposta e di quella esposta. In questo studio è stata riscontrata una differenza statisticamente significativa di rischio di sviluppare melanoma cutaneo tra i pazienti e i controlli per quanto riguarda il numero e la tipologia dei nevi. Variabili risultate altamente significative sono state, inoltre, il colore cutaneo costituzionale (misurato mediante colorimetria) e il colore chiaro degli occhi. Tutti gli altri fattori di rischio esaminati non hanno raggiunto un elevato grado di significatività statistica. Conclusioni. Come già suggerito anche da altri Autori, si concorda circa la necessità di associare la misurazione oggettiva del colore cutaneo alle valutazioni delle caratteristiche fenotipiche per una corretta valutazione del rischio di melanoma. PAROLE CHIAVE: Melanoma cutaneo - Fattori di rischio Misura del colore cutaneo. References 1. Desmond RA, Soong SJ. Epidemiology of malignant melanoma. Surg Clin North Am. 2003;83:1-29. 2. Koh HK. Cutaneous melanoma. N Engl J Med1991;325:171-82. 3. Swerlick RA, Suephy C. The melanoma epidemic. Arch Dermatol 1996;132:881-4. 4. Holman CD, James IR, Gattey PH. An analysis of trends in mortality from malignant melanoma of the skin in Australia. Int J Canc1980;26:703-9. Vol. 140 - N. 4 RUBEGNI 5. Rubegni P, Burroni M, Cevenini G, Perotti R, Dell’Eva G, Barbini P et al. Digital dermoscopy analysis and artificial neural network for the differentiation of clinically atypical pigmented skin lesions: a retrospective study. J Invest Dermatol 2002;119:471-4. 6. 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Youl P, Aitken J, Hayward N, Hogg D, Liu L, Lassam N et al. Melanoma in adolescents: a case-control study of risk factors in Queensland, Australia. Int J Cancer 2002;98:92-8. 17. Kennedy C, Bajdik CD, Willemze R, De Gruijl FR, Bouwes Bavinck JN; Leiden Skin Cancer Study. The influence of painful sunburns and lifetime sun exposure on the risk of actinic keratoses, seborrheic warts, melanocytic nevi, atypical nevi, and skin cancer. J Invest Dermatol 2003;120:1087-93. 18. Whiteman DC, Whiteman CA, Green AC. Childhood sun exposure as a risk factor for melanoma: a systematic review of epidemiologic studies. Cancer Causes Control 2001;12:69-82. 19. Cristofolini M, Franceschi S, Tasin L, Zumiani G, Piscioli F, Talamini R et al. Risk factors for cutaneous malignant melanoma in a northern Italian population. Int J Cancer 1987;39:150-4. 20. Zanetti R, Franceschi S, Rosso S, Colonna S, Bidoli E. Cutaneous melanoma and sunburns in childhood in a southern European population. Eur J Cancer 1992;28A:1172-6. 21. Carli P, Biggeri A, Giannotti B. Malignant melanoma in Italy: risks associated with common and clinically atypical melanocytic nevi. J Am Acad Dermatol 1995;32(5 Pt 1):734-9. 22. Landi MT, Baccarelli A, Calista D, Pesatori A, Fears T, Tucker MA et al. Combined risk factors for melanoma in a Mediterranean population. Br J Cancer 2001;85:1304-10. 23. Brenner AV, Lubin JH, Calista D, Landi MT. Instrumental measurements of skin color and skin ultraviolet light sensitivity and risk of cutaneous malignant melanoma: a case-control study in an Italian population. Am J Epidemiol 2002;156:353-62. 24. Fargnoli MC, Piccolo D, Altobelli E, Formicone F, Chimenti S, Peris K. Constitutional and environmental risk factors for cutaneous melanoma in an Italian population. A case-control study. Melanoma Res 2004;14:151-7. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 379 G ITAL DERMATOL VENEREOL 2005;140:381-7 Photodynamic therapy of actinic keratoses with methyl-aminolevulinate (METVIX®) R. ROSSI, L. MAVILIA, I. GHERSETICH, T. LOTTI Aim. Actinic keratoses (AKs) are considered one of the most common cutaneous intraepithelial neoplastic disorders. Some authors have recently proposed to define AK as a keratinocyte intraepithelial neoplasia with 3 possible steps of evolution towards squamous cell carcinoma. According to others, AK is a true neoplasia from the very beginning. It has been calculated that 60% of subjects with skin from I to III phototype over 40s present at least one AK. This diffuse and progressing condition requires a prompt diagnosis and an adequate treatment. The prevalence of AKs increases with age, with sun exposure and is regulated by different factors, including immunosurveillance and low phototype. The more risk factors there are, the more diffuse the lesions which required multiple therapies. Up to now both medical (5-FU cream, imiquimod cream, retinoids etc.) and surgical (cryosurgery, laser surgery, diclofenac gel, etc.) treatments have been used to treat AK with variable results. Photodynamic therapy (PDT) seems to represent a new, effective and well tolerated therapy for the treatment of AKs with excellent cosmetic results and long term follow up in terms of efficacy. In this paper the efficacy of PDT treatment has been evaluated with special attention to the employment of methyl-ester of aminolevulinic acid (MAL), which is a prodrug recently introduced in our country. Methods. We have treated 100 Caucasian patients (70 males, 30 females) with a skin phototype ranging from 1 to 3 according to the Fitzpatrick classification, for a total of 170 AKs of the face and scalp. Results. 15 days after the treatment showed complete healing in 114 lesions of the face (82.4%) and in 44 lesions of the scalp (78%). 84% of the more superficial and less squamous keratosis (grade I) presented a complete response against 80% of Address reprint requests to: Dott. R. Rossi, U.O. Struttura Dermatologica Complessa Fisioterapia Dermatologica, Dipartimento di Scienze Dermatologiche, Via Della Pergola 58, 50121 Firenze. E-mail: [email protected] Vol. 140 - N. 4 Unit of Dermatological Physiotherapy Department of Dermatological Sciences University of Florence, Florence, Italy grade II lesions. The general response to the first treatment was 77%. We showed 90% of complete healing with excellent compliance and cosmetic results. Conclusion. This study has shown that metylaminolevulinate PDT is an effective, safe and well tolerated treatment for AKs, which could probably be considered the treatment of choice for this very common and emerging cutaneous disorder. PDT is also a promising treatment modality with a good potential for future development in different fields, such as T cell lymphoma, acne, localized eczema, and human papillomavirus infection. KEY WORDS: Skin neoplasms - Actinic keratoses - Photodynamic therapy - Methyl-aminolevulinate. A ctinic keratoses (AKs) are considered one of the most common cutaneous intraepithelial neoplastic disorders. Yantos et al.1 have recently proposed defining AK as a keratinocyte intraepithelial neoplasia with 3 possible steps in their evolution towards squamous cell carcinoma. According to others, AK is a true neoplasia from the very beginning.2, 3 It has been calculated that 60% of subjects over-40s showing a skin with I to III phototype present at least one AK and that this percentage increases to 80% in the over-60s.4 In patients with diffuse signs of photocarcinogenesis AKs may undergo difficult therapeutic management, especially if those subjects for professional rea- GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 381 ROSSI PHOTODYNAMIC THERAPY OF ACTINIC KERATOSES WITH METHYL-AMINOLEVULINATE (METVIX) sons or life style are chronically photoexposed.5, 6 In these cases, lesions are usually widespread and tend to recur. Available topical treatments usually have bad compliance, surgical treatments are usually considered too invasive for patients. Thus a new and less invasive approach has become a hoped-for therapeutic option. In cases of patients with diffuse signs of photocarcinogenesis, photodynamic therapy (PDT) is considered the treatment of choice. PDT, a treatment modality involving the use of a photosensitizing agent, oxygen, and light of a specific wavelength to produce controlled cell death, has gained increasing popularity in the treatment of premalignant and malignant skin lesions.7 This treatment offers the potential advantage of reduced scarring and improved cosmetic outcome compared with conventional treatments. PDT using topical 5-aminolevulinic acid (ALA), a precursor of the active endogenous photosensitizer protoporphyrin IX, is effective in the treatment of Aks.8-13 In contrast with systemic photosensitizers, persistent skin photosensitization seems not to be a problem for topical ones. Numerous studies have led to the approval of this therapy for dermatological purposes, which was obtained in 1999 from the USA FDA for a topical formulation (Levulan® Kerastick, DUSA Pharmaceuticals, Tarrytown, NY) 14, 15 and more recently in Europe and Italy (March 2004) for methylaminolevulinate (MAL) (Metvix®, PhotoCure, Oslo, Norway). The aim of the present study was to evaluate the efficacy and tolerability of topical MAL- PDT (recently approved in our country) in the treatment of AKs.16-18 Materials and methods We evaluated 100 Caucasian patients (70 males and 30 females) of average age 68 (range 32-93) and Fitzpatrick skin phototype ranging from 1 to 3 (Table I). These subjects presented one or more AKs, for a total of 170 lesions of the face (114/170, 67%) and/or the scalp (56/170, 33%). The lesions were not pigmented and of grade 1 (119/170, 70%) or moderate (grade 2) (51/170, 30%). Lesions was classified as follows:19 Grade 1: easily visible, slightly palpable Grade 2: easily visible, palpable Grade3: frankly visible and very hyperkeratosic. 382 TABLE I.—Baseline characteristics of patients. Sex Male Female Age (years) Mean Range Skin type (Fitzpatrick skin type) I II III Total no. of lesions Lesion location Face Scalp No. of lesions per grade Grade I (AK thin) Grade II (AK moderate) 70 (70%) 30 (30%) 68 (32-93) 10% 30% 60% 170 114/170 (67%) 56/170 (33%) 119/170 (70%) 51/170 (30%) Treatment Each lesion was prepared before treatment to facilitate access of the cream and to ensure that illumination was not blocked. Scales and crusts were removed by a small dermal curette and the surface of the lesion was scraped gently. The intention of this very gentle curettage was to remove scales and crusts without bleeding. A thick layer of 160 mg/g MAL cream (Metvix®, Photocure ASA, Oslo, Norway) (approximately 1 mm) was applied to each lesion and 5 mm of surrounding tissue and covered with an occlusive dressing (Tegaderm, 3M Health care, St Paul, MN, USA) and covered with gauze to avoid photo exposure and to prevent the accidental activation of the cream (photobleaching). After 180 min lesions were examined by fluorescence with a light emitting diode (LED) lamp (DICAM-UV® - Alpha Strumenti, Milan, Italy) with UV at 405 nm irradiation to better appreciate the AKs lesions and the penetration of the cream (Figure 1). After 3 h (from the beginning) dressings were removed and lesions treated with non-coherent red light. We employed a device which uses so called LED light. This lamp (Aktilite PDT - Model CL128- Photocure ASA, Oslo, Norway) has an average wavelength of 630 nm, light dose 37J/cm2, light intensity 70100 mW/cm2. It illuminates areas from 80 to 180 mm at a distance from 50 to 80 mm (Figure 2). After PDT treatment, all lesions were treated with topical antibiotic ointments until complete healing (approximately 1 week). Some patients requested burning and stinging dur- GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 PHOTODYNAMIC THERAPY OF ACTINIC KERATOSES WITH METHYL-AMINOLEVULINATE (METVIX) ROSSI Figure 1.—DICAM UV - Alpha Strumenti, Milan, Italy. ing the treatment; in these cases we pretreated the area with EMLA for 1 h before treatment in the next therapeutic session. The first control was performed 15 days after the first treatment. At this point clinically cleared lesions were included in the follow up program (control every 3 months), while not completely cleared lesions were treated again following the same algorithm. Results All selected patients (100 patients presenting with 170 lesions, spread for 67% over the face, 33% on the Figure 2.—Aktilite “ PDT- Model CL128 - Photocure ASA, Oslo, Norway, scalp) were treated after informed consent, with a ses- distributed by Galderma. sion of MAL-PDT. 15 days after the treatment we showed complete healing in 114 lesions of the face (82.4%) and in 44 bility. All results were highly satisfactory from an lesions of the scalp (78%). 84% of the more superfi- esthetic point of view. None of the patients underwent local intralesional cial and less squamous keratosis (grade I) (Figure 3A, 3B) presented a complete response against 80% of anesthesia. grade II lesions (Figure 4A, 4B). The general response to the first treatment was 77%. Discussion and conclusions Over the lesions which did not completely heal a second treatment was performed. Three months after The ideal treatment for AKs should be effective, the second treatment 10 more lesions of the face and well tolerated and have an excellent cosmetic out5 more lesions of the scalp were healed. In conclusions we showed a total healing of 153 of come, particularly in exposed areas such as the face. The fashion tendency to photoexposure even in the 170 treated lesions (90%) (Table II) with a higher response for grade I lesions of the face. After 6 months winter months (sun beds, tropical trips...) have determined a significant reduction in the average time needof follow up there were no recurrencies. All patients enjoyed good compliance and tolera- ed for the development of AKs. Vol. 140 - N. 4 GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 383 ROSSI PHOTODYNAMIC THERAPY OF ACTINIC KERATOSES WITH METHYL-AMINOLEVULINATE (METVIX) Figure 3.—A) Grade I Ak before treatment with MAL-PDT. B) Complete response 6 months after the first MAL-PDT session. Figure 4.—A) Actinic keratosis of the nose before MAL-PDT treatment. B) Response 15 days after MAL-PDT session. The pathogenetic role of ultraviolet rays in the induction and progression of AKs is proved both on experimental and epidemiologic models. AKs are also known as solar keratoses. The term solar is more specific because it refers to a variety of rays. Even among sun rays, action spectrum evaluations indicate that ultraviolet B rays (290-320 nm) are the most damaging, while UVA rays (320-400 nm) can augment the damaging effects of UVB rays. AKs are very often associated with other aspects of photodamage such as actinic elastosis, teleangiec- 384 tasias, wrinkles and solar lentigos.20 Five percent to 20% of these lesions will progress in 10-25 years into squamous cell carcinomas.2 Immunosurveillance can modulate the progression of AKs towards neoplastic lesions in single subjects. Grafted or immunocompromised patients usually have widespread AKs with a more rapid progression towards malignancy. AKs are usually seen as multiple lesions in sunexposed areas (face, dorsa of the hands, bald portions of the scalp in men). Usually the lesions measure less GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 PHOTODYNAMIC THERAPY OF ACTINIC KERATOSES WITH METHYL-AMINOLEVULINATE (METVIX) ROSSI TABLE II.—Lesion response rate by lesion grade and location 6 months after the first and the second PDT session. Lesion location Face Scalp Lesion grade Grade I (AK thin) Grade II (AK moderate) Complete response MAL-PDT After 1 Sessiom 114/170 (67%) 56/170 (33%) 94/114 (82.4%) 44/56 (78%) 10/20 (104/114) 91% 5/12 (49/56) 87.5% 119/170 (70%) 51/170 (30%) 100/119 (84%) 38/51 (80%) 138/170 (77%) 9/19 (109/119) 91.5% 6/13 (44/51) 86% 153/170 (90%) than 1 cm in diameter. They are erythematous, are often covered by adherent scales, and except in their hypertrophic form, show little or no infiltration. Some solar keratoses are pigmented and show peripheral spreading, making clinical differentiation from lentigo maligna difficult. Occasionally, lesions show marked hyperkeratosis and have the clinical appearance of cutaneous horns. Histologically AKs are squamous cell carcinoma in situ. However, biologically, the lesions are still benign; invasion into the dermis, if present at all, is limited to the most superficial portion of the papillary dermis. In the typical histological pattern the epidermis is thickened and shows irregular downward proliferation, which is limited to the uppermost dermis and does not represent frank invasion. Most keratinocytes show a loss of polarity and a disorderly arrangement. Some of these cells show pleomorphism and atypia of their nuclei, which appear large, irregular and hyperchromatic.21 PDT is a treatment modality involving the administration of a photosensitizing compound and the accumulation of the sensitizer molecules in the target cells, followed by selective irradiation of the lesion with visible light with wavelength preferentially between 600 and 700 nm, in order to achieve a deep penetration in the tissue. Basically, photodynamic action requires the presence and interaction of 3 components: photosensitizer, light and oxygen. The initiating step of the photosensitizing mechanism is the absorption of a light photon by the sensitizer, causing a promotion of the drug molecule from its ground state to the extremely unstable excited singlet state. The singlet excited photosensitizer either decays back to the ground state, resulting in the emission of light in the form of fluorescence, or undergoes intersystem crossover to the longer lived triplet excited state by electron spin conversion. The in situ generation of singlet oxygen via the type II pathway Vol. 140 - N. 4 After 2 sessions appears to play a central role in photodynamic cytotoxicity because of the highly efficient interaction of the O2 species with different biomolecules.22, 23 The tissue damaging effect is realized via several pathways: i. cell necrosis and apoptosis of dysplastic cells; ii. microcirculation arrest: damage of endothelial cells promotes thrombus formation and consequent vascular neological stasis which also contributes to tumor ablation. iii. inflammation in the exposed tissue iv. induction of host immune response. MAL is a new topical photosensitizer that may offer advantages over ALA in terms of improved skin penetration as a result of enhanced lipophilicity and greater selectivity for neoplastic cells than other ALA-induced porphyrins.24-27 In addition, as the cellular uptake mechanisms for these agents differ, the intensity of pain may be lower during PDT using MAL than ALA. The good cosmetic results which are obtained due to selective tissue destruction and mobilization of the organism’s proper immune response is one of the main PDT advantages. From the pharmacological point of view, sensitizers show low toxicity and almost no interaction with other medications, making PDT a safe treatment modality. Many other treatments for AKs are available, but most of them present side effects, and they do not assure the same esthetic results.28-30 Cryosurgery is generally limited to patients with only a very limited number of lesions where hypopigmentation can resist after treatment. Curettage has potential complications of scarring and infection, and local anesthesia is required before the procedure. Topical application of 5-fluorouracil cream has the disadvantages of prolonged erythema and exudation as part of the treatment and recovery, lack of patient compliance, morbidity, only partial effectiveness in removing deep or hyperkeratotic lesions, and the potential for GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 385 ROSSI PHOTODYNAMIC THERAPY OF ACTINIC KERATOSES WITH METHYL-AMINOLEVULINATE (METVIX) exacerbation of other cutaneous conditions such as acne rosacea.6 PDT has gained increasing popularity in the treatment of premalignant or malignant skin lesions. The treatment offers the advantages of reduced scarring and improved cosmetic outcome compared with conventional treatments. The use of gentle curettage before MAL-PDT and the occlusion of the drug before illumination probably contribute to the efficacy of the treatment.31 Moreover with this treatment local intralesional anesthesia is not required and the stinging sensation is usually limited to the treated area and can be easily treated with refrigerating devices which are usually present in the most modern and sophisticated PDT lamps. The intensity of itching or pain seem to be lower during PDT using MAL instead of ALA, because of the different cellular uptake mechanisms for these agents. From a practical perspective, MAL PDT can be usefully and safely integrated into clinical practice. In conclusion, in this study we have shown that MAL PDT is an effective, safe and well tolerated treatment for AKs, which could be probably considered the treatment of choice for this very common and emerging cutaneous disorder. PDT is also a promising treatment modality with a good potential for future development in different fields, such as T cell lymphoma, acne, localized eczema, and human papillomavirus infections.32-43 Acknowledgement.—Mrs. S. Lombardi and Mrs. C. Izzap are gratefully acknowledged for their technical assistance. Riassunto Terapia fotodinamica con Metil-aminolevulinato (METVIX®) nel trattamento delle cheratosi attiniche Obiettivo. La cheratosi attinica (CA) o cheratosi solare rappresenta la più comune neoplasia cutanea circoscritta. Alcuni Autori hanno recentemente proposto di definire la CA una “neoplasia intraepiteliale cheratinocitaria” con 3 gradi di evoluzione verso il carcinoma squamocellulare. Secondo altri, la CA è una vera e propria neoplasia fin dall’inizio. È stato calcolato, infatti, che il 60% dei soggetti a basso fototipo (I-III) oltre i 40 anni di età presenta almeno una cheratosi solare. Questa patologia rappresenta, inoltre, la principale condizione per lo sviluppo dei carcinomi a cellule squamose e richiede, perciò, una rapida diagnosi e un efficace trattamento. La prevalenza della CA aumenta con l’età e con l’esposizione a fattori di rischio (elioesposizione) e varia in presenza di fattori predisponenti (immunosoppressione). In questi casi le lesioni sono spesso diffuse e recidivanti e neces- 386 sitano di trattamenti multipli. Le CA devono sempre essere trattate. Oggi sono disponibili terapie sia mediche (crema al 5-FU, peeling medio-profondi, imiquimod crema, retinoidi orali, interferone a2b) sia chirurgiche (elettrochirurgia, escissione chirurgica, laser-chirurgia, criochirurgia, dermoabrasione). La terapia fotodinamica rappresenta una recente efficace acquisizione per il trattamento delle CA, ben tollerata e con eccellenti risultati cosmetici. Può, inoltre, risultare particolarmente utile per la scarsa invasività e la possibile ripetibilità che la contraddistinguono. In questo lavoro è stata valutata l’efficacia di tale terapia con particolare riguardo all’utilizzo del metil-estere dell’ALA, profarmaco fotosensibilizzante di recentissima introduzione nel nostro Paese. Metodi. Sono stati trattati 100 pazienti (70 di sesso maschile, 30 di sesso femminile) di razza caucasica e fototipo 1 e 2 o 3 secondo la scala di Fitzpatrick, per un totale di 170 CA, localizzate al volto e al cuoio capelluto. Risultati. Dopo 15 giorni dal primo trattamento è stata evidenziata una risposta completa (completa regressione clinica della lesione) in 114 lesioni del volto (82,4%) e in 44 lesioni del cuoio capelluto (78%). In base al grado di evoluzione delle cheratosi, l’84% delle lesioni più superficiali e meno squamose (grado I ) ha presentato risposta completa contro l’80% di quelle di grado medio (grado II). La risposta complessiva al primo trattamento è risultata pari al 77%. Sono state ottenute risposte di guarigione completa nel 90% dei casi, risultato sovrapponibile o superiore se comparato ai trattamenti convenzionali. Conclusioni. In questo studio, la terapia fotodinamica con metil-aminolevulinato si è dimostrata una terapia efficace e ben tollerata per il trattamento delle CA; i risultati ottenuti, pertanto, sembrano rimarcare il potenziale ruolo della terapia fotodinamica come trattamento di prima scelta per questa patologia molto comune e in costante aumento. Lo scenario è, inoltre, destinato ad arricchirsi rapidamente in quanto studi pilota sono in corso per il trattamento fototerapeutico di patologie anche molto diverse tra loro, come il linfoma primitivo cutaneo a cellule T e a cellule B, alcune forme di acne e di eczema, l’ipertricosi, il lichen sclerosus et atrophicans e le infezioni da papillomavirus. 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Milano: Masson Editore; 2003. p.138-47. 28. Szeimies RM, Karrer S, Radakovic-Fijan S, Tanew A, Calzavara-Pinton PG, Zane C et al. Photodynamic therapy using topical methyl-5-aminolevulinate (Metvix) is as efficacious as cryotherapy in actinic keratosis, but with superior cosmetic results and high patient satisfaction: a prospective, randomised study. J Am Acad Dermatol 2002;47:258-62. 29. Freeman M, Vinciullo C, Francis D, Spelman L, Nguyen R, Fergin P et al. A comparison of photodynamic therapy using topical methyl aminolevulinate (Metvix) with single cycle cryotherapy in patients with actinic keratosis: a prospective, randomized study. J Dermatol Treat 2003;14: 99-106. 30. Horn M, Wolf P, Wulf HC, Warloe T, Fritsch C, Rhodes LE et al. Topical methyl aminolaevulinate photodynamic therapy in patients with basal cell carcinoma prone to complications and poor cosmetic outcome with conventional treatment. Br J Dermatol 2003;149:1242-9. 31. Morton CA. Methyl aminolevulinate (Metvix) photodynamic therapy-practical pearls. J Dermatol Treat 2003;14:23-6. 32. Coors EA, Von der Driesh P. Topical photodynamic therapy for patients with therapy-resistant lesions of cutaneous T-cell lymphoma. J Am Acad Dermatol 2004;50:363-7. 33. Orenstein A, Haik J, Tamir J, Winkler E, Trau H, Malik Z et al. Photodynamic therapy of cutaneous lymphoma using 5-aminolevulinic acid topical application. Dermatol Surg 2000;26:765-9. 34. Rittenhouse-Diakun K, van Leegoed H, Morgan J, Hryhorenko E, Paszkiewicz G, Whitaker JE et al. The role of trasferrin receptor (CD71) in photodynamic therapy of activated and malignant lymphocytes using the hem precursor delta-aminolevulinic acid (ALA). Photochem Photobiol 1995;61:523-8. 35. Edstrom DW, Portwit A, Ros AM. Photodynamic therapy with topical 5-aminolevulinic acid for mycosis fungoides: clinical and histological response. Acta Derm Venereol 2001;81:184-8. 36. Stables GI, Stringer MR, Robinson DJ. The treatment of cutaneous T cell lymphoma by topical aminolevulinic acid photodynamic therapy. Br J Dermatol 1997;137(S50):51. 37. Markham T, Sheahan K, Collins P. Topical 5-aminolevulinic acid photodynamic therapy for tumor-stage mycosis fungoides. Br J Dermatol 2001;144:1262-3. 38. Mori M, Mavilia L, Rossi R, Campolmi P, Cappugi P, Pimpinelli N. La terapia fotodinamica nel trattamento dei linfomi primitivi cutanei. G Ital Dermatol Venereol 2005;140:123-7. 39. Hongcharu W, Taylor CR, Chang Y, Aghassi D, Suthamjariya K, Anderson RR. Topical ALA-photodynamic therapy for the treatment of acne vulgaris. J Invest Dermatol 2002;115:183-92. 40. Hillemanns P, Untch M, Prove F, Baumgartner R, Hillemanns M, Korell M. Photodynamic therapy of vulvar lichen sclerosus with 5aminolevulinic acid. Obstet Gynecol 1999;93:71-4. 41. Fabbrocini G, Di Costanzo MP, Riccardo AM, Quarto M, Colasanti A, Roberti G et al. Photodynamic therapy with topical 5-aminolaevulinic acid for the treatment of plantar warts. J Photochem Photobiol B Biology 2001;61:30-4. 42. Stender IM, Na R, Fogh H, Gluud C, Wulf HC. Photodynamic therapy with 5-aminlaevulinic acid or placebo for recalcitrant foot and hand warts: randomised double-blind trial. Lancet 2000;355:963-6. 43. Ross EV, Romero R, Kollias N, Crum N, Anderson RR. Selectivity of protoporphyrin IX fluorescence for condylomata after topical application of 5-aminolaevulinic acid: implications for photodynamic treatment. Br J Dermatol 1997;137:736-42. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 387 G ITAL DERMATOL VENEREOL 2005;140:389-95 Childhood atopic dermatitis The relationship between parental and dermatologist assessment of disease severity E. MAZZOTTI 1, C. DI PIETRO 2, S. MASTROENI 1, A. PROVINI 3, M. PARADISI 3, S. TABOLLI 2 Aim. The instruments to measure the severity of atopic dermatitis (AD) are time consuming, so their use is limited in routine clinical practice. The self administered eczema area severity index (SA-EASI) was developed and validated for a caregiver’s self assessment of the severity of child’s AD. The aim of this study was to assess the relationship between the SA-EASI and SCORing atopic dermatitis (SCORAD) tools. Methods. Thirty-five patients with AD admitted at the Pediatric Unit of a Dermatological Hospital in Rome, Italy, and their parents, participated in the study. The severity of the disease has been assessed at admission, by parents using the SA-EASI, and by dermatologists using the SCORAD, independently. Results. Evidence of convergent validity was provided by high correlation between SA-EASI and SCORAD (Rank Spearman rs=0.71; P<0.001). Conclusion. Both instruments are useful in daily clinical practice and in the research on outcomes. The parents received no training in the measurement of eczema and no training in the use of the SA-EASI instrument itself. SAEASI, moreover promotes the involvement of families with an affected child. KEY WORDS: Atopic dermatitis - Child - Measures. This report is part of a broader study examining the effects of a patient/parental education programme on medical and psychosocial outcomes. This study was supported by a grant from the Italian Ministry of Health. Received: September 15, 2004. Accepted for publication: July 6, 2005. Address reprint requests to: Dott.ssa E. Mazzotti, Istituto Dermopatico dell'Immacolata, IDI-IRCCS, Via dei monti di Creta 104, Rome, Italy. E-mail: [email protected]. Vol. 140 - N. 4 1Epidemiology Unit, Istituto Dermopatico dell'Immacolata IDI-IRCCS, Rome, Italy 2Health Service Research Unit, Istituto Dermopatico dell'Immacolata, IDI-IRCCS, Rome, Italy 3Pediatric Unit, Istituto Dermopatico dell'Immacolata, IDIIRCCS, Rome, Italy A topic dermatitis (AD) is a chronic skin condition with a significant quality of life (QoL) impact. Psychological stress, quality of family life, financial problems and social well-being are related to the severity of AD in children.1-4 Essential to the study of the impact of AD on QoL is the measurement of disease severity. Different scoring systems have been developed to determine the severity of AD.5-11 Recently, more objective scores like that using permeability barrier function and stratum corneum hydratation with computer-assisted estimates for extent disease,12 or the specific software to evaluate automatically the extension of the involved area 13 have been developed. Despite better precision and reproducibility of objective measures are known, clinicians and researchers use more classical instruments.14-16 The SCORing atopic dermatitis (SCORAD),17 one of the best validated systems,18-20 is based on objective signs (e.g. extension) and subjective symptoms (e.g. pruritus and sleep loss). The instrument is suited for clinical trials, but is too time consuming for routine clinical use. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 389 MAZZOTTI CHILDHOOD ATOPIC DERMATIS Generally, the use of self-assessed severity indices in dermatology is restricted to adult patients.21, 22 Recently, an instrument for a caregiver’s self-assessment of the severity of his/her child’s AD, the selfadministered eczema area and severity index (SAEASI) has been developed.23 The original study evidenced that “caregivers can accurately assess their child’s cutaneous disease severity in a valid fashion using the SA-EASI”.23 Aims of our study are to assess the performance of the SA-EASI italian version and to measure the relationship between parental and dermatologist assessment of AD severity. Materials and methods Subjects A total of 35 inpatients, 16 males and 19 females, aged 2 months to 17 years, were recruited from the Pediatric Unit of a dermatological hospital in Rome, Italy. Only patients with a diagnosis of AD were enrolled in the study. The exclusion criteria were: other concomitant severe diseases; parents not available to complete the SA-EASI. Instruments The SA-EASI is a one-page instrument allowing caregivers of children with AD to measure disease severity. To estimate the surface area involved a linedrawing silhouette of the body (front and back) was presented to the caregivers and they have to shade the areas currently affected by AD. Based on the silhouette shading, an investigator not directly involved in patient evaluation assigned a value, corresponding to 0-100% body surface area (BSA) involvement, for each of the following 4 areas: head, upper extremities, trunk, and lower extremities. To the BSA involved for each of the 4 body regions was assigned a proportional score as defined on a seven-point ordinal scale: 0, no eruption; 1, ≤9%; 2, 10-29%; 3, 30-49%; 4, 5069%; 5, 70-89%; 6, 90-100%. Each area score was then multiplied by a factor assigned to the corresponding body area on the SA-EASI scoring sheet. The multiplier varied according to body region and the child’s age. SA-EASI weights the involvement of the head, upper extremities, trunk, and legs as 10%, 20%, 30%, and 40% of the total BSA, respectively, 390 for children aged above 7 years, roughly consistent with the “rule of nines”.24 For children <7 years old, a modification was used: BSAs were 20% for the head, 20% for the upper extremities, 30% for the trunk and 30% for the lower extremities.24 Finally the 4 products were summed to obtain the total area score. The second part of the one-page SA-EASI instrument consisted of five 100-mm visual analogue scales (VASs). The VAS consists of a continuous line on which the caregivers make a mark to show the average severity of the AD lesions. The VASs enabled caregivers to describe the redness, thickness, dryness, number of scratches and itchness of an average AD lesion. On each VAS, extremes and intermediate levels were labelled with anchor marks at equivalent intervals along the VAS line. For example, the VAS for redness contains the following word descriptions: no redness, slightly pink, pink, red, and dark red. Severity scoring was calculated from an equation.23 The percentual value, no proportional score, of BSAs were used in this study. Translation and adaptation were authorized by the author. In the Italian version we changed the figure from adult to child, and the intermediate levels on each VAS were marked by signs of 3-mm (heigh) at intervals of 10-mm. The SCORAD is a scoring system based on the assessment of severity by dermatologists. The complete system is called SCORAD index 17 and also includes the assessment of subjective symptoms (pruritus, sleep loss) on a VAS. The extent of lesions is scored by applying the rule of nine after drawing the lesions on an evaluation form like that of SA-EASI. The intensity is determined by grading each of the 6 items on a scale from 0 to 3 (erythema, edema/papulation, oozing/crust, excoriation, lichenification, and dryness). Each item should be scored on the most representative area for a given intensity item. Finally the total score is the sum of extent/5+7*intensity/2 in a standardized way. Owing to this formula extent accounts for about 25% and intensity for about 75% of the total score. The range of the objective SCORAD lies between 0 and 83. Based on the objective SCORAD, the severity of AD can be classified into mild (<15), moderate (between 15 to 40), and severe (>40). In this study we adopted the version without the assessment of subjective symptoms (pruritus, sleep loss).17 Both instruments were completed immediatly after the admission. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 CHILDHOOD ATOPIC DERMATIS MAZZOTTI Statistical analysis Correlation between total SCORAD and SA-EASI score and between BSAs and severity was calculated using the Rank Spearman’s correlation. Stability was calculated using the Rank Spearman’s correlation between 2 tools, delayed 48 h, for each instrument. BSA scores are based on the following formulas: BSASA-EASI=(0.1*Ah)+(0.2*Au)+(0.3*At)+(0.4*Al) BSASCORAD=(9*Ah)+(18*Au)+(37*At)+(36*Al) (Ah, head area score; Au, upper extremities area score; At, trunk area score; Al, legs area score) All analysis were performed using PC-STATA.25 Results The investigated population (N=35; 51% females) with ages ranging from 2 months to 17 years (mean±SD=7.7±5.07; median=8) was assessed. Children were affected by moderate (61.6%) and severe (38.4%) AD according to objective SCORAD. Mean SCORAD score was 38.8+13.03 (median= 36.1), ranging between 15.2 and 70.3. A positive correlation was observed between total SA-EASI score and objective SCORAD (Rank Spearman’s rs=0.71 P<0.0001) and also the extent, according to the rule of nine, (rs=0.68 P<0.0001). Positive correlations, ranged from 0.59 to 0.70, were observed between singular body area (Ah, rs=0.61; Au, rs=0.61; At, rs=0.59; Al, rs=0.70; P<0.0001). The intensity items were poorly correlated. Between redness and erythema (rs=0.42 P=0.0013), scratches and excoriation (rs=0.43 P=0.0010), between dryness (rs=0.29 P=0.061). Other correlations were less than 0.10. TABLE I.—Psychometric features (mean, standard deviation-sd-, median, range) of total SCORAD score and total SA-EASI score. At admission (time 1) and at discharge (time 2). (N=23). Time 1 Total SCORAD score Mean (sd) 36.9 (11.55) Median 35 Range 21.8-68.5 Total SA-EASI score Mean (sd) 10.2 (8.24) Median 8.4 Range 0.16-30.24 *delta=time 1-time 2 Vol. 140 - N. 4 Time 2 Delta* 16.0 (13.24) 14 0-57.4 19.5 (12.20) 18.8 -10.11--45.38 2.5 (5.07) 1.0 0-22.57 7.7 (7.1) 6.1 1.25-22.6 A second administration was proposed to parents of 23 children (13 females and 10 males) and to the dermatologist. The lenght of 48 h was choosen for balancing the effect of recall bias. Patients showed mild (52.4%), moderate (42.9%), and severe (4.8%) AD, according to objective SCORAD, 48-72 h after admission (Table I). The negative delta value observed for SCORAD range is a result of disease worsening for a patient between 2 times observations. Discussion To measure AD severity SA-EASI has been proved to be equivalent to SCORAD. The modified Italian SA-EASI version was easily understandable and managed by all involved parents. The results of this preliminary study are relevant showing parents’ ability to assess, in a reliable and similar way to dermatologist, the extension and severity of AD in their children using this tool. All correlation showed a convergence and had similar magnitude. As expected the tools showed differences for what concern specific characteristic of lesions, peculiar of each instrument, however such discrepancies had no influence about the total score. Conclusions The SA-EASI is identified as an instrument useful to assess the AD manifestations (intensity and extension) in an easy and reliable way. It does not request specific training or health personnel and can be used by patient or caregiver to monitor the disease. The SA-EASI could be considered a communication tool between parents and physicians. The physician can be able to follow the response to treatments. The SCORAD allows the dermatologist to assess the disease in ambulatory care, where time is limited, and in chronic patients in their follow up. In comparison to SA-EASI, SCORAD is a more precise instrument. The dermatologist with his personal experience can identify different clinical manifestations, obviously reported in a wider range of the severity assessed. The total scores of both instruments report different patient status. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 391 MAZZOTTI CHILDHOOD ATOPIC DERMATIS In clinical practice it should be useful to adopt both tools in a hospital setting, in day-hospital or in ambulatory setting, and, for patient/caregiver, at home. SA-EASI moreover participate in the empowerment of families with an affected child. However, other studies with more patients are necessary to confirm the tools capability to monitor changes in the disease during the follow up. Acknowledgments.—The authors thank all the families for participating in the study and their perseverance in completing the tools. References 1. Aziah MS, Rosnah T, Mardziah A, Norzila M. Childhood atopic dermatitis: a measurement of quality of life and family impact. Med J Malaysia 2002;57:329-39. 2. Ben-Gashir MA, Seed PT, Hay RJ. Are quality of family life and disease severity related in childhood atopic dermatitis? J Eur Acad Dermatol Venereol 2002;16:455-62. 3. Warschburger P, Buchholz HTH, Petermann F. Psychological adjustment in parents of young children with atopic dermatitis: which factors predict parental quality of life? Br J Dermatol 2004;150:30411. 4. Ben-Gashir MA, Seed PT, Hay RJ. Quality of life and disease severity are correlated in children with atopic dermatitis. Br J Dermatol 2004;150:284-90. 5. Clendenning WE, Clack WE, Ogawa M, Ishizaka K. Serum IgE studies in atopic dermatitis. J Invest Dermatol 1973;61:233-6. 6. Ring J, Senter T, Cornell RC, Arroyave CM, Tan EM. Plasma complement and histamine changes in atopic dermatitis. Br J Dermatol 1979;100:521-30. 7. Zachary CB, MacDonald DM. Quantitative analysis of T lymphocytes subsets in atopic eczema, using monoclonal antibodies and flow cytometry. Br J Dermatol 1983;108:411-22. 8. Queille-Roussel C, Raynaud F, Saurat JH. A prospective computerized study of 500 cases of atopic dermatitis in childhood. Acta Derm Venereol Suppl 1985;114:87-92. 9. Costa C, Rilliet A, Nicolet M, Saraut JH. Scoring atopic dermatitis: the simpler the better? Acta Derm Venereol 1989;69:41-5. 10. Hanifin J. Standardized grading of subjects for clinical research studies in atopic dermatitis: workshop report. Acta Derm Venereol Suppl 1989;144:13-4. 11. Abrams BB. Atopic dermatitis: elements in clinical study design and analysis. Acta Derm Venereol Suppl 1989;144:15-9. 12. Sugarman JL, Fluhr JW, Fowler AJ, Bruckner T, Diepgen TL, Williams ML. The objective severity assessment of atopic dermatitis score: an objective measure using permeability barrier function and stratum corneum hydration with computer assisted estimates for extent of disease. Arch Dermatol 2003;139:1417-22. 13. Tripodi S, Panetta V, Pelosi S, Pelosi U, Boner AL. Measurement of body surface area in atopic dermatitis using specific software (ScoradCard(c)). Pediatr Allergy Immunol 2004;15:89-92. 14. Frederiksson AJ, Peterssonn DC. Severe psoriasis: oral therapy with a new retinoid. Dermatologica 1978;157:238-44. 15. Bahmer FA, Schafer J, Schubert HJ. Quantification of the extent and the severity of atopic dermatitis: the ADASI score. Arch Dermatol 1991;127:1239-40. 16. Kezawa Z, Ikebe T, Ogura H, Odajima H, Kurosaka F, Sase K et al. Clinical effect of hypoallergenic rice HRS-1 in a atopic dermatitis. Jpn J Allergol 1991;40:633-42. 17. European Task Force on Atopic Dermatitis. Severity scoring of atopic dermatitis: the SCORAD index. Consensus report of the European task force on atopic dermatitis. Dermatology 1993;186:23-31. 18. Kunz B, Oranje AP, Labreze L, Stalder JF, Ring J, Taieb A. Clinical validation and guidelines for the SCORAD index: consensus report of the European Task Force on Atopic Dermatitis. Dermatology 1997;195:10-9. 19. Oranje AP, Stalder JF, Taieb A, Tasset C, de Longueville M. Scoring of atopic dermatitis by SCORAD using a training atlas by investigators from different disciplines. ETAC Study Group. Early Treatment of the Atopic Child. Pediatr Allergy Immunol 1997;8:28-34. 20. Wolkerstorfer A, De Waard Van Der Spek FB, Glazenburg EJ, Mulder PGH, Oranje AP. Scoring the severity of atopic dermatitis: three item severity score as a rough system for daily practice and as a prescreening tool for studies. Acta Derm Venereol 1999;79:356-9. 21. Feldman SR, Fleischer AB Jr, Reboussin DM, Rapp SR, Exum ML, Clark AR et al. The self-administered psoriasis area and severity index is valid and reliable. J Invest Dermatol 1996;106:183-6. 22. Fleischer AB Jr, Rapp SR, Reboussin DM, Vanarthos JC, Feldman SR. Patient measurement of psoriasis disease severity with a structured instrument. J Invest Dermatol 1994;102:967-9. 23. Housman TS, Patel MJ, Camacho F, Feldman SR, Fleischer AB jr, Balkrishnan R. Use of the Self Administered Eczema Area and Severity Index by parent caregivers: results of a validation study. Br J Dermatol 2002;147:1192-8. 24. Hanifin JM, Thurston M, Omoto M, Cherill R, Tofte SJ, Graeber M, the EASI Evaluator group. The eczema area and severuty index (EASI): assessment of reliability in atopic dermatitis. Exp Dermatol 2001;10:11-8. 25. StataCorp. 1999. Statistical Software: Release 6.0. College Station, TX: Stata Corporation. La dermatite atopica infantile. Confronto tra la misura di gravità del dermatologo e quella del genitore L a dermatite atopica (DA) è una patologia cronica che ha un impatto significativo sulla qualità della vita (quality of life, QoL), non soltanto del paziente ma anche dei suoi familiari: a una maggiore gravità clinica della DA corrisponde un più elevato livello di stress psicologico, maggiori difficoltà 392 nelle relazioni familiari e la necessità di disporre di maggiori risorse, economiche e sociali, per fronteggiarla 1-4. Per la valutazione dell’impatto che la patologia ha sui diversi aspetti della QoL la misura della gravità diviene un fattore cruciale. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 CHILDHOOD ATOPIC DERMATIS MAZZOTTI Sono stati proposti diversi metodi per la misura della gravità della DA 5-11. Tra i contributi più recenti ci sono la misura del funzionamento della permeabilità della barriera e dell’idratazione dello strato corneo che si avvalgono di algoritmi computerizzati per la stima dell’estensione del problema 12, o la valutazione automatica, con software dedicati, dell’estensione delle lesioni cutanee 13. Sebbene sia nota la maggior precisione e la riproducibilità di misure oggettive, i clinici e i ricercatori utilizzano strumenti più classici 14-16. L’introduzione dello SCORing atopic dermatitis (SCORAD, European Task Force on Atopic Dermatitis) 17, uno dei sistemi meglio validati che combina insieme la valutazione di alcuni criteri oggettivi (ad esempio l’estensione delle lesioni) e soggettivi (ad esempio prurito, sonno perso), ha rappresentato un valido criterio di riferimento per la misura della gravità della DA 18-20. Benché utilizzato nella ricerca clinica, richiede, tuttavia, tempi troppo lunghi per la compilazione e ciò ne limita l’utilizzo nella pratica clinica di routine. Inoltre gli strumenti di valutazione di gravità in dermatologia sono stati generalmente rivolti a pazienti adulti 21, 22. Solo recentemente è stato sviluppato il self administered eczema area and severity index (SA-EASI) 23 che consente ai genitori la valutazione della gravità della DA dei figli. I risultati riportati nello studio originale 23 hanno evidenziato che “i genitori, utilizzando il SA-EASI, possono valutare in modo accurato la gravità della patologia cutanea dei figli”. Scopo di questo contributo è stato valutare la performance della versione in italiano del SA-EASI, confrontando la misura della gravità della DA effettuata dal genitore con quella effettuata dal dermatologo. Materiali e metodi Soggetti Hanno partecipato allo studio 35 pazienti, 16 di sesso maschile e 19 di sesso femminile, di età compresa tra 2 mesi e 17 anni, ricoverati presso la divisione di dermatologia pediatrica dell’IDI-IRCCS di Roma, e almeno uno dei loro genitori. Sono stati inclusi pazienti con diagnosi di DA confermata da un dermatologo esperto. Sono stati esclusi i pazienti con patologie concomitanti gravi e/o i cui genitori non erano disponibili o non erano in grado di compilare il SAEASI. Strumenti Il SA-EASI è uno strumento di una pagina che consente al genitore del bambino affetto da DA di misurare la gravità della patologia del figlio. È articolato in 2 sezioni, la prima relativa alla localizzazione e all’estensione della dermatite, la seconda relativa alle caratteristiche cliniche, oggettive e soggettive, della dermatite stessa. Nella prima sezione sono rappresentate 2 silhouette di un bambino ideale, una vista da una prospettiva frontale, l’altra posteriore. Il genitore Vol. 140 - N. 4 deve tratteggiare con una penna le aree della silhouette corrispondenti all’eczema sul corpo del proprio figlio. La stima dell’area della superficie corporea (body surface area, BSA) coinvolta è ottenuta applicando un algoritmo a partire dalla valutazione percentuale (0-100) che un ricercatore, che non ha visto il paziente, fa dell’area indicata dal genitore, separatamente per la rappresentazione anteriore e posteriore e per 4 distinti distretti corporei: testa, arti superiori, tronco, arti inferiori. I valori percentuali vengono trasformati in punteggi proporzionali, definiti su una scala ordinale a 7 punti, dove 0 corrisponde a “nessun eczema”, 1 corrisponde a una superficie coinvolta del ≤9%, 2 equivale a 10-29%, 3 a 30-49%, 4 a 50-69%, 5 a 70-89%, e 6 a 90-100%. Ogni punteggio di area viene moltiplicato per uno specifico peso (0,1 per la testa, 0,2 per gli arti superiori, 0,3 per il tronco, 0,4 per gli arti inferiori) e i prodotti sono poi sommati per ottenere il punteggio totale (BSA). Per i soggetti con età inferiore ai 7 anni di età viene applicato un algoritmo modificato in cui i pesi corrispondenti ai distretti corporei sono rispettivamente 0,2 per la testa, 0,2 per gli arti superiori, 0,3 per il tronco, 0,4 per gli arti inferiori. In entrambi i casi la funzione applicata è consistente con la “regola del nove” 24. La seconda sezione è costituita da 5 scale analogo visive (visual analogue scales, VAS) di 100-mm. La VAS è costituita da una linea continua su cui il genitore deve segnare il punto che corrisponde alla gravità media di una lesione della DA. Sulla linea i livelli estremi e intermedi sono posti a distanze equivalenti. Le scale VAS consentono al genitore di descrivere l’intensità media di arrossamento (nessun rossore, lievemente rosa, rosa, rossa, rosso scuro), di ispessimento (da non ispessita a molto), di secchezza (da non secca a estremamente secca), di lesioni da graffiamento (da nessun graffio a molti graffi), di prurito (da nessun prurito a prurito severo). Il punteggio di gravità è ottenuto applicando un’equazione che combina i punteggi derivati dalle 2 sezioni 23. In questo studio è stata utilizzata una versione in cui i pesi specifici per distretto corporeo sono moltiplicati direttamente per i punteggi percentuali di area. L’autorizzazione per tradurre, adattare e utilizzare lo strumento in lingua italiana è stata gentilmente concessa dall’Autore. Dal riferimento originale l’immagine di un uomo è stata modificata in quella di un bambino e le 5 scale, esattamente di 100 mm, sono state graduate con linee di medesima altezza, di 3 mm, collocate a una distanza di 10 mm l’una dall’altra. È stata effettuata una retro traduzione per verificare la congruità della versione italiana con quella in inglese. Lo SCORAD 17 è uno strumento per la valutazione della gravità clinica della DA completato dal dermatologo. È articolato in 3 sezioni. La prima è inerente alla valutazione dell’estensione e alla localizzazione della dermatite. La seconda è relativa alla valutazione dell’intensità di: eritema, edema/papule, siero/crosta, escoriazioni, lichenificazione e secchezza; quest’ultima è valutata sulle aree non coinvolte dalla dermatite, mentre le altre caratteristiche sono valutate sull’area maggiormente interessata dall’eritema. La terza sezio- GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 393 MAZZOTTI CHILDHOOD ATOPIC DERMATIS Le correlazioni sono state calcolate tra i punteggi totali SAEASI e SCORAD, e separatamente tra estensione, distretti corporei e gravità. Come indice di correlazione è stato calcolato il coefficiente ρ di Spearman (rs). La stabilità è stata misurata, separatamente per i 2 strumenti, in 23 soggetti con una seconda somministrazione a distanza di 48 h. Le valutazioni, riportate in percentuale, sono state fatte separatamente per distretto corporeo (testa, arti superiori, tronco, arti inferiori) e piano d’osservazione (frontale, dorsale). Sono stati, quindi, applicati gli specifici algoritmi per la misura delle BSA: BSASA-EASI=(0,1*Ah)+(0,2*Au)+(0,3*At)+(0,4*Al) BSASCORAD=(9*Ah)+(18*Au)+(37*At)+(36*Al) (Ah= area testa, Au= area arti superiori, At= area tronco, Al= area arti inferiori( Per l’analisi statistica è stato utilizzato il software PCSTATA 25. na= 8) hanno partecipato allo studio. Il 61,6% sono risultati affetti da DA moderata (SCORAD tra 15 e 40) e il 38,4% da DA grave (SCORAD >40). Il range di variazione del punteggio totale SCORAD al momento del ricovero è compreso tra 15,2 e 70,3, con un punteggio medio di 38,8 (ds=13,03) e mediano di 36,1. Per valutare le caratteristiche del SA-EASI, come misura della gravità della DA, sono stati confrontati i punteggi ottenuti con quelli derivati dallo SCORAD. Sono stati calcolati gli indici di correlazione tra i 2 punteggi totali, tra i punteggi relativi alle misure di BSA e tra i punteggi relativi ai 4 distretti corporei. Tutti gli indici sono risultati soddisfacenti. Tra i punteggi totali la correlazione è risultata 0,71 (P<0,0001), e solo leggermente inferiore tra le 2 misure di BSA (rs=0,68; P<0,0001). Le correlazioni tra ogni coppia dei 4 distretti corporei variano tra 0,59 e 0,70 (superficie testa rs=0,61; superficie estremità superiori rs=0,61; superficie tronco rs=0,59; superficie estremità inferiori rs=0,70; P<0,0001). Dal confronto delle caratteristiche specifiche delle lesioni emergono relazioni basse che suggeriscono una limitata concordanza tra le misure; tra arrossamento ed eritema l’indice di relazione è rs=0,42 (P=0,0013), tra graffi e escoriazione rs=0,43 (P=0,0010), tra secchezza rs=0,29. Per le caratteristiche di ispessimento e prurito, considerate dal SAEASI, e quelle di lichenificazione, presenza di crosta/siero e infiammazione o formazione di papule dello SCORAD, per le quali non vi è corrispondenza tra i 2 strumenti, le correlazioni sono inferiori a 0,10. Per valutare la capacità dei 2 strumenti di registrare un cambiamento nello stato clinico del paziente è stato proposto al dermatologo e ai genitori di un gruppo di 23 soggetti, 13 di sesso femminile e 10 di sesso maschile, di età compresa tra 3 mesi e 17 anni (media 8,03, ds 5,22, mediana 8,11), di compilare il SA-EASI e lo SCORAD al momento del ricovero e a distanza di 48 h o più. La durata dell’intervallo minimo è stata scelta per bilanciare l’effetto della possibile distorsione dovuta al ricordo della prova precedente che può verificarsi quando l’intervallo tra le 2 è troppo breve. Nella Tabella I sono riportate le caratteristiche dei punteggi totali dei 2 strumenti al tempo 1 e al tempo 2. Nella Tabella I, la differenza, delta, tra il punteggio al momento del ricovero e quello successivo evidenzia un miglioramento della sintomatologia durante il ricovero, sia nei punteggi SCORAD sia in quelli SA-EASI. Il punteggio differenziale negativo nel range dello SCORAD identifica un soggetto che tra i 2 momenti della misura ha avuto un aggravamento dell’area interessata dalla patologia. Il 52,4% dei soggetti presentano a distanza di pochi giorni dal ricovero un quadro di gravità lieve (SCORAD<15), il 42,9% ancora moderata, e il 4,8% grave. Risultati Discussione Trentacinque soggetti (51% di sesso femminile) di età compresa tra 2 mesi e 17 anni (media= 7,7; ds= 5,07; media- Il SA-EASI si è dimostrato uno strumento equivalente allo SCORAD per misurare la gravità della DA infantile. ne è relativa ai sintomi soggettivi di prurito e perdita di sonno negli ultimi 3 giorni. Nella prima sezione sono rappresentate 2 silhouette di un bambino ideale, una vista da una prospettiva frontale, l’altra posteriore. Il dermatologo deve tratteggiare con una penna le aree della silhouette corrispondenti all’eczema sul corpo del bambino. La stima della BSA coinvolta è ottenuta applicando un algoritmo a partire dalla valutazione percentuale (0-100) che un ricercatore, che non ha visto il paziente, fa dell’area indicata dal clinico, separatamente per la rappresentazione anteriore e posteriore e per 4 distinti distretti corporei: testa, arti superiori, tronco, arti inferiori. Successivamente i punteggi di area sono moltiplicati per uno specifico peso (9 per la testa, 18 per gli arti superiori, 36 per il tronco, 37 per gli arti inferiori). Valori diversi sono proposti per i soggetti con meno di 2 anni di età 17. Infine i prodotti sono sommati per ottenere il punteggio BSA. Il punteggio totale è derivato dall’applicazione della formula estensione/5+7intensità/2 in cui l’estensione pesa per il 25% e l’intensità per il 75% del punteggio totale. Il range teorico del punteggio è compreso tra 0 e 83. La gravità della DA è classificata in lieve (<15), moderata (tra 15 e 40) e grave (>40). In questo studio è stata utilizzata la versione che esclude il conteggio dei criteri soggettivi relativi al prurito e al sonno perso 17. Entrambe le valutazioni sono state effettuate al ricovero. Il dermatologo (AP), dopo aver visitato il bambino e compilato lo SCORAD, consegnava al genitore il SA-EASI. Analisi statistica 394 GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 CHILDHOOD ATOPIC DERMATIS MAZZOTTI La versione italiana adattata è risultata facilmente e immediatamente comprensibile a tutti i genitori coinvolti. I risultati di questo studio rappresentano un importante contributo preliminare in quanto evidenziano la capacità del genitore di valutare in modo accurato, e sufficientemente sovrapponibile a quello del dermatologo, l’estensione e la gravità della DA del proprio figlio utilizzando uno strumento strutturato. La valutazione fatta dal genitore non si discosta da quella fatta dal dermatologo, tutte le correlazioni indicano una sostanziale convergenza e sono del medesimo ordine di grandezza. Come atteso i 2 strumenti mostrano delle differenze per quanto riguarda le caratteristiche specifiche delle lesioni peculiari di ogni strumento, tuttavia quest’assenza di parallelismo non sembra influenzare i punteggi totali. Conclusioni In conclusione, sembra che i risultati di questo studio indirizzino verso l’individuazione di uno strumento, il SAEASI, che ha il vantaggio - comune a molti strumenti autosomministrati - di non richiedere l’intervento di personale specializzato, che non necessita di addestramento, che consente, attraverso una rapida e facile compilazione, di valutare le manifestazioni di intensità ed estensione della patologia. Il SA-EASI potrebbe essere lo strumento di comunicazione, sulla patologia, tra paziente (o genitore) e medico curante, e rappresentare, per quest’ultimo, il mezzo per seguire l’andamento della patologia. L’altro strumento, lo SCORAD, presenta caratteristiche analoghe, e il suo impiego nella routine consentirebbe al clinico una valutazione più puntuale e oggettiva, non soltanto in quei contesti nei quali il tempo a disposizione del medico è poco, ad esempio le visite ambulatoriali, ma anche nel rapporto con il paziente cronico, seguito nel tempo. Rispetto al SA-EASI, lo SCORAD si caratterizza come una misura più accurata in quanto il dermatologo con la sua esperienza professionale è in grado di differenziare le diver- Vol. 140 - N. 4 se presentazioni cliniche della patologia. Tuttavia il SAEASI presenta il vantaggio secondario di coinvolgere attivamente, nella valutazione e nel monitoraggio, la famiglia del bambino affetto da patologia cronica. I punteggi di entrambi gli strumenti riflettono il diverso stato clinico del paziente anche se saranno necessari altri studi, su campioni più numerosi, per confermare la capacità di registrare cambiamenti nella patologia, sia nel tempo, sia come risposta ai trattamenti. Riassunto Obiettivo. Gli strumenti per misurare la gravità della dermatite atopica (DA) richiedono tempo sia per la compilazione, sia per l’attribuzione del punteggio e sono, quindi, poco utilizzati nella pratica clinica abituale. Il self administered eczema area severity index (SA-EASI) è stato sviluppato e validato per la valutazione, da parte dei genitori, della gravità della DA dei figli. Lo scopo di questo studio era analizzare la relazione tra SA-EASI e SCORing atopic dermatitis (SCORAD). Metodi. Hanno partecipato allo studio 35 pazienti, ricoverati presso la divisione pediatrica di un ospedale dermatologico di Roma, e almeno uno dei loro genitori. La gravità della patologia è stata valutata, al momento del ricovero in ospedale, separatamente dal genitore, con il SA-EASI, e dal dermatologo, con lo SCORAD. Risultati. La validità concorrente è espressa dalla correlazione tra i punteggi totali dello SCORAD e del SA-EASI (ρ di Spearman=0,71; P<0,001). Conclusioni. Entrambi gli strumenti sono utili nella pratica clinica quotidiana e nella ricerca sugli outcome. Il SAEASI ha il vantaggio di non richiedere l’intervento di personale specializzato e non necessita di addestramento; favorisce, inoltre, un maggior coinvolgimento dei familiari. Parole chiave: Dermatite atopica - Estensione - Gravità Misura dell’accordo - SA-EASI - SCORAD - Validità concorrente. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 395 REVIEWS G ITAL DERMATOL VENEREOL 2005;140:397-406 Extracellular matrix protein 1: a newly discovered glycoprotein with an important role in skin biology I. CHAN 1, T. HAMADA 2, N. OYAMA 3, V. WESSAGOWIT 1, J.A. McGRATH 1 Extracellular matrix protein 1 (ECM1) is a glycoprotein found in many tissues, including skin. First discovered in 1994, its function in skin biology was largely unknown until 2002 when it was identified as the candidate gene/protein for the autosomal recessive disease, lipoid proteinosis. This inherited disorder is characterised clinically by skin and mucosal infiltration and scarring, and histologically by disruption or duplication of basement membrane, as well as widespread deposition of hyaline material in the dermis. Over 30 pathogenic mutations in the ECM1 gene have been characterised, with recurrent mutations, ancestral alleles, genotype-phenotype correlation and new diagnostic techniques now established for this rare genodermatosis. Further insight into the role of ECM1 in human skin was revealed in 2003 with the discovery of circulating autoantibodies against the ECM1 protein in the sera of most patients with lichen sclerosus, a common chronic inflammatory condition that shares some clinicopathological features with lipoid proteinosis. These autoantibodies have been characterised and the immunodominant epitope isolated, and a new ELISA test for lichen sclerosus is currently being evaluated. Protein-protein interaction studies have identified that ECM1 binds to the major heparan sulphate proteoglycan, perlecan, as well as to matrix metalloproteinase 9, epidermal growth factor, and legumain. These findings, in combination with the lipoid proteinosis and lichen sclerosus data, suggest that ECM1 has a key role in several aspects of epidermal differentiation, maintaining dermal architecture, and regulating basement membrane composition. Clearly, the newFunding from the Charitable Foundation of Guy’s and St Thomas’ Hospitals and the British Skin Foundation for several of the original studies referred to in this review is gratefully acknowledged. Address reprint requests to: J. McGrath, Genetic Skin Disease Group, St John’s Institute of Dermatology, St Thomas’ Hospital, Lambeth Palace Road, London SE1 7EH, UK. E-mail: [email protected] Vol. 140 - N. 4 1Genetic Skin Disease Group St John’s Institute of Dermatology Division of Skin Sciences Guy’s, King’s College and St Thomas’ Hospitals’ Medical School, St Thomas’ Hospital, London, UK 2Department of Dermatology Kurume University School of Medicine, Kurume, Japan 3Department of Dermatology Fukushima Medical University School of Medicine Fukushima, Japan ly discovered glycoprotein ECM1 has an important function in skin biology. KEY WORDS: Gene mutation - Autoantibodies - Lipoid proteinosis - Lichen sclerosus - Epidermis - Dermis - Basement membrane. The discovery of extracellular matrix protein 1 E xtracellular matrix protein 1 (ECM1) is a glycoprotein that was first discovered in 1994. In a study relevant to bone matrix biology, Mathieu et al. analysed the proteins secreted by a clonal osteogenic stromal cell line, MN7, derived from mouse bone marrow, using two-dimensional polyacrylamide gel electrophoresis, Western blotting and microsequencing.1 Amongst several proteins identified, a novel glycosylated 85-kDa protein with an average isoelectric point of 5.7 was isolated.1 This protein was initially desig- GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 397 CHAN EXTRACELLULAR MATRIX PROTEIN 1 nated p85 based on its size, but was also named ECM1 because it was discovered amidst various other connective tissue proteins including type I collagen, osteonectin, cathepsin and sialo bone protein.1, 2 The human homologue was later identified in 1997.3, 4 Most of the early functional studies on ECM1 concentrated on its role in tissues other than skin, and involvement of ECM1 in bone and cartilage development, angiogenesis and certain malignancies, was demonstrated. For example, ECM1 was found to be a negative regulator of endochondral bone formation, inhibiting alkaline phosphatase activity and mineralisation.5 ECM1 was also shown to be able to stimulate blood vessel endothelial cell proliferation (in culture), and to promote angiogenesis (in chicken embryos).6 Presence of ECM1 was also demonstrated in the stroma of 2 human breast cancer cell lines, MDA-435 and LCC15.6 Additionally, increased ECM1 expression, revealed by microarray experiments, has been reported in cartilage formation, dendritic cell differentiation and maturation, and in grade I, II and IV glioblastoma multiformes.7-9 Some experimental data for a potential role for ECM1 in the skin, notably in epidermal differentiation, were also postulated,2, 10 but the key role of ECM1 in multiple aspects of cutaneous biology was not immediately apparent. Protein structure of extracellular matrix protein 1 The ECM1 protein consists of 3 isoforms, ECM1a, ECM1b and ECM1c, of 540, 415 and 559 amino acids, respectively. ECM1 contains a signal peptide of 19 amino acids followed by 4 functional domains: a cysteine-free N-terminus, 2 tandem repeats and a C-terminus.2, 3, 11 The latter 3 domains contain numerous cysteine residues, arranged in a specific manner. The cysteine distribution and structure in humans is almost identical to its mouse counterpart, containing 28 cysteine residues, although there is just one amino acid less in the human protein.3 Significantly, the cysteine-containing domains all have the typical CC-(X7-10)C arrangement that is capable of forming protein double loops involved in protein-protein interactions.2, 3 ECM1 contains 1 double-loop domain within each of the 2 tandem-repeats and 1 in the C-terminal domain.11 Different double loops can have varying binding affinities, increasing the potential for interactions with a vari- 398 ety of biological ligands.12 The CC-(X7-10)C motif is also present in the serum albumin family of proteins and shows structural similarities to the Endo 16 calcium-binding protein of sea urchin.2, 13 The specific motif may enable ECM1 to serve as a transporter protein or to be involved in binding growth or differentiation factors.10 Indeed, yeast-two-hybrid studies have shown that ECM1 can bind to several other proteins, including the major heparan sulphate proteoglycan, perlecan, matrix metalloproteinase 9 (MMP-9, type IV collagenase), epidermal growth factor (EGF), and legumain.11, 14 The ECM1 protein also contains 3 Nglycosylation sites for protein kinase C and several phosphorylation sites for casein kinase II.3 ECM1 also contains a calcium-binding domain, which is present in the ECM1a and ECM1c isoforms but not in ECM1b.2, 10 Gene structure and expression of extracellular matrix protein 1 The ECM1 gene has been mapped to chromosome 1q21.2 3, 4 and it has 3 known splice variants, ECM1a, ECM1b and ECM1c.3, 11 ECM1a is encoded by a 10exon gene, whereas ECM1b lacks exon 7 and ECM1c contains an additional exon 5a within intron 5. Exon 5a is homologous to the sixth mouse exon that was initially thought to be absent in the human gene.11 ECM1a is the most widely expressed splice variant. It is found in various tissues including skin, liver, small intestines, lung, ovary, prostate, testis, skeletal muscle, pancreas and kidney, but gene expression is greatest in placenta and heart. By contrast, ECM1b has a much more restricted expression pattern, being detectable only in tonsils and keratinocytes.3 The full pattern of ECM1c expression has yet to be determined, but in skin it accounts for approximately 15% of total ECM1 RNA.11 Apart from the functions identified for the ECM1 protein, sequence analysis also predicts that ECM1 is a positive regulator of the I-κB kinase/NF-κB cascade. The precise localisation of the ECM1 gene maps just centromeric to the epidermal differentiation complex region,10 a locus that contains a cluster of 3 families of genes involved in epidermal differentiation.3 Nevertheless, ECM1 may have a role in terminal keratinocyte differentiation, as suggested by studies demonstrating expression of ECM1a within basal keratinocytes and ECM1b in suprabasal cells.10 GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 EXTRACELLULAR MATRIX PROTEIN 1 CHAN Figure 1.—Skin and mucous membrane features of lipoid proteinosis in (A) a 36-year-old man with severe inflammation, erosions and thickening of the oral mucosa and skin infiltration and scarring on his face, and (B) a 10-year-old boy with infiltration of the lips and tethering of the tongue with a thickened frenulum and reduced tongue movement. There is also papular infiltration of his facial skin. Extracellular matrix protein 1 gene mutations in lipoid proteinosis Lipoid proteinosis (OMIM 247100), also known as Urbach-Wiethe disease or hyalinosis cutis et mucosae, is a rare autosomal recessive disorder typified by generalised thickening and scarring of the skin and mucosae (Figure 1).15, 16 Other characteristic clinical features include beaded eyelid papules, waxy yellow skin papules and nodules, and most notably, a hoarse voice from infancy.16 Increased skin scaling and thickening occurs in regions exposed to mechanical friction including elbows, hands and knees. In 2002, genomewide linkage analysis using genomic DNA from consanguineous families with lipoid proteinosis, was reported.17 Screening mapped lipoid proteinosis to a 2.3-cM interval on the long arm of chromosome 1, at 1q21.2.17 A candidate gene approach was then used, presupposing that, as in many other recessive genodermatoses, the lipoid proteinosis gene product would show reduced expression in dermal fibroblasts compared to normal control fibroblasts. Following this rationale, the gene for lipoid proteinosis was identified as ECM1. Sequencing of genomic DNA from 6 consanguineous families disclosed the presence of homozygous loss-of-function mutations (nonsense, frameshift or internal deletions) in all cases.17 Reduced ECM1 protein expression in lipoid proteinosis skin was also noted.17 Thereafter, the molecular basis of lipoid proteinosis has been determined in over 50 Vol. 140 - N. 4 patients world-wide and 31 different pathogenic mutations (including unpublished data) have been identified (Figure 2).18-22 Mutations have been identified in every exon, apart from exon 5a. The majority of mutations occur in exons 6 and 7, with 9 mutations occurring in exon 7, and 10 mutations occurring in exon 6. Most of the mutations are nonsense or frameshift mutations, presumably resulting in truncation of the ECM1 protein, and/or low levels of the corresponding mRNA through nonsense-mediated mRNA decay mechanisms. In addition, there are 4 missense mutations: V10G in exon 1 and F167I,18 F167L and L210P in exon 6 (including unpublished data). All 4 of these amino acid changes represent substitution of one hydrophobic neutral amino acid by another, but the relative sizes of the amino acids are different and therefore the functional conformation of ECM1 may be altered. All these missense changes have been excluded as rare polymorphisms and the substituted amino acids are highly conserved residues. The missense mutation V10G occurs at the start of the ECM1 gene in the region coding for the signal peptide, whereas F167I, F167L and L210P all occur in the first tandemly-duplicated domain of ECM1. A donor splice site mutation, 80+1G>A, has also been identified (unpublished data). Initial genotype-phenotype correlation suggested that mutations occurring outside exon 7 were associated with a slightly more severe mucocutaneous phenotype, but this has not been borne out in more detailed GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 399 CHAN EXTRACELLULAR MATRIX PROTEIN 1 Q95X/1432delA F167L Q32X 80+1G>A V10G Q114X 501insC 541 del3 ins16 2 3 4 5 C220G/R476X 1253delC R243X 5a 1 1019delA Q276X Q346X 735delTG W359X R243X 6 7 L210P Q197X 542insAA/R243X W160X/F167I R53X 735delTG 243delG 507delT E248X 785delA 892delC 8 9 10 1163-bp deletion H1190insC Figure 2.—Schematic representation of all known pathogenic mutations in the ECM1 gene in lipoid proteinosis. Double arrows indicate homozygous mutations; joined arrows depict compound heterozygosity. analyses.18 Specifically, considerable inter-individual variability has been shown, for example in 29 South African subjects, all with the same homozygous mutation, Q276X, in exon 7.20 This mutant allele was thought to have been propagated by a German settler (from Cologne) to the Northern Cape in the 1650s.20, 23-25 Other mutated ancestral ECM1 alleles have also been identified, for example 501insC in Northern Europe and W359X in Scotland. The mutation 507delT, however, appears to be a hotspot mutation, having occurred on different genetic backgrounds in 2 Thai brothers, a Canadian Iranian family, a Japanese individual, and an Indian girl with lipoid proteinosis.18, 21 New diagnostic test for lipoid proteinosis Lipoid proteinosis can be difficult to diagnose in early life. With time, it can usually be diagnosed clinically but the early manifestations of lipoid proteinosis are protean and may overlap with other diseases, including subtypes of porphyria. Having identified ECM1 as the lipoid proteinosis gene, however, it is now possible to screen DNA in suspected cases to establish the diagnosis, notwithstanding that this may 400 be time-consuming and not readily available in many diagnostic laboratories. Alternatively, one complementary approach to the diagnosis of many recently characterised genodermatoses has involved skin immunohistochemistry. Indeed, many of the severe, usually autosomal recessive, single gene disorders typically involve loss-of-function mutations leading to reduced or absent expression of the encoded protein. Such changes can often be detected through diminished immunohistochemical labelling of skin sections using an antibody to the corresponding protein. This approach has proved to be very useful in the rapid diagnosis of other genodermatoses such as epidermolysis bullosa, lamellar ichthyosis, Netherton’s syndrome and Kindler syndrome.26, 27 To develop an immunohistochemical test for lipoid proteinosis, a rabbit polyclonal antibody to human ECM1 was raised against the oligopeptide SGDTENAKGQGEQGSTG, encoding the carboxyl-terminal of the human ECM1 protein.28 Immunolabelling with this antibody was reduced in lipoid proteinosis skin, confirming its usefulness as a diagnostic probe (Figure 3). However, the pattern of labelling was also able to provide clues as to where the pathogenic mutation might lie.28 Specifically, attenuated but not absent labelling suggested that the pathogenic mutation was in exon 7, whereas GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 EXTRACELLULAR MATRIX PROTEIN 1 CHAN Figure 3.—Immunohistochemical labelling of (A) normal control skin (B) skin from a patient with lipoid proteinosis caused by the homozygous mutation Q276X in exon 7 of the ECM1 gene, using a polyclonal anti-ECM1 antibody. In (A), there is intracellular and cell-surface labelling in the lower epidermis, particularly in basal keratinocytes and to lesser extent, in suprabasal keratinocytes. (B) shows attenuated, though still detectable, immunostaining. (Bar = 50 µm). absence of staining indicated a mutation elsewhere in ECM1. Thus, lipoid proteinosis can now be diagnosed quickly and reliably by skin immnuohistochemistry. Extracellular matrix protein 1 autoantibodies in lichen sclerosus Lichen sclerosus is a chronic inflammatory disorder of skin of unknown aetiology.29, 30 Its features are variable, including white papules and plaques, skin atrophy and scarring, mostly in genital skin but also involving extragenital sites (Figure 4). The prevalence of lichen sclerosus is estimated to be up to 1 in 300 with a ratio of affected females to males of approximately 10:1.30 There is a strong association with autoimmune diseases such as vitiligo, alopecia areata, thyroid disease and pernicious anaemia and there is a positive association with HLA class II antigen DQ7.30, 31 This suggests that part of the disease aetiology or pathological process in lichen sclerosus may involve generation of autoantibodies to one or more antigens in skin. The possibility of ECM1 representing a putative target for humoral immunity in lichen sclerosus is highlighted by the dermatopathological abnormalities in this disorder. Notably, the histology of lichen sclerosus includes hydropic generation of basal ker- Vol. 140 - N. 4 atinocytes, a homogeneous appearance of collagen within the upper dermis (hyalinisation) and disruption of basement membranes. Collectively, many of the alterations in the epidermis, dermis and dermal blood vessels show some histological overlap with lipoid proteinosis, raising the possibility that ECM1 may be a target antigen in lichen sclerosus. To investigate this further, immunoblotting, using a full-length fusion protein for ECM1, was used to demonstrate presence of circulating autoantibodies to ECM1 in the sera of most patients with lichen sclerosus (74%, as compared with 7% of controls).32 These antibodies were present at low titre, since indirect immunofluorescence microscopy was positive in only one case. However, when the lichen sclerosus sera were affinity purified (i.e. concentrated approximately 25 times), a specific pattern of skin immunostaining was identified. This staining pattern was very similar to the appearances of skin labelling with an antibody to ECM1. Moreover, demonstration that the lichen sclerosus sera were targeting ECM1 was confirmed by ablation of labelling following preabsorption of the sera with recombinant ECM1 protein.32 Further immunoblotting studies with fragments of ECM1 recombinant protein were able to show that lichen sclerosus sera react with multiple ECM1 epitopes, the immunodominant epitope being between GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 401 CHAN EXTRACELLULAR MATRIX PROTEIN 1 Figure 4.—Atrophic shiny, pale white plaques of extragenital lichen sclerosus on the trunk of (A) a 38-year old man and (B) a 65-year-old woman. amino acids 359 and 559 within the distal second tandem repeat and the carboxyl-terminus of ECM1.33, 34 The anti-ECM1 IgG subclass is predominantly IgG2, with almost 90% of sera containing IgG2 anti-ECM1 autoantibodies, either alone or in combination with other subclasses.33 These antibodies are not just an epiphenomenon since they are not observed in other sclerosing or basement membrane diseases.32 Furthermore, passive transfer studies with affinity-purified lichen sclerosus IgG autoantibodies injected intradermally into the ears of neonatal mice have shown that, compared to control injected sites, lichen sclerosus IgG injection causes the mice to scratch their ears. Macroscopically, at 28 days, there is swelling and erythema, and microscopically there is oedema, a patchy mononuclear inflammatory cell infiltrate in the dermis, focal pigmentary incontinence in the superficial dermis and dilatation of some superficial blood vessels.34 These changes do not fully recapitulate the full histological features of lichen sclerosus (i.e. no hyalinosis) but are fully consistent with early histological changes seen in this disease. New ELISA test for lichen sclerosus High throughput diagnostic ELISA measurement of serum autoantibodies has become an established part of the investigation of several autoimmune skin disorders, such as pemphigus and bullous pemphigoid.35 Moreover, the antibody titres determined by ELISA in these diseases may have clinical implications for optimal patient management. To assess whether there 402 might be any correlation between anti-ECM1 antibody titres and disease parameters in lichen sclerosus, a diagnostic ELISA was recently reported.34 The protein for the ELISA in this study was based on the immunodominant epitope, i.e. the distal second tandem repeat and carboxyl-terminus of ECM1. This ELISA test exhibited a high sensitivity of 80% (76 of 95 sera were positive) and a high specificity of 93.7% in discriminating lichen sclerosus from normal controls and other autoimmune basement membrane or sclerosing diseases, thus establishing this ELISA as a useful diagnostic test (Figure 5).34 Clinically, higher ELISA titres correlated with more longstanding disease, with cases that were refractory to treatment, and with cases complicated by squamous cell carcinoma.34 This ELISA, therefore, may be useful in detecting individuals with lichen sclerosus who need more aggressive immunosuppressive therapy or who may be at risk for disease complications, such as malignancy or extensive scarring. Detection of the antibodies by ELISA may also provide a means of assessing the potential benefits of new treatments for lichen sclerosus, such as immunoadsorption therapies in which recombinant ECM1 protein might be used to remove circulating anti-ECM1 antibodies from the sera of patients with lichen sclerosus. Currently, however, no prospective studies examining how the anti-ECM1 antibody fluctuates with disease activity and time in individual patients have been reported, and these will be extremely important in establishing the potential usefulness of the ECM1 antibody ELISA in clinical practice. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 EXTRACELLULAR MATRIX PROTEIN 1 CHAN Arbitrary unit of ELISA (405 nm) 1.2 EPIDERMAL DIFFERENTIATION 1.0 0.8 0.6 0.4 0.2 ADHESION ECM1 Cut off (0.328) 0.0 Normal (n=161) LS (n=95) SLE (n=70) BP (n=72) SSc (n=15) Figure 5.—ELISA for anti-ECM1 antibodies assessed in 95 patients with lichen sclerosus (LS), as well as 318 control subjects, comprising 161 normal volunteers, and 70 systemic lupus erythematosus (SLE), 72 bullous pemphigoid (BP), and 15 systemic sclerosis (SSc) individuals. These data show that the assay is highly sensitive and specific for lichen sclerosus. MMP-9 ECM1 The function of extracellular matrix protein 1 in human skin ECM1 PERLECAN BINDING TO COLLAGENS ELASTIC The discovery that loss-of-function mutations in FIBRES ECM1 result in lipoid proteinosis provided the first GROWTH clinical indication of the possible relevance of the BLOOD FACTORS ECM1 protein to skin adhesion, epidermal differentiVESSEL ation, wound healing, scarring, angiogenesis and baseENDOTHELIAL CELL ment membrane integrity. Histologically, skin from PROLIFERATION patients with lipoid proteinosis shows hyperkeratosis, basement membrane thickening at the dermal-epider- Figure 6.—Illustration of the possible functions of ECM1 in human skin mal junction and around blood vessels and adnexal biology. epithelia, as well as presence of hyaline material in the dermis.16 This suggests that a lack of ECM1 may influence the normal pattern of epidermal differenti- ation and, therefore, the epidermal atrophy seen in ation, as well as disrupting dermal physiology. It is lichen sclerosus may be due to changes in the dynamplausible that one of the main functions of ECM1 in the ics of normal keratinocyte maturation.10, 32 In the derdermis is to act as some form of biological glue, help- mis, the basement membrane thickening and hyaline ing to regulate basement membrane and interstitial appearance to collagen could reflect perturbations in collagen fibril macro-assembly and growth factor bind- the normal binding of ECM1 to proteoglycans, such as ing.19 In lipoid proteinosis, the glue is defective or perlecan.11, 32 Notably, co-localisation between ECM1 simply missing, leading to dysregulation of dermal and perlecan has been demonstrated in skin basement homoeostasis and clinical features of skin infiltration membranes, in dermal blood vessels, and surroundand scarring.19 The clinical features seen in lichen ing adnexal epithelia and it has been shown that the carsclerosus may also help illustrate the function of ECM1 boxy-terminus of ECM1 interacts with the EGF-like in human skin, i.e. antibodies to ECM1 disrupt the modules flanking the LG2 subdomain of perlecan normal function of the protein and thereby illustrate domain V.11 Perlecan is also known to bind to what the ECM1 protein normally protects against or fibronectin, laminin, type IV collagen, fibulin 2, dysprevents. ECM1 has a role in keratinocyte differenti- troglycan, platelet-derived growth factor 7 and fibrob- Vol. 140 - N. 4 GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 403 CHAN EXTRACELLULAR MATRIX PROTEIN 1 ECM1 gene ECM1 protein 5a 1 23 45 6 7 8 9 10 N CCysteine tandem tandem free repeat repeat terminal domain domain 1 2 C Signal peptide Inherited mutations Acquired autoantibodies sation.39 Legumain and MMP-9 interact with ECM1 only at the 2 tandem repeat domains, while EGF only interacts at the cysteine-free and C-terminal regions of ECM1.14 Collectively, disruption of these protein-protein interactions with ECM1 in either lipoid proteinosis or lichen sclerosus may provide an explanation for some of the clinicopathological abnormalities in both these conditions (Figures 6, 7). Indeed, similar disruption of basement membrane at the dermal-epidermal junction and around dermal blood vessels has been identified in both disorders.40 Conclusion Lipoid proteinosis Lichen sclerosus Figure 7.—Illustration of the ECM1 gene and protein and the corresponding disease associations. Loss-of-function mutations in the ECM1 gene result in lipoid proteinosis whereas autoantibodies to ECM1 are found in the sera of most patients with lichen sclerosus. In both these disorders, light microscopy reveals a hyaline appearance to the papillary dermis with dilated superficial blood vessels. last-growth factor binding protein.11, 36 Disruption to these normal associations could account for the hyaline abnormalities seen in lichen sclerosus.32 Perhaps disruption of normal expression of ECM1 in blood vessels could also explain the ecchymoses seen in lichen sclerosus. Moreover, the link between ECM1 expression and certain malignant tumours might provide a partial explanation for the increased incidence of squamous cell carcinoma in lichen sclerosus.25 ECM1 also interacts with legumain, MMP-9, and EGF.14 Legumain is a protease linked to aspects of epidermal differentiation;37 EGF is a critical component of several signalling cascades, including calcium response pathways;38 and MMP-9 is a metalloproteinase with a key role in basement membrane and interstitial collagen remodelling as well as vasculari- 404 It is becoming clear that ECM1 has an important role in the anatomy and biology of normal human skin. Although a precise role has yet to be fully elucidated, several clues to its function have been highlighted by its disease associations in the rare genodermatosis, lipoid proteinosis, and the common acquired inflammatory skin disorder, lichen sclerosus, and in its protein-protein interactions with several important regulators of epidermal and dermal homeostasis. Riassunto Proteina 1 della matrice extracellulare: una nuova glicoproteina fondamentale nella biologia cutanea La proteina 1 della matrice extracellulare (ECM1) è una glicoproteine presente in molti tessuti, compresa la cute. Scoperta nel 1994, il suo ruolo nella biologia della cute è stato in gran parte sconosciuto fino al 2002 quando è stato identificato il gene/proteina responsabile della proteinosi lipode, malattia a carattere autosomico recessivo. Dal punto di vista clinico è caratterizzata da infiltrazioni e lesioni cicatriziali a livello della cute e delle mucose, e da un punto di vista istopatologico, da distruzione e duplicazione della membrana basale con numerosi depositi di sostanza ialina nel derma. Sono state identificate più di 30 mutazioni del gene di ECM1, mutazioni ricorrenti, alleli ancestrali, correlazioni fra genotipi e fenotipi; sono state pertanto messe a punto nuove strategie diagnostiche. Un nuovo passo avanti nelle conoscenze del ruolo di ECM1 nella biologia cutanea è stato fatto nel 2003 con la scoperta di autoanticorpi circolanti contro ECM1 nel siero di pazienti affetti da lichen sclerosus, una malattia infiammatoria cronica che condivide molti aspetti anatomopatologici e clinici con la proteinosi lipoide. Sono stati caratterizzati gli autoanticorpi e isolati gli epitopi immunomodulanti; attualmente viene utilizzato un nuovo test ELISA per la diagnosi del lichen sclerosus. Studi di GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 EXTRACELLULAR MATRIX PROTEIN 1 CHAN valutazione dell’interazione proteine/proteine hanno dimostrato che ECM1 lega a perlecan, proteglicano eparan solfato, alle proteine della matrice metalloproteinasi 9, fattori di crescita dell’epidermide e legumain. Questi risultati, insieme ai dati di correlazione fra la proteinosi lipoide e il lichen sclerosus, suggeriscono che ECM1 gioca un ruolo fondamentale in molti aspetti della differenziazione dell’epidermide, nel mantenimento dell’architettura del derma e nel regolare la composizione della membrana basale. Pertanto la glicoproteina ECM1 ha un ruolo fondamentale nella biologia cutanea. PAROLE CHIAVE: Geni, mutazioni - Autoanticorpi - Proteinosi lipoide - lichen sclerosus - Epidermide - Derma - Membrana basale. References 1. Mathieu E, Meheus L, Raymackers J, Merregaert J. Characterization of the stromal osteogenic cell line MN7: identification of secreted MN7 proteins using two-dimensional polyacrylamide gel electrophoresis, western blotting and microsequencing. J Bone Miner Res 1994;9:903-13. 2. Bhalerao J, Tylzanowski P, Filie JD, Kozak CA, Merregaert J. Molecular cloning, characterization and genetic mapping of the cDNA coding for a novel secretory protein of mouse. Demonstration of alternative splicing in skin and cartilage. J Biol Chem 1995;270:16385-94. 3. Smits P, Ni J, Feng P, Wauters J, van Hul W, Boutaibi ME et al. The human extracellular matrix gene 1 (ECM1): genomic structure, cDNA cloning, expression pattern and chromosomal localization. Genomics 1997;45:487-95. 4. Johnson MR, Wilkin DJ, Vos HL, de Ortiz Luna RI, Dehejia AM, Polymeropoulos MH et al. Characterization of the human extracellular matrix protein 1 gene on chromosome 1q21. Matrix Biol 1997;16: 289-92. 5. Deckers MM, Smits P, Karperien M, Ni J, Tylzanowski P, Feng P et al. Recombinant extracellular matrix protein 1 inhibits alkaline phosphatase activity and mineralization of mouse embryonic metatarsals in vitro. Bone 2001;28:14-20. 6. Han Z, Ni J, Smits P, Underhill CB, Xie B, Chen Y et al. Extracellular matix protein 1 (ECM1) has angiogenic properties and is expressed by breast tumor cells. FASEB J 2001;15:988-94. 7. Sekiya I, Vuoristo J, Larson B, Prockop DJ. In vitro cartilage formation by human adult stem cells from bone marrow defines the sequence of cellular and molecular events during chondrogenesis. Proc Natl Acad Sci USA 2002;99:4397-402. 8. Le Naour F, Hohenkirk L, Grolleau A, Misek DE, Lescure P, Geiger JD et al. Profiling changes in gene expression during differentiation and maturation of monocyte-derived dendritic cells using both oligonucleotide microarrays and proteomics. J Biol Chem 2001;276: 17920-31. 9. Rickman D, Bobek M, Misek D, Kuick R, Blaivas M, Kurnit DM et al. Distinctive molecular profiles of high-grade and low-grade glinomas based on oligonucleotide microarray analysis. Cancer Res 2001;61:6885-91. 10. Smits P, Poumay Y, Karperien M, Tylzanowski P, Wauters J, Huylebroeck D et al. Differentiation-dependent alternative splicing and expression of the extracellular matrix protein 1 gene in human keratinocytes. J Invest Dermatol 2000;114:718-24. 11. Mongiat M, Fu J, Oldershaw R, Greenhalgh R, Gown AM, Iozzo RV. Perlecan protein core interacts with extracellular matrix protein 1 (ECM1), a glycoprotein involved in bone formation and angiogenesis. J Biol Chem 2003;278:17491-9. Vol. 140 - N. 4 12. Kragh-Hansen U. Structure and ligand binding properties of human serum albumin. Dan Med Bull 1990;37:57-84. 13. Godin RE, Urry LA, Ernst SG. Alternative splicing of the Endo16 transcript produces differentially expressed mRNAs during sea urchin gastrulation. Dev Biol 1996;179:148-59. 14. Terlizzi J, Li K, Aho A, Fujimoto N, Oyama N, Hamada T et al. Characterization of ECM1 protein interactions by yeast-two-hydrid system. J Invest Dermatol 2004;22:A38. 15. Urbach E, Wiethe C. Lipoidosis cutis et mucosae. Virchows Arch Path Anat 1929;273:285-319. 16. Hamada T. Lipoid proteinosis. Clin Exp Dermatol 2002;27:624-9. 17. Hamada T, McLean WHI, Ramsay M, Ashton GH, Nanda A, Jenkins T et al. Lipoid proteinosis maps to 1q21 and is caused by mutations in the extracellular matrix protein 1 gene (ECM1). Hum Mol Genet 2002;11:833-40. 18. Hamada T, Wessagowit V, South AP, Ashton GH, Chan I, Oyama N et al. Extracellular matrix protein 1 gene (ECM1) mutations in lipoid proteinosis and genotype-phenotype correlation. J Invest Dermatol 2003;120:345-50. 19. Chan I, El-Zurghany A, Zendah B, Benghazil M, Oyama N, Hamada T et al. Molecular basis of lipoid proteinosis in a Libyan family. Clin Exp Dermatol 2003;28:545-8. 20. van Hougenhouck-Tulleken W, Chan I, Hamada T, Thornton H, Jenkins T, McLean WH et al. Clinical and molecular chracterization of lipoid proteinosis in Namaqualand, South Africa. Br J Dermatol 2004;151:413-23. 21. Chan I, Sethuraman G, Sharma VK, Bruning E, Hamada T, McGrath JA. Molecular basis of lipoid proteinosis in two Indian siblings. J Dermatol 2004;31:764-6. 22. Chan I, Bingewar G, Patil K, Nayak C, Wadhwa SL, McGrath JA. An Indian child with lipoid proteinosis resulting from a recurrent frameshift mutation (507delT) in the extracellular matrix protein 1 (ECM1) gene. Br J Dermatol 2004;151:726-7. 23. Heyl T. Genealogical study of lipoid proteinosis in South Africa. Br J Dermatol 1970;83:338-40. 24. Gordon H, Gordon W, Botha V. Lipoid proteinosis in an inbred Namaqualand community. Lancet 1969;1:1032-5. 25. Strassberger E. The Rhenish Mission Society in South Africa 18301950. South Africa: C. Struik (Pty) LTD; 1969. 26. McGrath JA, Eady RA. The role of immunohistochemistry in the diagnosis of the non-lethal forms of junctional epidermolysis bullosa. J Dermatol Sci 1997;14:68-75. 27. Ashton GHS, McLean WHI, South AP, Oyama N, Smith, FJD, AlSuwaid R et al. Recurrent mutations in Kindlin-1, a novel keratinocyte focal contact protein, in the autosomal recessive skin fragility and photosensitivity disorder, Kindler syndrome. J Invest Dermatol 2004;122:78-83. 28. Chan I, Oyama N, Hamada T, Bhogal BS, Black MM, Hamada T et al. Rapid diagnosis of lipoid proteinosis using an anti-extracellular matrix protein 1 (ECM1) antibody. J Dermatol Sci 2004;35: 151-3. 29. Powell J, Wojnarowska F. lichen sclerosus. Lancet 1999;353:1777-83. 30. Tasker GL, Wojnarowska F. lichen sclerosus. Clin Exp Dermatol 2003;28:28-33. 31. Neill SM, Tatnall FM, Cox NH. Guidelines for the management of lichen sclerosus. Br J Dermatol 2002;147:640-9. 32. Oyama N, Chan I, Neill SM, Hamada T, South AP, Wessagowit V et al. Autoantibodies to extracellular matrix protein 1 in lichen sclerosus. Lancet 2003;362:118-23. 33. Chan I, Oyama N, Neill S, Wojnarowska F, Black MM, McGrath JA. Characterization of autoantibodies to extracellular matrix protein 1 (ECM1) in lichen sclerosus. Clin Exp Dermatol 2004;29: 499-504. 34. Oyama N, Chan I, Neill S, South AP, Wojnarowska F, Kawakami Y et al. Development of antigen-specific ELISA for circulating autoantibodies to extracellular matrix protein 1 (ECM1) in lichen sclerosus. J Clin Invest 2004;113:1550-9. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 405 CHAN EXTRACELLULAR MATRIX PROTEIN 1 35. Ishii K, Amagai M, Hall RP, Hashimoto T, Takayanagi A, Gamou S et al. Characterization of autoantibodies in pemphigus using antigenspecific enzyme-linked immunosorbent assays with baculovirusexpressed recombinant desmogleins. J Immunol 1997;159:2010-7. 36. Dunlevy JR, Hassell JR. Heparan sulphate proteoglycans in basement membranes: perlecans, agrin and collagen XVIII. In: Iozzo RV editor. Proteoglycans; structure, biology and molecular interactions. New York: Marcel Dekker Inc; 2000.p.275-336. 37. Zeeuwen PL. Epidermal differentiation: the role of proteases and their inhibitors. Eur J Cell Biol 2004;83:761-73. 406 38. Uyemura T, Takagi H, Yanagida T, Sako Y. Single-molecule analysis of epidermal growth factor signaling that leads to ultrasensitive calcium response. Biophys J 2005;88:3720-30. Epub 2005 Mar 4. 39. Sung HJ, Johnson CE, Lessner SM, Magid R, Drury DN, Galis ZS. Matrix metalloproteinase 9 facilitates collagen remodeling and angiogenesis for vascular constructs. Tissue Eng 2005;11:267-76. 40. Kowalewski C, Kozlowska A, Chan I, Gorska M, Wozniak K, Jablonska S et al. Three-dimensional imaging reveals major changes in skin microvasculature in lipoid proteinosis and lichen sclerosus. J Dermatol Sci 2005;38:215-24. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 G ITAL DERMATOL VENEREOL 2005;140:407-16 The histologic diagnosis of early mycosis fungoides: frequent problems, sporadic solutions E. J. GLUSAC Recent advances in the diagnosis of mycosis fungoides, including T cell receptor gene rearrangement studies, have improved our ability to diagnose this challenging condition. However, it is clear that clonality alone does not equate with malignancy. Despite the aide of ancillary studies, mycosis fungoides remains one of the most difficult dermatologic diagnoses to establish. The histopathologic diagnosis of mycosis fungoides remains within the realm of clinical/histologic/molecular correlation. As such, histologic analysis of early mycosis fungoides remains a key factor in the diagnosis of this challenging condition. It is well known that mycosis fungoides can mimic and be mimicked by a variety of other dermatologic conditions, most of which are inflammatory. A wide variety of studies have well characterized the histologic findings of mycosis fungoides, however, we know much less about which histologic criteria are most specific for this disorder. This article will address myriad of difficulties encountered in the histopathologic diagnosis of mycosis fungoides and then review individual criteria used to establish this disorder, with emphasis on criterion specificity. KEY WORDS: Mycosis fungoides, diagnosis - Mycosis fungoides, histology - Mycosis fungoides, classification. What is the most difficult diagnosis to establish in dermatopathology? P atch Stage mycosis fungoides (MF) is arguably the most difficult dermatopathologic diagnosis to establish histopathologically. There is literature to substantiate this impression. Approximately a decade ago, Address reprint requests to: E. J. Glusac, MD, Yale University School of Medicine, Dermatopathology Laboratory, 5031 LMP, P.O. Box 208059, New Haven, CT 06520-8059. E-mail: [email protected] Vol. 140 - N. 4 Department of Pathology and Dermatology Yale University School of Medicine, New Haven, CT, USA studies performed by members of European Organization for Research and Treatment of Cancer (EORTC) demonstrated accuracy in the diagnosis in MF likened to a coin toss.1 Three expert pathologists reviewed 73 MF biopsies admixed with controls on 2 different occasions. MF was identified correctly on both occasions 50% of the time.1 Forty percent of control cases were identified correctly on both occasions. Other studies seemed to appear more optimistic but, on closer review, were equally disheartening. In a study similar to that of the EORTC, Olerud et al.2 were able to correctly diagnose or suggest MF 92% of the time (60% of MF biopsies diagnostic of MF; 32% consistent with or suspicious for MF). Review of the control group in this study revealed significant problems, however. Fifty two percent of control biopsies were called suspicious for MF, and 6% were called MF outright. These results suggest that if we lower our threshold for the diagnosis of MF, we will significantly over-diagnose this condition. Given the implications of overtreatment, insurance coverage issues, psychological well being of patients, and a variety of other matters, it is arguably more important not to over-diagnose MF than to under-diagnose it. Some authors who have addressed discordance in the diagnosis of MF have argued that similar inaccuracies are seen in most diseases and in most organ sys- GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 407 GLUSAC THE HISTOLOGIC DIAGNOSIS OF EARLY MYCOSIS FUNGOIDES: FREQUENT PROBLEMS, SPORADIC SOLUTIONS tems.3 A variety of references are typically cited in support of this premise. One frequently cited reference, authored by Rosai, addressed diagnostic discrepancies in the histopathology of the intraductal breast carcinoma.4 However, a careful review of this manuscript reveals disagreement between benign and malignant diagnoses (the type of discrepancy we see in MF studies) in less than 1/4 of cases. Frequently cited papers in pulmonary and in gynecologic pathology demonstrate even less significant discrepancies when compared with MF.5, 6 And, for all the difficulties encountered in histopathologic interpretation of melanocytic lesions, interrelator agreement is still much better with these lesions than with MF.7 Why so difficult? There are at least 8 important reasons why MF is so difficult to diagnose. 1) MF is a clinicopathologic diagnosis.8-10 The history we often receive is rule out MF. It is often unclear in such cases whether the clinician believes the patient likely has MF or, rather, an inflammatory disease, with MF at the bottom of the differential diagnosis. A variety of other conclusions are also possible. 2) Treatment alters the histopathologic features of MF. At the time of biopsy, patients have often failed standard treatments for a presumed exanthem, including topical steroids. It should be noted that topical steroids appear to diminish or erase the epidermotropic features of MF.11 3) MF has a wide variety of clinical and histopathologic variants, granulomatous, folliculotropic, verrucous, bullous, hyperpigmented and hypopigmented to name a few.12 While the diagnosis of standard patch stage MF is difficult enough, the existence of a variety of challenging variants complicates the matter. 4) Clinically, early MF often looks more like a rash than a neoplasm.13 Certainly, other neoplasms can occasionally resemble exanthems; sporadic examples of Bowen’s disease (squamous cell carcinoma in situ) show an appearance that resembles eczema. Fortunately, these 2 diseases do not resemble one another under a microscope. Most neoplasms are mimicked by other neoplasms, e.g. malignant melanoma and pigmented basal cell carcinoma. Fortunately, these 2, at least, do not resemble each other microscopically. 408 5) Mycosis fungoides very often looks like a rash histologically as well. As such, a clinical misdiagnosis may be supported by a congruous if inaccurate histologic impression. This can readily occur, as the cells of early MF often do not show significant morphologic differences from those of inflammatory conditions.11, 14 6) Mycosis fungoides does not resemble merely a few different inflammatory processes under the microscope; it resembles many. Shapiro et al., in an analysis of 222 MF biopsies, demonstrated that virtually every inflammatory pattern developed by Wallace Clark and A. Bernard Ackerman can be seen in MF.8 The most common patterns encountered are psoriasiform, lichenoid and psoriasiform/lichenoid. Less common patterns include superficial perivascular, superficial perivascular and interstitial, vacuolar, psoriasiform/spongiotic, spongiotic/psoriasiform/lichenoid, nodular, superficial and deep perivascular and interstitial, diffuse and folliculitic. Rare patterns include spongiotic, vasculitic, vesicular and panniculitic. With this in mind, I am sometimes asked how one becomes good at diagnosing MF. My answer is “by becoming good at everything in the differential diagnosis” (inflammatory skin disorders mostly). Often, one can only rule out MF by making another diagnosis. 7) MF resembles inflammatory disorders not to a small degree but, frequently, to a large degree. Lymphocytes typically infiltrate the basal layer in MF, interacting with Langerhans cells.11, 14, 15 This feature is seen in a wide variety of other inflammatory conditions and is, in fact, typical of some of them, including lichen sclerosis et atrophicus.16, 17 8) There is no agreed upon absolute criteria for the diagnosis of early MF. The concept of MF underwent a paradigm shift in the late 1970’s.11, 14, 18 Previously thought of as a rare, relentless, fatal lymphoma, MF became accepted as a lymphoma which usually behaves in an indolent fashion, marked by patches, with progression to tumor stage disease or fatal disease in a minority of patients. It is really a tale of 2 diseases. In fact, were a particular British author able to write about this disorder he might have said (my changes in italics): “It was the best of diseases, it was the worst of diseases, it was the diagnosis of wisdom, it was the diagnosis of foolishness... we has all criteria before us, we had nothing before us, we were all going direct to Heaven, we were all going direct the other way”.19 Histopathologists generally demonstrate a high GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 THE HISTOLOGIC DIAGNOSIS OF EARLY MYCOSIS FUNGOIDES: FREQUENT PROBLEMS, SPORADIC SOLUTIONS degree of accuracy in the diagnosis of tumor stage MF. Clinically and histopathologically, the presence of a neoplasm is evident at this stage. And whether these tumors retain their epidermotropic capacity or whether they lose it, as they often do, a diagnosis of tumor stage MF can generally be made. And whether the tumor is comprised of small to medium size convoluted lymphocytes or large transformed ones, a diagnosis of the tumor stage of this lymphoma can usually be established.18 Plaque stage disease, though not as simple, is generally not so laden with difficulties as patch stage disease.20 One sees, by definition, involvement of the reticular dermis in plaque stage disease. This is usually accompanied by the epidermal and papillary dermal changes typical of MF. In difficult examples of plaque stage disease, ancillary studies, including immunohistochemistry and gene rearrangement studies, are often helpful.21-23 The diagnosis of patch stage disease, as mentioned, is entirely a different matter. Regarding the literature on histopathologic diagnosis of early MF, it is fair to say that there is good news and bad news. The good news is that some studies have conveyed that accuracy in the diagnosis of MF does increase with experience.2, 3 The bad news is that most studies suggest that we have not yet established adequate criteria to diagnose or dismiss MF consistently.1-3 Magic bullet? The next logical question to ask is “If histopathology is inadequate to diagnose early MF, what ancillary studies can identify this challenging condition?” The first to come to mind is immunophenotyping. It is readily accessible and commonly used, but how useful is it? Early hopes centered on increased CD4:CD8 ratio, but it has been subsequently demonstrated that most inflammatory disorders are CD4 predominant, and, of course, some examples of cutaneous T cell lymphoma are CD8 positive. Immunophenotyping is clearly helpful in plaque and tumor stage MF, where loss of the common T cell antigens CD2, CD3 and/or CD5 may be seen.21, 22 Such losses are not seen, however, in patch stage disease. Loss of Leu-8 was originally thought to be helpful in patch stage disease, but its value has now been dismissed.21, 22, 24 Loss of CD7 (Leu-9) is sometimes still touted as useful,25 but loss of CD7 can be seen as frequently in inflammatory conditions as in early MF.21, 22, 24 Additionally, it is Vol. 140 - N. 4 GLUSAC important to bear in mind that immunohistochemical analysis of T cell infiltrates relies upon identifying antigen loss. It must be kept in mind that, even with adequate controls, it is more difficult to be certain about absence of staining than positive staining. At Yale University and at some other MF centers, immunohistochemistry is generally reserved until after a diagnosis of cutaneous T cell lymphoma has been established. It is then employed to identify aggressive subsets of cutaneous T cell lymphoma, such as gamma/delta lymphoma or aggressive variants of CD8 positive lymphoma. Less controversial is investigation for T cell receptor gene rearrangements (TCR) via polymerase chain reaction (PCR). It is important to keep a variety of caveats in mind, however, in the interpretation of TCR results. It should be noted that a significant percentage of patients with indubitable patch stage MF will not demonstrate a clone via PCR.23, 26-28 Furthermore, investigation of patients without indubitable MF is fraught with even greater difficulty. Patients described as having parapsoriasis,29 pre-MF 26 or as borderline 26 can show clonality rates of significantly less than 50%. It is also important be aware that clones can be identified in disorders that we do not categorize as malignant. A few of these include cutaneous lymphoid hyperplasia,29, 30 pityriasis lichenoides et varioliformis acuta,31-33 pigmented purpuric eruption,34 lichen sclerosis,35 and even lichen planus.36 As such, it is fair to say that there is no magic bullet. It is also likely fair to say that the gold standard for the diagnosis of MF remains in the realm of clinical/histologic/molecular correlation. As such, histopathology remains a key factor. With this in mind, and given the coin toss like status of histopathologic diagnosis of MF, it is fair to ask: “Do we know the histologic criteria for MF?” I think that we do. There have been many excellent descriptive studies regarding MF.8, 14, 37, 38 From such studies we know a great deal about histologic features of MF, but we know less regarding which criteria are most specific. To be more accurate in the diagnosis of MF, we must know the relative specificity of various criteria employed. We can only establish specificity with controlled, blinded studies of MF versus controls. Importantly, there must also be a gold standard against which the histopathologic features of MF can be analyzed as independent variables. Given that the current gold standard remains within the realm of clinical/histo- GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 409 GLUSAC THE HISTOLOGIC DIAGNOSIS OF EARLY MYCOSIS FUNGOIDES: FREQUENT PROBLEMS, SPORADIC SOLUTIONS Figure 1.—Lymphocytes within the epidermis which are larger than those within the dermis has been shown to be a specific criterion for MF. logic/molecular correlation, it is difficult to avoid circular reasoning in histologic studies of MF e.g. biopsies are placed into an MF arm of a study, at least in part, due to the fact that they originally exhibited histologic features of MF.18 With these limitations in mind, at least 3 studies have attempted to address the specificity of various criteria for MF. Each involved blinded reviews of MF and control cases. I will refer to these as the Stanford study,15 the EORTC study 39 and the International Society for Cutaneous Lymphoma (ISCL) study (Burg G, Cerroni L, Glusac EJ, Guitart J, Haeffner AC, Sander CA et al. International Society for Cutaneous Lymphoma-Early Mycosis Fungoides Study Project; Zurich, May 1999, manuscript sumitted). The Stanford study was the largest of the 3, involving 64 MF biopsies and 47 controls.15 This study emulated a practice-like scenario. The MF cases were biopsies sent in to rule out MF that subsequently proved to be MF, as judged by the clinician involved in the study, via analysis of disease progression and of ancillary tests (immunophenotyping and/or gene rearrangement studies). Control cases were biopsies sent in to rule out MF from patients who proved to have another disorder as judged by these same means. The ISCL study, with 33 MF biopsies and 33 control biopsies had, arguably, the purest MF group (Burg et al. submitted). It employed early biopsies from patients who subsequently progressed to tumor stage disease and/or died of disease. The control group included a wide variety of inflammatory disorders. I had the good TABLE I.—Criteria for mycosis fungoides. Stanford 15 7-9 µ Convoluted Lymphocytes LargeER Interepidermal Lymphocytes Convoluted lymphocytes Haloed lymphocytes Pautrier’s Microabscesses Disproportionate Exocytosis Basilar*** Lymphocytes Pagetoid Dystribution Papillary dermal fibrosis EORTC 39 MF CTL MF __ __ 100%** 20%* 67%* 59%* 0% 32% 13% __ __ __ 37%* 2% 58%* 28% __ 67%* 23% 46%** — 61%* — 49% 33%** 33%** 4%** ISCL (submitted) CTL MF CTL __ __ 17% 53% 13% 3% 12% 0% 17% 7% 37% 6% 0% 17% 3% 0% 100% 0% 67% 0% 63% 8% __ __ __ 0% __ * 2+ or greater (of 4) ** tiny collections of 4 cells in 42% *** Defined variously, see text Stanford = Stanford University study ISCL = International Society for Cutaneous Lymphoma study EORTC = European Society for Research and Treatment of Cancer study MF = Mycosis Fungoides CTL = Control Cases 410 GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 THE HISTOLOGIC DIAGNOSIS OF EARLY MYCOSIS FUNGOIDES: FREQUENT PROBLEMS, SPORADIC SOLUTIONS GLUSAC fortune to be involved in the Stanford 15 and ISCL studies (Burg et al. submitted). The third study, performed by the EORTC, involved 24 MF biopsies.39 This study evaluated initial biopsies from patients who subsequently presented with indubitable MF via subsequent biopsy and clinical course. The EORTC study had a strong control group of 13 distinct MF mimics. In the remainder of the article, I will focus on the findings of these 3 studies. Criteria for mycosis fungoides LargER intraepidermal lymphocytes Lymphocytes in the epidermis which are larger Figure 2.—Strictly defined, Pautrier’s microabscesses such as these have than those within the dermis (largER intraepidermal been shown to be a specific feature of MF. lymphocytes) were evaluated by the Stanford 15 and ISCL (Burg et al. submitted) studies (Figure 1). This finding was seen in 17% to 20% of MF biopsies and in 0 to 3% of controls (Table I). This criterion is a very interesting one. It highlights the fact that, early in the course of MF, neoplastic cells tend to home to the epidermis. It also suggests that much of what we see in a MF biopsy is reaction pattern to tumor rather than tumor per se. This connotes that a variety of other frequently employed criteria may represent reaction to pattern to tumor in large part.18 Papillary dermal fibrosis and band-like infiltrate come to mind. The presence of LargER intraepidermal lymphocytes is not a sensitive criterion; however, it is an important one. It is important because it is a relatively specific criterion for MF, a disease with few specific criteFigure 3.—Pseudo-Pautrier’s microabscesses are composed primarily of ria. Langerhans cells. They often show a flask-shaped configuration, opening on to the epidermal surface. Pautrier’s microabscesses Pautrier’s microabscesses, another important criterion, is also relatively specific but insensitive for MF. How often one identifies Pautrier’s microabscesses depends on how one defines the term. The EORTC study defined it as “sharply marginated clusters of atypical lymphoid cells... that were closely opposed to one another with uniform cytologic features... with no plasma or fibrin deposition or significant cytopathic changes in the surrounding keratinocytes” 39 (Figure 2). Defined so rigorously, Pautrier’s microabscesses were found in only 4% of early MF biopsies by the EORTC group.39 It should be noted that this study also had a category termed “tiny collections” of up to 4 cells with- Vol. 140 - N. 4 in the epidermis. Such collections were seen in 42% of early MF biopsies, approximating the percentage of Pautrier’s microabscesses seen in the Stanford study (37%), which accepted 4 cell clusters.15 Most other studies of Pautrier’s microabscesses, including the ISCL study (Burg et al. study submitted), have identified them in approximately 20% of early MF biopsies.8, 37 It is important to bear in mind that collections of cells within the epidermis resembling Pautrier’s microabscesses can be seen in inflammatory conditions.40-42 These are usually comprised of Langerhans cells. Many such pseudo-Pautrier’s microabscesses have a flask shaped appearance, opening onto the epi- GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 411 GLUSAC THE HISTOLOGIC DIAGNOSIS OF EARLY MYCOSIS FUNGOIDES: FREQUENT PROBLEMS, SPORADIC SOLUTIONS Figure 4.—Disproportionate exocytosis (lymphocytes in the epidermis associated with relative paucity of spongiosis) is a subjective finding indicative of MF. Figure 6.—Lymphocytes at all levels of the epidermis (pagetoid distribution) is indicative of MF but is rarely seen in early biopsies of this condition. mitted) subjectively evaluated this criterion, and both found it to be useful. Basilar lymphocytes dermal surface 40 (Figure 3). Such collections will label with CD1a and S100.41 They are typical of spongiotic processes but can also be seen in MF.41 Basilar lymphocytes have been likened to strings of pearls or toy soldiers. Neoplastic lymphocytes in MF typically infiltrate the basal layer, in contiguity with Langerhans cells.11 When basilar lymphocytes are florid, a diagnosis of MF is likely.14 But how much is enough? In the Stanford study, 1-5 lymphocytes in the basal layer per 20X field was a statistically significant and relatively sensitive if poorly specific discriminator.15 In the ISCL study 4 contiguous lymphocytes within the basal layer (Figure 5) was an insensitive criterion (17% of MF cases), but it was almost perfectly specific (Burg et al. study submitted). The EORTC study found that several contiguous rete (not further specified) involved by basilar lymphocytes was seen in approximately half of MF cases and, surprisingly, in no control specimens.39 Disproportionate exocytosis Pagetoid distribution Disproportionate exocytosis describes intraepidermal lymphocytes, associated with a relative paucity of spongiosis 14 (Figure 4). It is a criterion that is uniformly relied upon in scanning any seemingly inflammatory skin biopsy. Though important, this criterion is difficult if not impossible to quantify. Both the Stanford study 15 and ISCL studies(Burg et al. study sub- A pagetoid distribution of lymphocytes (Figure 6) (lymphocytes seen at all levels of the epidermis) was not seen in any MF case or any controls in the ISCL study (Burg et al. submitted). This is not surprising, as a pagetoid distribution is generally considered a finding of more advanced MF. Again, however, there is unexpected data from the EORTC study, where page- Figure 5.—Four or more contiguous lymphocytes within the basal layer has been shown to be a feature indicative of MF. 412 GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 THE HISTOLOGIC DIAGNOSIS OF EARLY MYCOSIS FUNGOIDES: FREQUENT PROBLEMS, SPORADIC SOLUTIONS GLUSAC Figure 7.—Medium-large convoluted lymphocytes that approximate the width of basilar keratinocytes are features strongly supportive of MF. Figure 8.—Lymphocytes with halos or vacuoles around them are a partially artifactual feature that is supportive of MF. toid distribution was seen in 1/3 of MF biopsies and in no control specimens.39 Haloed lymphocytes Medium-large convoluted lymphocytes The EORTC study evaluated for the presence of medium to large (7-9 µ) lymphocytes with convoluted outlines (Figure 7). Such cells approximate the width of basilar keratinocyte nuclei. In the EORTC study, such cells were present within the epidermis of all 24 MF biopsies and within the dermis of 22 of 24 MF biopsies.39 This feature was found in only one of 13 control specimens, and, then, only in the epidermis of that case. This data is very striking, and it is curious. It would appear to imply that a diagnosis of MF can be made routinely and reliably. Other studies performed by members of this same group1 and other groups 2 do not appear to support that supposition. Nonetheless, medium-large convoluted lymphocytes is an important criterion. Its combination of size and convolution has crystalized existing elements in the literature and provided a useful histologic benchmark (width of basilar keratinocyte nuclei) for comparison. The Stanford 15 and ISCL studies (Burg et al. submitted) did not evaluate for the above criterion, but, rather, for convoluted nuclei alone. Each found convoluted nuclei to be a significant, if imperfect discriminator. It should be noted that the evaluation of nuclear convolutions is a highly subjective endeavor 43 and requires excellent histopathologic sections. Vol. 140 - N. 4 Haloed lymphocytes are defined as lymphocytes within the epidermis with a vacuole around them evident at relative low magnification (Figure 8). The cause of halos is not precisely known. They are not typically seen in frozen sections of MF and do not contain mucin.37, 44 They are thought to be, in large part, an artifactual phenomenon. They are possibly the result of contraction of the more abundant cytoplasm of the neoplastic lymphocytes and/or poor cohesion of neoplastic lymphocytes to surrounding keratinocytes.14, 38 Haloing does vary amongst laboratories. In the Stanford study, performed on biopsies processed in the Stanford University histopathology laboratory, it was the strongest histologic discriminator between MF and control cases.15 In the ISCL study (Burg et al. study submitted), performed on biopsies from a variety of European laboratories, it was an insensitive (13%) but completely specific discriminator. Papillary dermal fibrosis While papillary dermal fibrosis (Figure 9) has been touted as a key feature of patch stage MF, none of the 3 studies in question found it to be a useful discriminator between MF and control cases. It may be a marker simply of disease chronicity. In fact, the EORTC study found papillary dermal fibrosis much more frequently in control cases than early MF biopsies.39 It should be noted that the criterion assessed in each of these studies was simply papillary dermal fibrosis, as GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 413 GLUSAC THE HISTOLOGIC DIAGNOSIS OF EARLY MYCOSIS FUNGOIDES: FREQUENT PROBLEMS, SPORADIC SOLUTIONS Figure 9.—Thickened, wiry collagen bundles within the papillary dermis are frequently seen in late patch state MF; however, this feature has not been shown to be a significant discriminator between MF and controls. opposed to lymphocytes splayed between thickened papillary dermal collagen bundles. The latter criterion may still be a useful discriminator, especially in late patch stage MF. Criteria clusters? It is well known that no single histologic criterion can establish a diagnosis of MF. It is poorly known at this time whether specific criteria clusters can be useful in the evaluation of MF. The Stanford study did find that moderate disproportionate exocytosis in combination with at least one haloed lymphocyte for 20X field was specific for MF.15 While I doubt that this feature is specific for MF, it is likely a criteria cluster that merits attention. Sézary syndrome Regarding the leukemic variant of cutaneous T cell lymphoma, Sézary syndrome it is fair to say that histologic diagnosis goes from difficult to more difficult. Buechner and Winkelmann’s classic treatise on this condition showed that only 15% of cases showed an epidermotropic pattern.45 Shapiro et al. demonstrated increased spongiosis and diminished epidermotropism as compared to standard MF.8 Other studies have demonstrated diminished disproportionate exocytosis,46 diminished basilar lymphocytes,46 fewer convoluted lymphocytes,46 increased acanthosis 47 and diminished Pautrier’s microabscesses 47 as compared to MF. 414 Trotter et al. performed, arguably, the most thorough study of this condition.48 Their study involved 41 patients with Sézary syndrome, each with a clonal proliferation in the blood as identified by southern blot analysis. Only 38% of biopsies in this study showed an epidermotropic pattern with atypical lymphocytes. One third of patients showed chronic spongiotic changes and no histologic evidence of lymphoma on original or repeat biopsies. Of note, this group showed no better survival than other patients within the study. The authors suggest that, at least in some patients with Sézary syndrome, the exanthem may represent a non-specific chronic spongiotic response to a primarily leukemic process.48 In this review, I have presented a variety of statistics. In doing so, I do not mean to imply that the diagnosis of MF can be made via an equation-like scheme. These have been attempted in the past but are problematic.4951 Nonetheless, it is useful to be aware of the sensitivity and specificity of various criterion in MF, in order to integrate this knowledge into the necessarily gestalt fashion in which we must all establish a diagnosis. The gold standard for the diagnosis of MF arguably remains within the realm of clinical/histologic/molecular correlation. Familiarity with sensitivity and specificity of criteria for MF, in conjunction with extensive knowledge of disorders within the differential diagnosis of MF (inflammatory dermatopathology), should help us improve our accuracy in the histopathologic diagnosis of early biopsies of this condition. Riassunto Diagnosi istopatologica della micosi fungoide: frequenti i problemi, rare le soluzioni Recenti studi sulla diagnosi della micosi fungoide, compresi quelli riguardanti il riarrangiamento del gene del recettore delle cellule T, hanno migliorato le possibilità diagnostiche di questa patologia che ancora oggigiorno lancia numerose sfide al dermatologo. Tuttavia è chiaro il concetto che clonalità non significa necessariamente neoplasia maligna. Attualmente la micosi fungoide costituisce una delle patologie più difficili da diagnosticare. L’analisi istopatologica permette di eseguire studi e correlazioni da un punto di vista clinico, istologico e molecolare e come tale rimane fondamentale nella diagnosi di questa patologia. La micosi fungoide si può manifestare con caratteristiche simili ad altre patologie dermatologiche generalmente di tipo infiammatorio. Molti studi hanno riportato le caratteristiche istologiche della micosi fungoide, tuttavia non sappiamo ancora quali siano i criteri istologici più specifici per poter fare diagnosi. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 THE HISTOLOGIC DIAGNOSIS OF EARLY MYCOSIS FUNGOIDES: FREQUENT PROBLEMS, SPORADIC SOLUTIONS Questa review prenderà in considerazione tutte le difficoltà incontrate nella diagnosi istopatologica della malattia e i criteri individuali utilizzati, siano essi più specifici o meno. PAROLE CHIAVE: Micosi fungoide, diagnosi - Micosi fungoide, anatomia patologica, Micosi fungoide, classificazione. References 1. Santucci M, Burg G, Feller AC. Interrater and intrarater reliability of histologic criteria in early cutaneous T-cell lymphoma. Dermatol Clin 1994;12:323-7. 2. Olerud JE, Kulin PA, Chew DE, Carlsen RA, Hammar SP, Weir TW et al. Cutaneous T-cell lymphoma. Evaluation of pretreatment skin biopsy specimens by a panel of pathologists. Arch Dermatol 1992;128:501-7. 3. Santucci M, Biggeri A, Feller AC, Burg G. Accuracy, concordance, and reproducibility of histologic diagnosis in cutaneous T-cell lymphomaan EORTC Cutaneous Lymphoma Project Group Study. European Organization for Research and Treatment of Cancer. Arch Dermatol 2000;136:497-502. 4. Rosai J. Borderline epithelial lesions of the breast. Am J Surg Pathol 1991;15:209-21. 5. Feinstein AR, Gelfman NA, Yesner R. Observer variability in the histopathologic diagnosis of lung cancer. Am Rev Respir Dis 1970;101:671-84. 6. Ismail SM, Colclough AB, Dinnen JS, Eakins D, Evans DM, Gradwell E et al. Observer variation in histopathological diagnosis and grading of cervical intraepithelial neoplasia. BMJ 1989;298:1030-1. 7. Weinstock MA, Barnhill RL, Rhodes AR, Brodsky GL. Reliability of the histopathologic diagnosis of melanocytic dysplasia. The dysplastic nevus panel. Arch Dermatol 1997;133:953-8. 8. Shapiro PE, Pinto FJ. The histologic spectrum of mycosis fungoides/Sezary syndrome (cutaneous T-cell lymphoma). A review of 222 biopsies, including newly described patterns and the earliest pathologic changes. Am J Surg Pathol 1994;18:645-67. 9. Glusac EJ, Shapiro PE, McNiff JM. Cutaneous T-cell lymphoma. Refinement in the application of controversial histologic criteria. Dermatol Clin 1999;17:601-14. 10. Glusac EJ. Criterion by criterion, mycosis fungoides. Am J Dermatopathol 2003;25:264-9. 11. Ming M, LeBoit PE. Can dermatopathologists reliably make the diagnosis of mycosis fungoides? If not, who can? Arch Dermatol 2000;136:543-6. 12. LeBoit PE. Variants of mycosis fungoides and related cutaneous T-cell lymphomas. Semin Diagn Pathol 1991;8:773-81. 13. Zackheim HS, McCalmont TH. Mycosis fungoides: the great imitator. J Am Acad Dermatol 2002;47:914-8. 14. Sanchez JL, Ackerman AB. The patch stage of mycosis fungoides. Am J Dermatopathol 1979;1:5-26. 15. Smoller BR, Bishop K, Glusac EJ, Kim YH, Hendrickson M. Reassessment of histologic parameters in the diagnosis of mycosis fungoides. Am J Surg Pathol 1995;19:1423-30. 16. Fung MA, LeBoit PE. Light microscopic criteria for the diagnosis of early vulvar lichen sclerosus. A comparison with lichen planus. Am J Surg Pathol 1998;22:473-8. 17. Citarella L, Massone C, Kerl H, Cerroni L. Lichen sclerosus with histopathologic features simulating early mycosis fungoides. Am J Dermatopathol 2003;25:463-5. 18. Glusac EJ. Of cells and architecture: new approaches to old criteria in mycosis fungoides. J Cutan Pathol 2001;28:169-3. 19. Dickens CA. A tale of two cities. Reader’s Digest ed. Pleasantville, NY: The Readers Digest Association, Inc; 1859. 20. Lefeber WP, Robinson JK, Clendenning WE, Dunn JL, Colton T. Vol. 140 - N. 4 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. GLUSAC Attempts to enhance light microscopic diagnosis of cutaneous T-cell lymphoma (Mycosis Fungoides). Arch Dermatol 1981;117:408-11. Ralfkiaer E. Immunohistological markers for the diagnosis of cutaneous lymphomas. Semin Diagn Pathol 1991;8:62-72. Ralfkiaer E. Controversies and discussion on early diagnosis of cutaneous T-cell lymphoma. Phenotyping. Dermatol Clin 1994;12:329-34. Bachelez H, Bioul L, Flageul B, Baccard M, Moulonguet-Michar I, Verola O et al. Detection of clonal T-cell receptor gamma gene rearrangements with the use of the polymerase chain reaction in cutaneous lesions of mycosis fungoides and Sezary syndrome. Arch Dermatol 1995;131:1027-31. Payne CM, Spier CM, Grogan TM, Richter LC, Bjore CG, Cromey DW et al. Nuclear contour irregularity correlates with Leu-9-, Leu-8cells in benign lymphoid infiltrates of skin. An ultrastructural morphometric and quantitative immunophenotypic analysis suggesting the normal T-cell counterpart to the malignant mycosis fungoides/Ã(c)zary cell. Am J Dermatopathol 1988;10:377-89. Ormsby A, Bergfeld WF, Tubbs RR, Hsi ED. Evaluation of a new paraffin-reactive CD7 T-cell deletion marker and a polymerase chain reaction-based T-cell receptor gene rearrangement assay: implications for diagnosis of mycosis fungoides in community clinical practice. J Am Acad Dermatol 2001;45:405-13. Aston-Key M, Diss TC, Du MQ. The value of the polymerase chain reaction in the diagnosis of cutaneous T-cell infiltrates. Am J Surg Pathol 1997;21:743-7. Tok J, Szabolcs J, Silvers DN, Zhong J, Matsushima AY. Detection of clonal T-cell receptor gamma chain gene rearrangements by polymerase chain reaction and denaturing gradient gel electrophoresis (PCR/DGGE) in archival specimens from patients with early cutaneous T-cell lymphoma: correlation of histologic findings with PCR/DGGE. J Am Acad Dermatol 1998;38:453-60. Bergman R, Faclieru D, Sahar D, Sander CA, Kerner H, Ben-Aryeh Y et al. Immunophenotyping and T-cell receptor gamma gene rearrangement analysis as an adjunct to the histopathologic diagnosis of mycosis fungoides. J Am Acad Dermatol 1998;39:554-9. Staib G, Sterry W. Use of polymerase chain reaction in the detection of clones in lymphoproliferative diseases of the skin. Cancer Res 1995;139:239-47. Wood GS, Tung RM, Haeffner AC, Crooks CF, Liao S, Orozco R et al. Detection of clonal T-cell receptor gamma gene rearrangements in early mycosis fungoides/Sezary syndrome by polymerase chain reaction and denaturing gradient gel electrophoresis(PCR/DGGE). J Invest Dermatol 1994;103:34-41. Weiss LM, Wood GS, Ellisen LW, Reynolds TC, Sklar J. Clonal T-cell populations in pityriasis lichenoides et varioliformis acuta (MuchaHabermann disease). Am J Pathol 1987;126:417-21. Dereure O, Levi E, Kadin ME. T-Cell clonality in pityriasis lichenoides et varioliformis acuta. Arch Dermatol 2000;136:1483-6. Weinberg JM, Kristal L, Chooback L, Honig PJ, Kramer EM, Lessin SR. The clonal nature of pityriasis lichenoides. Arch Dermatol 2002;138:1063-67. Toro JR, Sander CA, LeBoit PE. Persistent pigmented purpuric dermatitis and mycosis fungoides: simulant, precursor, or both? A study by light microscopy and molecular methods. Am J Dermatopathol 1997;19:108-18. Lukowsky A, Munche JM, Sterry W, Audring H. Detection of expanded T cell clones in skin biopsy samples of patients with lichen sclerosus et atrophicus by T cell receptor-y. J Invest Dermatol 2000;115:254-9. Schiller PI, Flaig MJ, Puchta U, Kind P, Sander CA. Detection of clonal T cells in lichen planus. Arch Dermatol 2000;292:568-9. Nickoloff BJ. Light-microscopic assessment of 100 patients with patch/plaque-stage mycosis fungoides. Am J Dermatopathol 1988;10:469-77. Smith NP. Histologic criteria for early diagnosis of cutaneous T-cell lymphoma. Dermatol Clin 1994;12:315-22. Santucci M, Biggeri A, Feller AC, Massi D, Burg G. Efficacy of his- GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 415 GLUSAC 40. 41. 42. 43. 44. 45. 416 THE HISTOLOGIC DIAGNOSIS OF EARLY MYCOSIS FUNGOIDES: FREQUENT PROBLEMS, SPORADIC SOLUTIONS tologic criteria for diagnosing early mycosis fungoides. An EORTC Cutaneous Lymphoma Study Group Investigation. Am J Surg Pathol 2000;24:40-50. LeBoit PE, Epstein BA. A vase-like shape characterizes the epidermalmononuclear cell collections seen in spongiotic dermatitis. Am J Dermatopathol 1990;12:612-6. Candiago E, Marocolo D, Manganoni MA, Leali C, Facchetti F. Nonlymphoid intraepidermal mononuclear cell collections (Pseudo-Pautrier Abscesses) A morphologic and immunophenotypical characterization. Am J Dermatol 2000;22:1-6. Burkert KL, Huhn K, Menezes DW, Murphy GF. Langerhans cell microgranulomas (pseudo-pautrier abscesses): morphologic diversity, diagnostic implications and pathogenetic mechanisms. J Cutan Pathol 2002;29:511-6. Yeh YA, Hudson AR, Prieto VG, Shea CR, Smoller BR. Reassessment of lymphocytic atypia in the diagnosis of mycosis fungoides. Mod Pathol 2001;14:285-8. El Darouti M, Marzouk SA, Horn TD. Failure of detection of mucin in the clear halos around the epidermotropic lymphocytes in mycosis fungoides. J Cutan Pathol 2000;27:183-5. Buechner SA, Winkelmann RK. Sezary syndrome: a clinicopathologic study of 39 cases. Arch Dermatol 1983;119: 979-86. 46. Kohler S, Kim YH, Smoller BR. Histologic criteria for the diagnosis of erythrodermic mycosis fungoides and Sezary syndrome: a critical reappraisal. J Cutan Pathol 1997;24:292-7. 47. Kamarashev J, Burg G, Kempf M, Hess Smid M, Dummer R. Comparative analysis of histological and immunohistological features in mycosis fungoides and Sezary syndrome. J Cutan Pathol 1998;25:407-12. 48. Trotter MJ, Whittaker SJ, Orchard GE, Smith NP. Cutaneous histopathology of Sezary syndrome: a study of 41 cases with a proven circulating T-cell clone. J Cutan Pathol 1997;24:286-91. 49. Cooper KD. A scoring system based on differentially weighted criteria for establishing a standardized threshold for the diagnosis of early mycosis fungoides. In: Lambert WC, Giannotti B, van Vloten WA editors. Basic mechanisms of physiologic and aberrant lymphoproliferation in the skin. NATO ASI series A. New York: Plenum Press; 1994. p. 291. 50. Hoppe RT, Wood GS, Abel EA. Mycosis fungoides and Sézary syndrome: pathology, staging, and treatment. Curr Probl Cancer 1990;14:293-71. 51. Guitart J, Kennedy J, Ronan S, Chimiel JS, Hsiegh YC, Variakojs D. Histologic criteria for the diagnosis of early mycosis fungoides: proposal for a grading system to standardize the pathology reporting. J Cutan Pathol 2001;28:174-83. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 G ITAL DERMATOL VENEREOL 2005;140:417-33 Late Lyme disease, Chronic Lyme disease and post Lyme disease syndrome Clinical and laboratory analysis G. TREVISAN 1, S. ORTENZIO 1, S. BONIN 1, 2 Lyme borreliosis (LB) is a tick-borne spirochetosis caused by Borrelia burgdorfei (Bb) and transmitted by the bite of infected hard-bodies ticks of the genus Ixodes. LB is a multisystemic disease involving skin, joints, nervous system, but also heart and eyes could be involved. The diagnosis of LB is primarily based on clinical and epidemiological criteria, but also serological tests, histological evaluation and cultivation can provide useful supporting evidence. Late involvement of skin, nervous system, joints can also arise after a long period from the thick bite, even after its apparent eradication. In this review we describe different clinical syndromes related to Lyme disease. The hypothesis on the pathogenesis, the similarities with other diseases, the clinical and diagnostic fundamentals for the differential diagnosis and the proposed treatment are presented. KEY WORDS: Lyme Borreliosis - Late Lyme disease - Chronic Lyme disease, therapy - Resistant -Lyme arthritis - Post -Lyme disease. L yme borreliosis (LB) is a tick-borne spirochetosis caused by Borrelia burgdorfei (Bb) and transmitted by the bite of infected hard ticks of the genus Ixodes. LB is a multisystemic disease involving skin, joints, nervous system, but also heart and eyes could be involved.1, 2 LB is clinically subdivided into 3 stages, otherwise the disease could be classified in early and late LB.3 The diagnosis of LB is primarily based on clinical and epidemiological criteria, but also seroAddress reprint requests to: G. Trevisan, Unità Operativa di Dermatologia, Dipartimento di Scienze Cliniche, Morfologiche e Tecnologiche, Università degli Studi di Trieste, Ospedale di Cattinara, Strada di Fiume 447, 34149 Trieste, Italy. E-mail: [email protected] Vol. 140 - N. 4 1Operative Unit of Dermatology Department of Clinical, Morphological and Technological Sciences University of Trieste, Cattinara Hospital, Trieste, Italy 2International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy logical tests, histological evaluation and cultivation can provide useful supporting evidence. In routine patients management the diagnosis of LB is usually without problem for the early manifestation of the disease.3 Late involvement of skin, nervous system, joints can also arise after a long period from the thick bite,4 even after its apparent eradication.5 In some of these cases, it is possible to find higher IgM values.6 The pathogenetic mechanism of antibodies persistence is not clear. Treatment with antibiotics is beneficial for all clinical manifestations of LB. However, in the late stages of borreliosis, symptoms may persist despite extensive and repeated antibiotic treatment,7 even without objective signs of infection or biological markers.8 This event could be explained by an intracellular persistence of the Bb in tissues. According to this theory, it escapes to the host immunity system. The intracellular location of Bb could also explain the persistence of the Borrelia in the skin and joints.9-11 Some patients, after the antibiotic treatment, present inflammation of one or more joints.3, 12 Borrelia seems to be able to trigger some postinfectious syndromes, whose GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 417 TREVISAN LATE LYME DISEASE, CRONIC LYME DISEASE AND POST LYME DISEASE SYNDROME symptoms can persist also in absence of live spirochetes and thus do not respond to antibiotics. In the last years the attention is focused on several clinical features related to LB. These clinical manifestations could be similar to LB but also non-specific complaints and could be associated to the spirochete characteristics or to the immune response to it. These syndromes are: — late Lyme disease; — chronic Lyme disease; — treatment-resistant Lyme disease; — post-Lyme disease; — chronic fatigue syndrome. Late Lyme disease Late Lyme disease occurs in 7 months or more after the bite of the infected thick. Cutaneous manifestations are mainly atrophosclerodermia, while extracutaneous manifestations involve mainly joints and nervous systems.13 Skin manifestations Acrodermatitis chronica atrophicans (Pick-Herxheimer disease) Acrodermatitis chronica atrophicans (ACA) is a relatively frequent chronic skin manifestation of LB. ACA develops insidiously. It occurs from few months up to 1 year after the thick bite. Initially it is characterised by red-bluish discolourations, usually on extensor surface of extremities. The lesion could be uni or bilateral.14 The disease evolution is chronic. The lesions enlarge very slowly over months to years, after which the edema slowly vanishes and atrophy become gradually prominent. The skin becomes thin and wrinkled and the discoluration becomes violet. Sometimes the atrophy lesions develop and dermis, subcutis and muscles are affected. It has been also reported the presence of chronic ulcers and malignant transformation of the atrophic skin. Lesions can occur with itching or burning sensation, but also without any symptoms.14, 15 Other cutaneous manifestations are: lichen sclerosus et atrophicus,16, 17 general sclerodermia, atrophodermia of Pierini-Pasini, of nodular panniculitis of Pfeifer-Weber- Christian.15, 18 Atrophosclerodermic dermatitis such as lichen sclerosus et atrophicus, morphea, circumscribed sclero- 418 dermia, linear scleroderma, idiopathic atrophoderma of Pierini-Pasini, Parry-Romberg syndrome, Busckhe scleredema and eosinophil fasciitis of Schulmann has been reported as late Lyme disease manifestations, although the associations have not yet been established satisfactorily. In some cases of ACA borrelial isolation was recovered from skin biopsy specimens of ACA lesion of more than 10 years duration. In particular, morphea has been related several times to late stages of LB. Morphea, also known as localised scleroderma, is characterised by the induration of cutis and subcutis due to collagen deposition.16 Morphea is classified according to clinical characteristics and the depth of cutaneous tissue involved. Some of these are plaque, generalised, linear and morphea profunda. Contrary to systemic sclerosis, in the localised scleroderma there is no involvement of internal organs, digital sclerosis or the presence of Raynaud's phenomenon. Bb DNA was detected by PCR amplification in skin biopsies of some morphea patients.9 Musculoskeletal system:chronic arthritis (duration > 1 year) Lyme arthritis is often regarded as a onset manifestation of LB in North America. In Europe it is less frequent, but the clinical features of Lyme arthritis are similar both in Europe and North America. The onset could appear from few weeks to years after the thick bite. The course of Lyme arthritis is very variable, it is usually recurrent and can last for several years. The arthritis could become chronic or maintain intermittent attacks lasting from a few weeks to months.3 In the beginning the attacks of arthritis are frequent and short, then they may be longer. Patients present usually general tiredness.15 Features:15 — mono or oligoarthritis; — asymmetric; — frequent and intermittent attacks. Localization:15 — large joints are predominantly affected, most often the knee. Clinical features:15 — swelling; — cutaneous nodules; — loss of functionality; — no rigidity in the morning. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 LATE LYME DISEASE, CRONIC LYME DISEASE AND POST LYME DISEASE SYNDROME Examination:15 Rx of the affected joints could present: — articular effusion; — osteoporosis; — erosion of caput osseum; — calcification of peri-articular soft tissues; — subarticular bone cysts. In absence of antibiotic treatment a irreversible erosion of cartilage and bone could occur. Duration of the illness: in the absence of antibiotic treatment, the arthritis can last for almost 4 years. Duration and severity of the arthritis seems to be related to genetic features. Patients with HLA-DR4 and /or DR2 haplotype develop frequently chronic arthritis characterised by erosion and treatment failure.15, 19, 20 Most patients present negative tests for the detection of Borrelial DNA in synovial fluid, even if the arthritis persists.4 Lyme arthritis is among the most common chronic arthritis in children (about 33%). TREVISAN TABLE I.—Differential diagnosis. ACA: Acrocyanosis Phlebitis Venous insufficiency LES Chronic neuroborreliosis: Meningitis (viral or bacterial origin) Alzheimer’s disease Amyotrophic lateral sclerosis Parkinson's disease Face ague Lyme arthritis: Rheumatoid arthritis Juvenile rheumatoid arthritis Reactive arthritis (Reiter’s syndrome) LES CSF may show lymphocytic pleocytosis 10, 22 with intrathecal antibody production. Borrelia culture from CSF is positive in about 5% of the cases, while PCR is positive in about 50% of the cases. Nervous system Heart Encephalopathy with cognitive dysfunction: typically it is subacute or chronic. Representative manifestations are subtle memory and cognitive dysfunction. Physical examination is usually normal. Patients could be fretful and somnolent.15 Chronic encephalomyelitis: patients develop features that can resemble those seen in focal sclerosis. Unifocal or multifocal inflammatory disease at the central nervous system (brain, optic nerve, encephalic trunk, cerebellum) is normally slowly progressive and involves white matter more than grey-matter (rare). Typical manifestations are temporary or permanent focal deficit like hemiparesis, paraparesis, ataxia and aphasia.15 MRI may suggest a white-matter disease. Multi-infarctual encephalopathy: acute focal neurological deficit 16 could be present, some of these could be temporary (like a transient ischemic attack) or permanent (like a stroke). Axonal poli-neuropathy:21 most of the patients with late LB develop mild sensitive neuropathy (mostly associated with arthritis). Typical manifestations are peripheral intermittent paresthesia at the extremities. Electromyography shows a picture of normal nerve conduction. At the investigation cerebrospinal fluid (CSF) is typically normal in patients with late LB, sometimes Vol. 140 - N. 4 Myocardiopathy 23 with heart failure is very rare and could be the only reason for fatal outcome in patients with LB. Eyes Ocular problems in LB seem to be very rare. Eyes can be affected primarily as a result of the inflammation of the ocular tissues such as conjunctivitis, keratitis, iridocyclitis, retinal vasculitis, chorioiditis and optic neuropathy.3, 24 Prolonged intraocular inflammation can result in blindness. Chronic Lyme disease and differential diagnosis A summary scheme of differential diagnosis is shown in Table I. Signs that enable a reliable clinical diagnosis are related to early LB, they include erythema migrans, the typical LB skin lesion, Bell's palsy or arthritis (in particular mono and oligo-articular). On the contrary, the diagnosis of late LB with its chronic symptoms results sometimes ambiguous.25, 26 Some symptoms, such as persistent fatigue, arthral- GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 419 TREVISAN LATE LYME DISEASE, CRONIC LYME DISEASE AND POST LYME DISEASE SYNDROME gias, fibromyalgias and other alteration of the nervous system (neurocognitive and neuropsychiatric), are related to chronic Lyme disease.27, 28 However, case reports of patients have reported several non-specific complaints such as paresthesia, tremor, palpitation, tachycardia, equilibrium alteration, sweating, visual and gastrointestinal (irritable colon) disorders, frequent urination.25 Some investigators believe that Bb persistence could be the causative agent. The pathogenic mechanism of the chronic Lyme disease is not yet understood. There are controversial opinions about chronic and late Lyme disease. Some authors, in fact, consider them the same clinical event, while others describe them as 2 distinct clinical aspect of the disease.25 To support the second theory there is the presence in patients with late LB arthritis of characteristic features (swelling, cutaneous nodules,...), while in chronic LB these signs are absent. Moreover, patients with chronic LB are mostly not responsive to antibiotic treatment. Patients with late LB and high IgG present a better response to treatment in comparison with patients with lower IgG and symptoms of chronic LB. Typically, chronic LB patients are characterised by high IgM value and low IgG.6 Several reports support the increasing evidence that IgM reactivity is common in chronic, active disease. It is known that IgM reactivity may represent reactivation of latent disease or persistent infections in other chronic infections (e.g. cytomegalovirus and toxoplasma) and it is likely the case of LB.6 On the other side, the detection of IgM and IgG antibodies to individual Bb antigens (24kDa, 31 kDa, 34 kDa, but mainly 41 kDa) can provide a supporting evidence of an active phase of LB, but IgM response could also be induced by several condition of cross reactivity, including ehrlichial, cytomegalovirus and toxoplasma infections leading to false positive results. Some patients with chronic Lyme disease who responded to treatment, decreased the level of IgM and increased the level of IgG. The mechanism that leads to IgM persistence and lower IgG production remains unclear.6 The entity of chronic Lyme disease has been the subject of great controversy. Some authors have not approved the chronic form of Lyme disease assuming that the ongoing long-lasting symptoms could be related to psychiatric problems. The fact that a chronic LB exists is supported by published reports of epidemiologic studies that are related to the incidence of chron- 420 ic neuroborreliosis in 30-50% of patients developing fibromyalgia and chronic fatigue.29 Chronic LB may be present not only in different organs but also in different patterns. The pathophysiology of the chronic symptoms is not well understood, hypothesis are ranging from persisting infection to autoimmunity or a combination of the 2. Chronic Lyme disease is not fatal, but debilitating, characterised by persistent symptoms with cyclic recrudement of the disease. The variety of symptoms could be related to genetic factors that can contribute to the development of chronic disease. The incidence of asymptomatic patients has not been reported but there are evidences that some individuals, asymptomatic for months or years after the infection, can manifest chronic symptoms of the disease owing to provoking events such as trauma, pregnancy or psychological stress.25 There are many theories about the mechanism leading to chronic LB, it is known that viable Bb can persist for decades and cause late skin manifestation of ACA. Thus, the immunopathogenetic findings in ACA can serve as a model for studying the chronic course of LB. Recent findings indicate that the most important cells for antigen presentation, the epidermal Langherans cells, are invaded by Bb in early LB. Therefore, Langherans cells were stained immunohisochemically with different markers to investigate their functional activity. The number of Langherans cells CD1a positive was reduced in erythema migrans but was normal or slightly elevated in ACA. In both diseases there was also a marked downregulation of major hisocompatibility complex class II molecules on Langherans cells. This phenomenon might be a mechanism that protects against the presentation of autoantigens and may be the cause of impaired capacity of Langherans cells to eliminate Bb antigens, thus explaining the chronicity of LB.30 Other authors have postulated the autoimmune theory 31 for chronic LB. T cell recognition of self antigens is a key event in the pathogenesis of autoimmune diseases.32 To date, the initial events that trigger autoreactive T cells are unknown. The molecular mimicry hypothesis predicts that during an infection T cells that recognize both a microbial antigen and a related self peptide become activated and cause autoimmune disease. The hypothesis that the T cell response to one or more antigens of Bb is different in patients with treatment-responsive or treatment-resistant Lyme arthritis was tested. Results from this study demon- GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 LATE LYME DISEASE, CRONIC LYME DISEASE AND POST LYME DISEASE SYNDROME strated that treatment resistant patients presented some alterations in T cell antigen recognition associated with an up-regulation of HLA molecules. Other theories have been made on the pathogenesis of chronic LB ranging from the intacellular infection persistence to coinfection with other microorganisms (e.g. Babesia microti) or the existence of Bb species resistant to antibiotic treatment.33 Most of the clinical manifestations of LB are due to the local presence of the causative agent, Bb in the affected tissue. However, the precise means of tissue damage are not well understood and there is not proof that the organism, live or dead, is always present. An understanding of the complex interactions between the organism and the host can explain manifestations of the disease and the persistence of symptoms and signs after antibiotic treatment.34 Whether chronic LB represents continual infection or it is a post-Lyme disorder is currently unknown. Some authors have reported about post-Lyme syndrome (PLS) in patients who have developed persistent symptoms after antibiotic treatment. They include physic and mental fatigue, myalgias, athralgias, paresthesias or dysesthesias or memory and mood disturbances.8, 35 Related symptoms have been detected both in seropositive and seronegative patients. Even for PLS the mechanism of symptoms persistence is not understood. There are also limited informations regarding to the utility of extended antibiotic treatments for this disorder. Some authors reported that treatment over several months appears to be required to achieve significant improvement in most patients, but other results have shown that treatment with long course of antibiotics did not improve symptoms more than placebo.36 Patients with PLS report the following features, as summarised by the Centers for disease control and prevention (CDC):36 — diagnosis of LB in the past; — treatment with standard courses of antibiotics for established acute LB;37 — long-lasting symptoms (for months or even years). In particular, PLS is characterised by: — encephalopathy with memory or mood disturbances (at a short date); — athralgias; — musculoskeletal pain localised in the back and cervicalgias; — chronic fatigue. Vol. 140 - N. 4 TREVISAN Some risk factors to develop PLS have been reported, such as delayed treatment (over 1 year), high levels of serum immunoglobulin G antibodies and the presence of multiple bands in Western blots that seems to be related to aphasia. Athralgias, mostly persistent knee synovitis, in some cases is possibly related to the triggering of intrasynovial autoimmunity. For these patients, there is no evidence of Borrelia infection by culture or detection of Bb DNA in blood or spinal and synovial fluids. The IgG positivity is a common feature with chronic LB, so serological analysis is not a good tool to distinguish PLS from chronic LB.38 Different studies have reported on the antibiotic treatment failure in patients with PLS.36 In these cases hydroxychloroquine treatment seems to be effective.6, 39 Another clinical syndrome associated to LB is the treatment-resistant Lyme arthritis. In about 10% of patients with Lyme arthritis joint inflammation persists for months or even several years after the apparent eradication of the spirochete, Bb, from the joint with antibiotic treatment.5, 12, 40 A model of molecular mimicry has been proposed affecting genetically susceptible individuals to explain this treatment-resistant course. The majority of patients with treatment-resistant Lyme arthritis have HLA-DRB1*0401 or related alleles, and the severity and duration of their arthritis correlate with cellular and humoral immune responses to outer-surface protein A (OspA) of the spirochete. Using an algorithm, the immunodominant epitope of OspA presented by the DRB1*0401 molecule was predicted to be located at aa 165-173. In a search of the Genetics Computer Group gene bank, only one human protein was identified, lymphocyte function associated antigen-1 (hLFA-1), that had sequence homology with OspA(165-173)and predicted binding in the DRB1*0401 molecule. Synovial fluid T cells from most patients with treatment-resistant arthritis responded to both OspA and hLFA-1, whereas those from patients with other forms of chronic inflammatory arthritis did not. Molecular mimicry between a dominant T cell epitope of OspA and hLFA-1 may be an important factor in the persistence of joint inflammation in genetically susceptible patients with treatmentresistant Lyme arthritis.12, 40, 41 Several studies have been performed to identify possible sites of bacterial persistence in patient with treatment resistant Lyme arthritis. Among them, PCR analysis in DNA obtained from urine, synovial fluid and GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 421 TREVISAN LATE LYME DISEASE, CRONIC LYME DISEASE AND POST LYME DISEASE SYNDROME membrane demonstrated that Bb DNA was not detectable in synovial fluid after antibiotic treatment. However, in patients with ongoing or recurring Lyme arthritis after antibiotic treatment a negative Bb PCR in synovial fluid or urine does not exclude a persisting infection. In these patients, in fact synovial membrane could be positive for Bb PCR detection.11 It has also been reported in literature about B garinii seronegative arthritis.42 Chronic fatigue syndrome Chronic fatigue syndrome (CFS) is a poorly understood condition characterized by debilitating fatigue and associated symptoms lasting at least 6 months. Studies indicate that the illness is not simply a manifestation of an underlying psychiatric disorder, but rather is an illness characterized by activation of the immune system, various abnormalities of several hypothalamic-pituitary axes, and reactivation of certain infectious agents.43 Many searchers have investigated factors associated with CFS. Infections may play a part in ongoing symptomatology in a minority of patients but most of the agents proposed, such as EBV, enteroviruses, fungi (Candida albicans) and bacteria (Chlamydia pneumoniae) have not been found to be important in the etiology of the disease. The main feature of the disease is a debilitating fatigue reducing activity to less than 50% of the patient's premorbid activity for at least 6 months. The disease could be persistent or recurrent.44 In addition, other symptoms could be present (at least 4): — neurophysiological disorders such as lack of concentration and forgetfulness; — pharyngitis; — painful cervical or axillary lymphadenopathy; — mild fever or chills; — myalgias or muscle discomfort or pain; — arthralgias; — headache; — sleep disturbance; — prolonged generalised fatigue after usual levels of activity. Exclusion criteria: chronic ongoing psychiatric illness that preceded the development of chronic fatigue and other diagnosis to explain symptoms of CFS. Many physical illnesses may have fatigue as a symp- 422 TABLE II.—Chronic fatigue syndrome: differential diagnosis. Autoimmune diseases Localised infections Chronic inflammatory diseases (Sarcoidosis) Malignant tumors Chronic or sub-acute infections (Lyme) Neuromuscular diseases Endocrine diseases Parasitosis Mycosis Psychiatric disturbances HIV infection Side effects of long-lasting therapies tom. It is clearly important to exclude common cause of prolonged fatigue such as anemia. A good history and examination may point out the way to other potential causes such as arthritis and diabetes mellitus. Numerous tests have been suggested for a patient presenting prolonged fatigue. Referrals to a specialist infectious diseases clinic have not, however, found batteries of tests to be particularly helpful. In particular, for a differential diagnosis with LB the exclusion criteria are mainly related to laboratory tests such as serology and PCR analysis. Chronic fatigue syndrome: differential diagnosis In Table II is shown the differential diagnosis of CFS.44, 45 According to the clinical aspect of the disease it is difficult to distinguish among chronic LB, fibromyalgias and CFS, because each disorder describes symptomatic pain and fatigue.46 In comparison with LB, CFS and fibromyalgias present more generalised and debilitating symptoms, comprising weakness, strong headache, widespread muscle pain, arthralgia, musculoskeletal pain, symmetrical painful sites in characteristic areas, sleep disturbance and lack of concentration.47 On the other side, these patients don't present arthritis and previous diagnosis of LB, moreover they have normal neurological tests and they are more anxious and depressed in comparison with chronic neuroborreliosis patients. Patients with CFS and PLS share many features. The neuropsychiatric differences have been examined in these disorders to enhance understanding of how mood, fatigue, and cognitive performance interrelate in chronic illness. Despite the symptoms overlap, GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 LATE LYME DISEASE, CRONIC LYME DISEASE AND POST LYME DISEASE SYNDROME patients with PLS show greater cognitive deficits than patients with CFS compared with healthy controls. This is particularly apparent among patients with PLS who lack premorbid psychiatric illness.48 Diagnosis Diagnosis of LB is mainly based on clinical and epidemiological criteria, supported by laboratory tests.3, 15 In routine patient management with typical early skin lesion the diagnosis of LB in endemic area is purely clinical. In these cases laboratory testing is not necessary. For the other manifestations of LB laboratory support is essential to confirm the infection. In more complicated cases it is necessary to evaluate more aspects. They include the clinical picture, other preceding or concomitant diseases of the Lyme borreliosis complex, serodiagnostic results, CSF findings, demonstration of intrathecal specific antibody synthesis, results of PCR analysis, response to adequate antibiotic therapy and exclusion of other diseases. The significance of each of these criteria depends on the clinical involvement and on the stage of Lyme borreliosis. Laboratory diagnosis is possible with direct or indirect methods. Indirect methods to assess Bb antibodies in serum, synovial and cerebral fluids are serological tests. These comprise ELISA, immunofluorescence assays (IFA) and Western blotting. A two-step serological approach has been proposed to increase specificity. A positive or equivocal first test (ELISA or IFA) is followed on the same serum sample by an immunoblot test which can detect IgM and IgG antibodies to individual Bb antigens. Direct laboratory diagnosis is related to histological and immunohistochemical techniques, cultivation and hybridiastion using fresh tissues and biological fluids.15 Routine laboratory tests, including VES are usually normal. VES value is an important aspect for the differential diagnosis with LES and rheumatoid arthritis. Seronegativity does not exclude LB diagnosis,49 as reported for Lyme arthritis.41 In suspected cases for late LB, a positive serology is fundamental.50 In about 75% of patients a negative ELISA and a positive Western blot was reported. Most patients with Lyme arthritis are IgG positive both in ELISA and Western blot. The appearance and evolution of IgM and IgG antibodies to Bb was investigated in patients with erythema migrans. The first immune response resulted against flagellin (41kDa) and OspC (24 kDa) in fact the most Vol. 140 - N. 4 TREVISAN frequent IgM bands were of 24 kDa (OspC) and 41 kDa. No IgG is typically detected in this phase of the disease. IgM to antigens of 60 and 66 kDa could be revealed with the further sero-conversion to IgG antibodies against 24 and 41 kDa antigens. With clinical manifestation of LB the presence of antibodies against 24 and 41 kDa antigens may be of assistance in confirming the diagnosis.6, 29, 51 The antigen of 41 kDa is not a characteristic only of Bb, but 24 kDa. Other antigens are characteristic of Bb such as 39, 83 or 93 kDa, The immune response to the recombinant outer surface protein A (OspA- 31 kDa) occurs about 1 year after the infection. With the resolution of symptoms the IgM level usually disappears or decreases but sometimes IgG can persist.6, 29. Some patients could be symptomatic despite negative western blot.52 The absence of immune response could be explained by the intracellular localisation of Bb, that in this way evades the immune system.25, 53, 54 Recently, a sensitive and specific ELISA was introduced in which the antigen is a 26-mer peptide within the sixth invariant region (IR6) of the Vlse (Variable major protein like sequence Expressed) outer-surface lipoprotein of Bb.55 The outer cell membrane of Bb contains many polypeptides, the most extensively studied are the outer surface proteins Osp. OspA and B proteins are expressed in vector, but not in vertebrate host. OspC and VlsE are expressed in vivo in vertebrate host.56 VlsE is an outer surface lipoprotein of Bb that undergoes antigenic variation through an elaborate gene conversion mechanism and is thought to play a major role in the immune response to Borellia in Lyme disease. The surface localization of the variable amino acid segments appears to protect the conserved regions from interaction with antibodies and hence may contribute to immune evasion.57, 58 VlsE has a predicted molecular mass of 34. Two invariable domains, one at the amino terminus and the other at the carboxyl terminus, encompass together approximately one-half of this molecule's length. The remainder is composed of a central variable domain that contains 6 variable regions (VRs) and 6 invariable regions (IRs). These 2 types of regions are interspersed with each other, and each constitutes about one-half of the variable domain's length. The coding sequence of VlsE contains 1 vls cassette region in the middle and 2 noncassette regions. Most sequence differences among the vls cassettes are confined within 6 highly variable regions.57, 58 DNA segments of the silent cassettes are able to recombine GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 423 TREVISAN LATE LYME DISEASE, CRONIC LYME DISEASE AND POST LYME DISEASE SYNDROME in an apparently random manner into the vlsE cassette region throughout the course of infection. Sequence results are consistent, with roughly 6 to 11 recombination events with multiple silent vls cassettes during the first 28 days of infection. The promiscuous recombination events at the vlsE site lead to extensive genetic and antigenic variation in VlsE variants.57, 58 Recombination events between the expressed and silent vmp genes lead to antigenic variation and thus evasion of the host immune response during the course of mammalian infection. Unlike the invariable regions of variable major protein, which are not antigenic in natural infections, the most conserved of the IRs, IR6, is immunodominant in Lyme disease patients. IR6 is exposed on the surface of VlsE, as assessed by immunoprecipitation experiments, but is inaccessible to Ab on the spirochete's outer membrane.58 VlsE thus significantly departs from the antigenic variation paradigm, whereby immunodominance is only manifest in variable portions. IR6 may contribute to divert the Ab response from other, perhaps protective regions of VlsE. The major limitation in serologic tests is that they do not reliably distinguish between active and past infection. With these tests the IgG and sometimes IgM response may persist for years after successful antibiotic treatment. A recent study reported that IgG antiVlsE antibody titres wane rapidly after successful antibiotic treatment in both humans and experimental animal.59 However, other authors reported that the Anti-VlsE response persisted for months or years after antibiotic treatment so their presence cannot be equated with spirochetal persistence in LB.60 Direct methods of laboratory diagnosis are particularly useful in the early phases of the disease when antibody titre is absent or low. Ideally, the detection of Bb by cultivation is the standard goal to prove the infection, but the sensitivity of this method is inadequate for diagnosis. Positive Bb colture results sometimes from skin biopsies, serum, synovial and cerebral fluids.15 PCR is a good tool for Bb detection especially for differential diagnosis of suspected LB.15 Therapy Antibiotic therapy is more effective in the early phases of the disease. The results of treatment depends not only on the location, extent, and duration of clinical manifestations but also on several other factors 424 including the choice of antibiotic, dosage, duration of treatment, potential or adverse effects and compliance.15 Amoxicillin and doxycycline are the treatment of first intention in early LB,3 but not for late disease. Some patients treated with standard courses of these antibiotics recover only partly. For other patients symptoms persist during and after treatment. In these cases longer duration of treatment has been proposed.36 Lyme arthritis could be treated with oral or parenteral antibiotic therapy (amoxicillin and doxycycline).4, 61 Treatment with these 2 antibiotics lasting even 28 days was reported with positive response. Nervous system involvement and Lyme carditis are usually treated with ceftriaxone and penicillin G or cefuroxime intravenously. Treatment for children consists of conventional antimicrobial therapy - either orally administered amoxicillin, doxycycline (major than 12 years old), erythromycin, or penicillin or intravenously administered ceftriaxone, better if children are older than 8 years.3, 4 In most patients with Lyme arthritis, antibiotic therapy is curative, but patients with persistent symptoms after the first cycle of antibiotic treatment could be retreated for 4 weeks with the previous oral therapy, otherwise for 2 weeks intravenously with ceftriaxone or cefotaxime. For patients treatment-resistant arthritis, in which antibiotic therapy is ineffective; arthroscopic synovectomy may be considered to reduce articular inflammation.4 In cases of neuroborreliosis with central nervous system involvement a lumbar puncture is recommended before antibiotic treatment with ceftriaxone and penicillin G or cefuroxime intravenously (2 g/die for 2-4 weeks).3, 4 The appropriate treatment of patients with chronic Lyme disese is a controversial clinical issue. With the possibility that chronic LB is due to persistent infection, the use of antibiotics with intracellular penetration capability such as macrolides and tetracycline is proposed.6, 39 The location of Bb in vivo is unknown, but increasing number of microbes with reactivation potential are located in lysosomes or other acidic endosomes. Macrolide alone has limited activity in acid environment. Usually, macrolides have not been used in the treatment of LB. A recent study reported the use of macrolides in conjunction with lysosomotropic agents which can alter the pH of acidic intacellular compartements such as lysosome. With this assum- GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 LATE LYME DISEASE, CRONIC LYME DISEASE AND POST LYME DISEASE SYNDROME tion, patients were treated with hydroxychloroquine as lysosomotropic agent.39, 62 Treatment over several months appears to be required to achieve significant improvement in most patients with chronic LB. In recent observations the duration of treatment appears to be closer to 12-18 months. However, some patients with chronic LB have no benefits from the use of this therapy. Even if late, chronic and post manifestations of Lyme disease have been identified, difficulties in diagnosis of late stages of Lyme disease persist due to low sensitivity of serological testing and late inclusion of Lyme disease in the differential diagnosis. A special attention to clinical spectrum and laboratory criteria is required in these cases. Based on experimental evidence and experience more additional work is needed to improve the understanding of the underlying pathophysiology of the disease, its diagnosis and treatment. Acknowledgements.—The authors wish to thank Dr. S. Miertusova for the English revision of the manuscript. References 1. Trevisan G, Cinco M. Lyme disease: a general survey. Int J Dermatol 1990;29:1-8. 2. Steere AC. Lyme disease. N Engl J Med 2001;345:115-25. 3. Hengge UR, Tannapfei A, Tyring SK. Lyme borreliosis. Lancet 2003;3:489-500. 4. Wormser GP, Nadelman RB, Dattwyler RJ, Dennis DT, Shapiro ED, Steere AC et al. Practice guidelines for the treatment of Lyme disease. Clin Infect Dis 2000;31 Suppl 1:1-14. 5. Carlson D, Hernandez J, Bloom BJ, Coburn J, Aversa JM, Steere AC. Lack of Borrelia burgdorferi DNA in synovial samples from patients with antibiotic treatment - resistant Lyme arthritis. Arthritis Rheum 1999;42:2705-9. 6. Donta S. The existence of chronic Lyme disease. Curr Treat Options Inf Dis 2001;3:261-2. 7. Preac-Mursic V, Weber K, Pfister HW, Wilske B, Gross B, Baumann A et al. Survival of Borrelia burgdorferi in antibiotically treated patients with Lyme borreliosis. J Infection 1989;17:355-9. 8. Steiner I. Treating post Lyme disease. Neurology 2003;60:1888-9. 9. Trevisan G, Stinco G, Nobile C, Bonin S, Stanta G. Detection of Borrelia burgdorferi in skin biopsies from patients with morphea by polymerase chain reaction. J Eur Acad Dermatol Venereol 1996;6:15. 10. Keller TL, Halperin JJ, Whitman M. PCR detection of Borrelia burgdorferi DNA in cerebrospinal fluid of Lyme neuroborreliosis patients. Neurology 1992;42:32-42. 11. Priem S, Burmester GR, Kamradt T, Wolbart K, Rittig MG, Krause A. Detection of Borrelia burgdorferi by polymerase chain reaction in synovial membrane, but not in synovial fluid from patients with persisting lyme arthritis after antibiotic theraphy. Ann Rheum Dis 1998;57:118-21. 12. Steere AC, Gross D, Meyer AL. Autoimmune mechanisms in antibiotic treatment - resistant Lyme arthritis. J Autoimmun 2001;16:263-8. 13. Wahlberg P, Granlund H, Nyman D, Panelius J, Seppala I. Late Lyme borreliosis: epidemiology, diagnosis and clinical features. Ann Med 1993;25:349-52. Vol. 140 - N. 4 TREVISAN 14. Trevisan G, Cattonar P, Nobile C, Perkan V. Dermatological manifestations of Lyme borreliosis. Acta Dermatovenereologica APA 1996;5:101. 15. Trevisan G, Stinco G. Dermatologia di importazione. In: Veraldi S, Rizzitelli G, Caputo R editors. Infezione da batteri: Borreliosi. Milano: Poletto; 2000. p. 2-21. 16. Kaya G, Berset M, Prins C, Chavaz P, Saurat JH. Chronic Borreliosis presenting with morphea and lichen sclerosus et atrophicus - like cutaneous lesions. A case report. Dermatology. 2001;202:373-5. 17. Trevisan G, Menni S, Stinco G, Nobile C, Pistritto G, Perin R. Lichen slerosus et atrophicus and Borrelia burgdorferi infection. Eur J Pediat Dermatol 1994;4:159. 18. Trevisan G. Atypical dermatological manifestations of Lyme borreliosis. Acta Dermatovenereologica 2001;10. 19. Kristoferistsch W, Mayr WR, Partsch H, Neumann R, Stanek G. HLADR in Lyme borreliosis. Lancet 1986;2:278. 20. Steere AC, Dwyer E, Winchester R. Association of chronic Lyme disease with HLA-DR4 and HLA-DR2 alleles. N Engl J Med 1990;323:219. 21. Duray PH. Clinical pathologic correlations of Lyme disease. Rev Infect Dis 1989; 11 Suppl 6: S1487-93. 22. Ogrinc K, Lotric-Furlan S, Maraspin V, Cimperman J, Ruzic- Sabljio, Strle F et al. Cerebrospinal fluid findings in patients with symptoms suggesting chronic Lyme borrliosis. Ien Klin Wochenschr 2002;114:535-8. 23. Cox J, Krajden M. Cardiovascular manifestations of Lyme disease. Am Heart J 1991;122:1449-55. 24. Bertuch AW, Rocco E, Schwartz EG. Lyme disease: ocular manifestations. Ann Ophtalmol 1988;20:376-8. 25. Donta S. Late and chronic Lyme disease. Med Clin N Am 2002;86: 341-9. 26. Vrethem M, Hellblom L, Widlund M, Ahl M, Danielsson O, Ernerud J et al. Chronic symptoms are common in patients with neuroborreliosis: a questionnaire follow- up study. Acta Neurol Scand 2002;106:205-8. 27. Elkins LE, Pollina DA, Scheffer SR, Krupp LB. Psychological states and neuropsychological performances in chronic Lyme disease. Appl Neuropsychol 1999;6:19-26. 28. Westervel HJ, McCaffrey RJ. Neuropsychological functioning in chronic Lyme disease. Neuropsychol Rev 2002;12:153-77. 29. Seltzer EG, Gerber MA, Cartter ML, Freudigman K, Shapiro ED. Long-term outcomes of persons with Lyme disease. JAMA 2000; 283:609-16. 30. Silberer M, Koszik F, Stingl G, Aberer E. Downregulation of class II molecules on epidermal Langerhans cells in Lyme borreliosis. Br J Derm 2000;143;786-94. 31. Behar SM, Porcelli SA. Mechanisms of autoimmune disease induction: the role of the immune response to microbial pathogens. Arthritis Rheum 1995;38:458-76. 32. Hemmer B, Gran B, Zhao Y, Marques A, Pascal J, Tzou A et al. Identification of candidate T - cell epitopes and molecular mimicry in chronic Lyme disease. Nat Med 1999;5:1346-7. 33. Sweeney CJ, Ghassemi M, Agger WA, Persing DH. Coinfection with Babesia microti and Borrelia burgdorferi in a western Wisconsin resident. Mayo Clin Proc 1998;73:338-41. 34. Sigal LH. Immunologic mechanisms in Lyme neuroborreliosis: the potential role of autoimmunity and molecular mimicry. Semin Neurol 1997;17:63-8. 35. Klempner MS. Controlled trials of antibiotic tratment in patients with post- treatment chronic lyme disease. Vector Borne Zoonotic Dis 2002;2:255-63. 36. Krupp LB, Hyman LG, Grimson R, Coyle PK, Melville P, Ahnn S et al. Study and treatment of post Lyme disease (STOP - LD). Neurology 2003;60:1923-30. 37. Datttwyler RJ, Halperin JJ, Volkman DJ, Luft BJ. Treatment of late Lyme boreliosis-randomized comparison of ceftriaxone vs Penicillin. Lancet 1988;2:1191-4. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 425 TREVISAN LATE LYME DISEASE, CRONIC LYME DISEASE AND POST LYME DISEASE SYNDROME 38. Weinstein A, Britchkov M. Lyme arthritis and post - Lyme disease syndrome. Curr Opin Rheumatol 2002;14:383-7. 39. Donta S. Macrolide therapy of chronic Lyme disease. Med Sci Monit 2003;9:PI136-42. 40. Guerau de Arellano M, Huber BT. Development of autoimmunity in Lyme arthritis. Curr Opinion Rheumatol 2002;14:388-93. 41. Akin E, Aversa J, Steere A. Expression of adhesion molecules in synovia of patients with treatment-resistant lyme arthritis. Infect Immun 2001;69:1774-80. 42. Dejmkova H, Hulinska D, Tezgova D, Pavelka K, Gatterova J, Vavrik P. Seronegative Lyme arthritis caused by Borrelia garinii. Clin Rheumatol 2002;21:330-4. 43. Craig T, Kakumanu S. Chronic fatigue syndrome: evaluation and treatment. Am Fam Physician 2002;65:1083-90. 44. Fukuda K, Staus SE, Hickie I, Sharpe MC, Dobbins JG, Komaroff A. The chronic fatigue syndrome: a comprehensive approach to its definition and study. International chronic fatigue syndrome study group. Ann Intern Med 1994;121:953-9. 45. Abbey S, Garfinkel P. Chronic fatigue syndrome and depression: cause, effect or covariate. Rev Infect Dis 1991;13 Suppl 1: S73-83. 46. Hsu VM, Patella SJ, Sigal LH: Chronic Lyme disease as the incorrect diagnosis in patients with fibromyalgia. Arthritis Rheum 1993;36: 1493-500. 47. Fallon BF. Differential diagnosis in Lyme disease. 12th International Conference on Lyme disease and other spirochetal and tick-borne disorders, 1999, 2-10 April, New York, NY. 48. Gaudino EA, Coyle PK, Krupp LB. Post-Lyme disease syndrome and chronic fatigue syndrome. Neuropsychiatric similarities and differences. Arch Neurol 1997;54:1372-6. 49. Dattwyler RJ, Volkman DJ, Luft BJ, Halperin JJ, Thomas J, Golightly MG. Seronegative Lyme disease. Dissociation of specific T- and Blymphocyte responses to Borrelia burgdorferi. N Engl J Med 1988;319:1441-6. 50. Dorward DW, Schwan TG, Garon CF. Immune capture and detection of Borrelia burgdorferi antigens in urine, blood, or tissues from infected ticks, mice, dogs, and humans. J Clin Microbiol 1991;29:1162-70. 51. Karlsson M. Aspects of the diagnosis of Lyme borreliosis. Scand J Infect Dis Suppl 1990;67:1-59. 52. Donta S. Tetracycline therapy for chronic Lyme disease. Clin Infect Dis 1997;25 Suppl 1:S52- 6. 53. Nanagara R, Duray PH, Schumacher HR Jr. Ultrastructural demonstration of spirochetal antigens in synovial fluid and synovial membrane in chronic Lyme disease: possible factors contributing to persistence of organisms. Hum Pathol 1996;27:1025-34. 54. Donta St. Treatment of chronic Lyme disease with macrolide antibiotics. 8th International Conference on Lyme Borreliosis, 1999, June 20-24, Munich, Germany. 55. Liang FT, Steere AC, Marques AR, Johnson BJ, Miller JN, Philipp MT. Sensitive and specific serodiagnosis of Lyme disease by enzimelinked immunosorbent assay with a peptide based on an immunodominant conserved region of Borrelia burgdorferi VlsE. J Clin Microbiol 1995;37:3990-6. 56. Ohnishi J, Piesman J, De Silva AM. Antigenic and genetic heterogeneity of Borrelia burgdorferi populations transmitted by icks. Proc Natl Acad Sci 2001;98:670-5. 57. Lawrenz MB, Hardham JM, Owens RT, Nowakowski J, Steere AC, Wormser GP et al. Human antibody responses to VlsE antigenic variation protein of Borrelia burgdorferi. J Clin Microbiol 1999;37:39974004. 58. Eicken C, Sharma V, Klabunde T, Lawrenz MB, Hardham JM, Norris SJ et al. Crystal structure of Lyme disease variable surface antigen VlsE of Borrelia burgdorferi. J Biol Chem 2002;277:21691-6. 59. Philipp MT, Bowers LC, Fawcett PT, Jacobs MB, Liang FT, Marques AR et al. Antibody response to IR6, a conserved immunodominant region of the VlsE lipoprotein, wanes rapidly after antibiotic treatment of Borrelia burgdorferi infection in experimental animals and in humans. J Infect Dis 2001;184:870-8. Epub 2001 Aug 30. 60. Peltoma M, McHugh G, Steere AC. Persistence of the antibody response to the VlsE sixth invariant region( IR6) peptide of Borrelia burgdorferi after successful antibiotic treatment of Lyme disease. J Infect Dis 2003;187:1178-86. 61. Cimmino MA, Accardo S. Long term treatment of chronic Lyme arthritis with benzathine penicillin. Ann Rheum Dis 1992;51:1007-8. 62. Maurin M, Benoliel AM, Bongrand P, Raoult D. Phagolysosolal alkalinization and the bactericidal effect of antibiotics: the Coxiella burnetii paradigm. J Infect Dis 1992;166:1097-102. Malattia di Lyme tardiva e cronica e sindrome post malattia di Lyme. Diagnosi clinica e di laboratorio L a borreliosi di Lyme (BL) è un’infezione multisistemica causata da Borrelia burgdorferi (Bb), una spirocheta trasmessa all’uomo dal morso di una zecca dura del genere Ixodes, che interessa principalmente la cute, le articolazioni, il sistema nervoso, il cuore e l’occhio 1, 2. La BL è in genere suddivisa in 3 stadi clinici, oppure in 1 stadio precoce e 1 tardivo 3. I criteri clinici ed epidemiologici, supportati da indagini sierologiche, istologiche e colturali, costituiscono le basi fondamentali per porre una diagnosi certa della malattia di Lyme, diagnosi che risulta essere relativamente semplice nelle forme precoci associate alla malattia 3. Tuttavia, manifestazioni cutanee, nervose, osteoarticola- 426 ri si possono manifestare anche dopo un lungo periodo dall’avvenuta trasmissione della spirocheta 4 o, addirittura, in seguito a un’apparente eradicazione della stessa 5. In alcuni casi, si associa un riscontro laboratoristico di elevati valori di IgM, espressione, come è noto, di attività di malattia, senza che sia stata fatta chiarezza sul probabile meccanismo patogenetico che causa tale persistente attività 6. In altri pazienti si segnala la persistenza dei sintomi, nonostante un ciclo completo di terapia antibiotica 7 adeguata e nonostante l’assenza di segni obiettivi o di marker biologici 8, il tutto legato, probabilmente, a una persistenza intracellulare della Bb che riesce così a eludere la risposta immunitaria dell’ospite o allo stesso tropismo della Bb che può per- GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 LATE LYME DISEASE, CRONIC LYME DISEASE AND POST LYME DISEASE SYNDROME sistere in siti privilegiati (cute 9, liquor 10, liquido sinoviale 11) ed eludere la risposta immunitaria. Una piccola percentuale di pazienti geneticamente predisposti, poi, si presenta resistente al trattamento, manifestando un’infiammazione articolare persistente nonostante l’apparente eradicazione della spirocheta 3, 12. Negli ultimi anni si è posta l’attenzione, quindi, su un complesso di sindromi cliniche correlate alla BL che presentano varie similitudini e sulla cui patogenesi sono state formulate e vagliate varie ipotesi, legate ora alle caratteristiche proprie della spirocheta ora alla risposta immunitaria nei confronti della stessa. Tali sindromi sono: — malattia di Lyme tardiva; — malattia di Lyme cronica; — artrite di Lyme resistente al trattamento; — post malattia di Lyme; — sindrome della stanchezza cronica. Malattia di Lyme tardiva La malattia di Lyme tardiva, definita anche “late Lyme disease” compare almeno 7 mesi dopo l’infezione: le manifestazioni cutanee sono prevalentemente atrofo-sclerodermiche e l’interessamento extracutaneo è di tipo sia articolare che neurologico 13. Manifestazioni cutanee Acrodermatite cronica atrofica (o malattia di Pick - Herxheimer) L’acrodermatite cronica atrofica (ACA) è una manifestazione cutanea classica della forma cronica e compare dopo mesi o anni. Inizia, insidiosamente, con una fase infiammatoria precoce sotto forma di placche eritemato-cianotiche, infiltrate a livello delle superfici estensorie delle estremità, specie in prossimità delle articolazioni uni o bilateralmente 14. Il decorso della malattia è cronico, le lesioni iniziali tendono progressivamente ad allargarsi coinvolgendo l’intera superficie acrale e tendono a divenire atrofiche. La cute diviene progressivamente liscia, sottile, trasparente e anelastica. L’atrofia può coinvolgere anche il sottocute e il tessuto muscolare sottostante con grave compromissione degli arti colpiti. Sono state descritte ulcerazioni croniche e trasformazioni maligne della cute atrofica. Possono essere presenti prurito o bruciore, ma la malattia può anche decorrere senza alcun sintomo 14, 15. Altre manifestazioni cutanee di tale forma sono: lichen sclero-atrofico 16, 17, sclerodermia generalizzata, atrofodermia di Pierini-Pasini, panniculite nodulare di Pfeifer-WeberChrstian 15, 18. Le dermatiti atrofosclerodermiche come il lichen sclerosus et atrophicus, la morfea, la sclerodermia in placche, la sclerodermia lineare, l’atrophoderma profundum di Pieri- Vol. 140 - N. 4 TREVISAN ni-Pasini, l’emiatrofia facciale di Parry-Romberg, lo scleredema di Busckhe e la fascite eosinofila di Schulmann sono state ripetutamente segnalate, quali manifestazioni tardive della BL, ma, mentre per l’ACA il rapporto con l’infezione da Bb sembra costante, con, in alcuni casi, l’isolamento della Bb da biopsie cutanee anche a 10 anni dall’insorgere della BL, per gli altri quadri atrofo-sclerodermici la correlazione con la BL è molto discussa 18. La morfea, in particolare, è stata più volte associata alla forma tardiva della BL; conosciuta anche come sclerodermia localizzata, è caratterizzata da un indurimento localizzato della cute e del tessuto sottocutaneo dovuto a un’eccessiva deposizione di collagene 16. I vari tipi di morfea si classificano in base alle caratteristiche cliniche e al grado di profondità del tessuto coinvolto. Essi includono forme a placca, generalizzate, lineari e forme più profonde. A differenza della sclerosi sistemica, nella forma localizzata non si hanno mai sclerodattilia, il fenomeno di Raynaud o coinvolgimento di organi interni. In alcuni pazienti, è stato possibile individuare tramite metodiche di polymerase chain reaction (PCR) il DNA della Bb in biopsie cutanee di pazienti con morfea 9. Sistema muscolo-scheletrico: artrite cronica (durata >1 anno) È la manifestazione clinica più caratteristica e più frequente nel Nord America, meno frequente in Europa 3. Può comparire qualche settimana ma, addirittura, anche dopo anni dall’inoculazione della Bb. Il disturbo può divenire cronico o intermittente, con attacchi che durano da un paio di settimane a qualche mese e poi si risolvono 3. L’intensità degli attacchi diminuisce con il tempo. Non è presente iperpiressia ma è comune un senso di affaticamento generale 15. Caratteristiche 15: — mono-oligosettorialità; — asimmetria; — attacchi frequenti. Sede 15: — l’articolazione del ginocchio risulta essere la più colpita; in genere sono interessate le grosse articolazioni. Clinica 15: — tumefazione; — marcata impotenza funzionale; — noduli cutanei; — assenza di rigidità al mattino. Esami strumentali 15: Radiografie delle articolazioni interessate dal processo patologico possono evidenziare: — versamento articolare; — osteoporosi; — erosioni capi ossei; — calcificazioni nei tessuti molli periarticolari; — cisti ossee sottoarticolari. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 427 TREVISAN LATE LYME DISEASE, CRONIC LYME DISEASE AND POST LYME DISEASE SYNDROME Se non viene trattata, può portare a una irreversibile erosione della cartilagine e dell’osso con conseguente danno permanente. Durata della malattia: se non è trattata, persiste per almeno 4 anni, fattori di tipo genetico influenzerebbero la durata e la severità dell’artrite. Soggetti con HLA-DR4 e/o DR 15, 19, 20 sembrano sviluppare con maggiore frequenza un’artrite cronica erosiva scarsamente responsiva alla terapia antibiotica e gli stessi possono avere una PCR negativa per la ricerca del DNA della Bb nel liquido sinoviale, nonostante la persistenza dell’infiammazione 4, per motivi che saranno descritti successivamente. L’artrite cronica viene considerata l’espressione più frequente di Lyme nei bambini (fino al 33%). Sistema nervoso Encefalopatia con soli difetti cognitivi. Manifestazioni tipiche sono disturbi della memoria (a breve termine) e delle funzioni intellettive. È presente irritabilità e sonnolenza 15. Encefalomielite cronica oscillante. Può simulare una sclerosi a placche per l’interessamento multifocale del sistema nervoso centrale (cervello, nervi ottici, tronco encefalico e cervelletto) il decorso e l’aspetto alla risonanza magnetica (multipli focolai nella sostanza bianca periventricolare). È caratterizzata da improvvisi deficit focali, transitori o permanenti, quali emiparesi, paraparesi, atassia e afasia 15. Encefalopatia multi-infartuale: possono essere presenti difetti neurologici focali 16 acuti transitori (tipo attacco ischemico transitorio) o permanenti (tipo ictus). Polineuropatia assonale 21: molti pazienti affetti da borreliosi tardiva (di solito in associazione con l’artrite) presentano una lieve neuropatia sensitiva, che si manifesta clinicamente con parestesie intermittenti agli arti. Gli studi elettromiografici rivelano un quadro multinevritico con velocità di conduzione nervosa generalmente nella norma. Esami effettuati sul liquido cerebrospinale mostrano nella neuroborreliosi cronica un liquor generalmente normale o con pleiocitosi 10, 22, con presenza di anticorpi anti Bb risultanti da una sintesi intratecale. Metodiche di PCR possono consentire l’isolamento genomico della spirocheta. Cuore La miocardiopatia dilatativa 23, considerata una complicanza molto rara, potrebbe essere la sola ragione di un outcome fatale nei pazienti con LB. Occhio Come conseguenza di una neuroborreliosi o di una condizione infiammatoria dell’occhio possono manifestarsi: cheratiti, episcleriti, iridocicliti, neurite ottica 3, 24. L’infiammazione intraoculare di lunga durata può portare a cecità. 428 Lyme cronico e diagnosi differenziale Nella Tabella I viene mostrato uno schema riassunto delle diagnosi differenziali. Mentre può essere relativamente semplice riconoscere disturbi associati alla malattia di Lyme quali l’eritema cronico migrante, la paralisi di Bell o l’artrite (specie se monoarticolare o oligoarticolare), può essere piuttosto difficoltoso riconoscere sintomi cronici associati alla malattia di Lyme 25, 26. Si definisce malattia di Lyme cronica un complesso di sintomi cronici correlati alla malattia di Lyme che consistono in senso di affaticamento generale con artralgie (senza alcun segno obiettivo), fibromialgie e altre disfunzioni del sistema nervoso (neurocognitive e neuropsichiatriche) 27, 28. Possono essere presenti, ma più raramente, sintomi associati, di tipo neurologico, quali parestesie, tremori, palpitazioni, tachicardia, alterazioni dell’equilibrio, sudorazione (a volte intensa), disturbi visivi, disturbi gastro-intestinali (sindrome del colon irritabile) e aumento della frequenza urinaria 25. Responsabile del corteo sintomatologico sarebbe la persistenza dell’infezione da Bb. Rimane ancora da distinguere, soprattutto per ciò che riguarda il processo patogenetico, tale forma dalla cosiddetta “late Lyme disease” descritta in precedenza; le 2 sindromi, infatti, non sono ancora perfettamente differenziate, anzi, spesso considerate sinonimi, costituirebbero, in realtà, 2 entità cliniche diverse 25. Le differenze principali consisterebbero nel fatto che la malattia di Lyme tardiva è caratterizzata, contrariamente alla forma cronica, da segni obiettivi di artrite (tumefazione, noduli cutanei...) che non sono presenti nell’altra forma e che la forma tardiva, oltre a essere più facile da diagnosticare, risulta molto più responsiva al trattamento rispetto alla cronica. Si è constatato, infatti, che pazienti affetti da malattia di Lyme tardiva con un’importante risposta anticorpale di tipo IgG rispondevano meglio alla terapia se confrontati con pazienti con sintomi cronici e con bassa risposta anticorpale IgG che necessitavano di terapia più prolungata. In genere, in molti pazienti affetti dalla forma cronica si possono ritrovare valori alti e persistenti di IgM associati a un titolo anticorpale IgG piuttosto limitato 6. La risposta anticorpale di tipo IgM contro le proteine specifiche di Bb (23Kd, 31Kd, 34Kd e soprattutto 41Kd) è indicativa di una fase attiva della malattia di Lyme. L’elevata presenza di titoli IgM nella forma cronica della malattia non è caratteristica solo della malattia di Lyme; ci sono esempi di altre infezioni che possono presentare un nuovo innalzamento delle IgM dopo l’infezione primaria in caso, ad esempio, di riattivazione della malattia (toxoplasmosi, citomegalovirus) 6. Nei casi di malattia di Lyme cronica che risponde al trattamento, si può assistere a un progressivo calo del titolo IgM a favore delle IgG. Rimane, tuttavia, ancora da stabilire il meccanismo che determina la persistente reattività delle IgM e la limitata reattività delle IgG, è molto probabi- GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 LATE LYME DISEASE, CRONIC LYME DISEASE AND POST LYME DISEASE SYNDROME le che sia coinvolto il processo di “switch” dalla risposta immunitaria di tipo IgM a quella di tipo IgG 6. Alcuni Autori negano l’esistenza di una forma cronica di malattia di Lyme, asserendo, piuttosto, che tali pazienti soffrano di disturbi psichiatrici; in realtà, studi epidemiologici confermano l’esistenza di una forma cronica di neuroborreliosi che si sviluppa dal 30% al 50% di pazienti che sviluppano una serie di disturbi spesso difficilmente distinguibili dalla fibromialgia e dalla sindrome della fatica cronica, considerando anche il fatto che entrambi questi disturbi possono essere conseguenti alla malattia di Lyme 29. I sintomi della malattia di Lyme cronica sono in genere persistenti con delle fasi di peggioramento che si verificano ciclicamente. Alcuni pazienti sono più sintomatici di altri, particolare che fa pensare a una differenza geneticamente determinata nella risposta all’infezione e al suo coinvolgimento sistemico. Si può, quindi, affermare che la malattia di Lyme cronica non è di certo una malattia distruttiva o fatale ma può essere fortemente debilitante. L’incidenza dell’infezione asintomatica non è stata valutata, ma è importante precisare che molti pazienti, sebbene asintomatici per lungo tempo, possono andare incontro a una riaccensione dell’infezione e, quindi, a una comparsa di sintomi della malattia di Lyme cronica mesi o anni dopo aver contratto l’infezione e ciò a causa di eventi scatenanti quali trauma, gravidanza, o stress di tipo psicologico 25. Non è noto il meccanismo responsabile della riattivazione della malattia e numerose sono le teorie sulla patogenesi della malattia di Lyme cronica. Le caratteristiche immunopatogenetiche presenti nell’ACA forniscono un modello di studio interessante per meglio comprendere la patogenesi di tale disturbo. Recenti studi indicano che le cellule di Langherans dell’epidermide sono invase dalla Bb già nelle forme precoci di malattia di Lyme. Tali cellule sono state studiate immunoistochimicamente con differenti marker per valutare la loro attività funzionale. Si è visto che le cellule di Langherans attive sono ridotte nell’eritema migrante ma sono normali o addirittura elevate nell’ACA. In entrambe le manifestazioni cliniche, c’è, inoltre, una downregulation di molecole di classe II del complesso maggiore di istocompatibilità sulle cellule di Langherans. Questo fenomeno di downregulation potrebbe essere una sorta di meccanismo protettivo che evita la presentazione di autoantigeni ma impedisce alle cellule di Langherans di eliminare Bb, da qui deriverebbe l’insorgenza della forma cronica 30. Secondo la teoria dell’autoimmunità 31, che è descritta meglio successivamente, esisterebbero dei linfociti T, nati dall’interazione con Bb, che mostrano un’alterazione nella loro capacità di riconoscimento dell’antigene 32 e potrebbero essere coinvolti nei sintomi neurologici e muscolo-scheletrici nelle varie fasi della malattia. Ci sarebbe, infatti, una delezione incompleta di tali linfociti autoreattivi nel timo e ciò, aggiunto alla up-regulation di molecole HLA, di co-stimolatori, di co-recettori provocherebbe il danno. Altre ipotesi che tentano di spiegare la patogenesi della Vol. 140 - N. 4 TREVISAN malattia di Lyme cronica citano l’esistenza di forme particolarmente attive di Bb che sfuggono al controllo degli antibiotici o suggeriscono che la forma cronica sia espressione di un danno causato dalla risposta dell’ospite nei confronti della spirocheta, o ancora che essa sia dovuta a una confezione di Bb con un altro microrganismo trasmesso dalla zecca Ixodes (Babesia microti, specie di Erlichia) 33. È noto che le manifestazioni cliniche della malattia di Lyme sono dovute alla presenza, a livello locale, dell’agente causale ma, a tutt’oggi, non è perfettamente noto il meccanismo di danno tissutale che deriva da un’interazione molto complessa tra il batterio in questione e il sistema immunitario dell’ospite 34. Alcuni pazienti mostrano una persistenza di sintomi, quali stanchezza fisica e mentale, mialgie, artralgie senza artrite, disestesie/parestesie, cefalea, vertigini, disturbi della memoria nonostante l’adeguato trattamento antibiotico 8, 35. Si parla, in questo caso, di “post-Lyme syndrome” (PLS). La persistenza di tali sintomi è stata riportata sia in pazienti privi di anticorpi IgG anti Borrelia sia in pazienti con titolo anticorpale positivo. Il meccanismo patogenetico che causa la persistenza di tali sintomi non è stato ancora chiarito e, anzi, la questione rimane piuttosto controversa. A causa dell’associazione di tali sintomi con l’infezione borreliosica alcuni pazienti sono stati sottoposti a terapie antibiotiche prolungate, spesso con il risultato che, alla sospensione del trattamento, avveniva la ricomparsa della sintomatologia precedentemente descritta 36. Un paziente si definisce affetto da PLS quando sono rispettati i seguenti criteri definiti dai Centers for disease Control and Prevention (CDC) 36: — documentata infezione di Lyme in passato; — ciclo completo di terapia antibiotica adeguata 37; — persistenza per mesi o anni dei sintomi precedentemente descritti. In particolare, la PLS si presenta con: — encefalopatia con disturbi della memoria a breve termine; — dolore muscolare con particolare coinvolgimento del dorso e della regione cervicale; — artralgie. Probabilmente i sintomi articolari, soprattutto alle ginocchia, sono causati dall’instaurarsi di un meccanismo autoimmunitario intrasinoviale. È importante considerare che, in tale forma, i sintomi non si associano ad alcun segno obiettivo né, tantomeno, a qualsiasi marker biologico. La ricerca della Borrelia nel sangue o nel liquor cerebri o sinoviae, tramite esame culturale o tecniche di amplificazione del DNA (PCR) della Borrelia risulta, infatti, negativa, mentre la sierologia può mantenersi positiva per alcuni anni dopo la guarigione dell’infezione borreliosica e, quindi, non consente di distinguere accuratamente tra la forma cronica e la PLS 38. Da vari studi descritti in letteratura emerge che, nella PLS, il trattamento antibiotico sistemico per via sia endovenosa che orale è inefficace 36 e, quindi, non necessario, mentre può essere efficace la terapia con idrossi-clorochina 6, 39. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 429 TREVISAN LATE LYME DISEASE, CRONIC LYME DISEASE AND POST LYME DISEASE SYNDROME Un’altra sindrome clinica associata alla malattia di Lyme è la cosiddetta “treatment-resistant Lyme arthritis”, un’artrite di Lyme che si manifesta nel 10% dei pazienti ed è caratterizzata da un’infiammazione articolare persistente che non risponde alla terapia antibiotica 12, 40. Tale infiammazione persiste per mesi o anni dopo l’apparente eradicazione della spirocheta 5. Studi di biologia molecolare hanno mostrato che tali pazienti hanno una predisposizione genetica dovuta a un aplotipo HLA - DRB1* 0401 e relativi alleli e che la severità e la durata di tale artrite sarebbe altamente correlata alla risposta immunitaria sia di tipo umorale sia cellulo-mediata nei confronti della proteina di superficie della Bb (Osp A). Ciò deriva dal fatto che, in questi pazienti, tale proteina, in particolare la sequenza di amminoacidi OspA165 - 173, ha una sequenza altamente omologa a un peptide contenuto in una proteina presente normalmente nell’uomo, la hLFA 1α (lymphocyte function associated antigen - 1alpha). I linfociti T del liquido sinoviale di questi pazienti con artrite resistente al trattamento e geneticamente predisposti determinano una risposta immunitaria nei confronti sia di OspA sia di hLFA - 1, ciò suggerisce che hLFA - 1α possa agire da parziale agonista e che abbia un ruolo importante per la persistenza dell’infiammazione articolare 12, 40, 41. Sono stati effettuati vari studi per individuare i possibili siti di persistenza della Bb in pazienti con forme resistenti al trattamento, comprese analisi di PCR per il DNA della spirocheta effettuate sulla membrana sinoviale in pazienti che mostravano risultati negativi per la PCR condotta su liquido sinoviale dopo il trattamento antibiotico. Ebbene, in alcuni casi, la PCR condotta sulla membrana sinoviale dava esito positivo suggerendo che, in forme di artrite di Lyme resistenti al trattamento antibiotico, la PCR negativa su liquido sinoviale non esclude la presenza intrarticolare della Bb 11. In letteratura sono state descritte anche forme di artrite sieronegativa da B. garinii 42. La sindrome da stanchezza cronica La sindrome da stanchezza cronica (chronic fatigue syndrome, CFS), nota anche come sindrome da stanchezza cronica e immunodeficienza, è una malattia caratterizzata da una persistente fatica cronica accompagnata da una serie di sintomi sistemici di tipo reumatologico, cognitivo e similinfettivo 43. Non è nota ancora la causa di tale sindrome, varie ricerche hanno confermato un possibile ruolo eziologico di alcune infezioni virali (compresi il virus dell’Epstein barr, enterovirus, virus poliomielitico), funghi (in particolare Candida albicans), batteri (Chlamydia pneumoniae), ma non c’è evidenza che essa possa essere provocata da un determinato e specifico agente eziologico. Colpisce tutte le razze, le etnie, senza distinzione di classe sociale; si rileva, comunque, una maggiore prevalenza per il sesso femminile tra la terza e la quarta decade di vita. La principale caratteristica della malattia è una stanchez- 430 za cronica che limita le attività precedenti la malattia del 50% per un periodo di almeno 6 mesi. La malattia può essere persistente o recidivante 44. Inoltre possono essere presenti altri sintomi (almeno 4): — compromissione della memoria e della concentrazione; — faringite; — dolorabilità dei linfonodi (cervicali o ascellari); — febbricola (o sensazione di febbre/brividi); — dolori muscolari (mialgia); — dolori articolari multipli (artralgia); — cefalee di nuovo esordio; — disturbi del sonno (ipersonnia o insonnia); — stanchezza successiva all’esercizio fisico. Criteri di esclusione: altra causa o diagnosi responsabile della stanchezza o dei sintomi accusati. In molti casi la valutazione della CFS si concentra sulla ricerca di una causa infettiva o di un’altra causa specifica per questa malattia. Molto importante è la diagnosi differenziale della malattia, che spesso è molto laboriosa, in quanto la stanchezza e gli altri sintomi generali si manifestano in molte altre malattie; in generale, si tratta di una diagnosi di esclusione, dopo aver effettuato un’anamnesi completa, un’attenta visita medica e, soprattutto, una serie di test laboratoristici che escludano altre cause 43. Nel caso della diagnosi differenziale con la malattia di Lyme esami sierologici e test diretti (PCR) ci permettono di porre diagnosi di esclusione. Diagnosi differenziale nella sindrome da stanchezza cronica Nella Tabella II è mostrata la diagnosi differenziale della CFS 444, 45. Soltanto sulla base clinica è arduo distinguere la malattia di Lyme cronica e la CFS o fibromialgia 46, questa difficoltà è dovuta al fatto che una piccola percentuale di pazienti in effetti sviluppa dolore cronico o la sindrome di affaticamento in associazione con o subito dopo la malattia di Lyme. Rispetto alla malattia di Lyme, la sindrome da fatica cronica o la fibromialgia tendono a produrre sintomi più generalizzati e disabilitanti, comprendenti notevole fatica, forte mal di testa, dolore muscolare scheletrico diffuso, punti dolorosi simmetrici in siti caratteristici, dolore e rigidità in molte articolazioni, disestesia diffusa, difficoltà di concentrazione e disturbi del sonno 47. I pazienti con la CFS o la fibromialgia non presentano evidenza di infiammazione articolare; hanno risultati normali nei test neurologici e hanno un grado di ansietà e depressione maggiore rispetto ai pazienti con neuroborreliosi cronica, non hanno storia di infezione di Lyme. La CFS mostra anche sintomi molto simili alla post-Lyme disease e, in alcuni studi, effettuati per valutare le differenze di ordine neuropsichiatrico, si è visto che i pazienti con PLS mostrano una deficienza cognitiva maggiore rispetto GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 LATE LYME DISEASE, CRONIC LYME DISEASE AND POST LYME DISEASE SYNDROME ai pazienti con CFS, e ciò avviene soprattutto e ovviamente in pazienti con PLS e con pregressi disordini di tipo psichiatrico 48. Diagnosi La diagnosi della malattia di Lyme si basa principalmente su criteri clinici ed epidemiologici con il supporto di indagini sierologiche, istologiche e colturali 3, 15. Quando l’affezione segue il suo decorso tipico, l’accertamento diagnostico risulta solitamente agevole; le indagini laboratoristiche non fanno altro che confermare l’ipotesi del clinico. Tuttavia, come nelle forme precedentemente descritte, la diagnosi può risultare estremamente difficile e raramente certa. Essa potrà essere il risultato della somma di molti indizi, quali provenienza da zona endemica, dato anamnestico positivo per puntura di zecca, sintomatologia clinica, che cercano di cogliere i criteri di diagnosi differenziale delle forme prima descritte, correlazione clinico temporale delle manifestazioni patologiche, sfumature del quadro clinico, esclusione di altre affezioni, positività, tecniche di amplificazione genica. Nella diagnostica di laboratorio della malattia di Lyme ci si avvale di metodiche dirette o indirette. La diagnosi indiretta si basa sulla ricerca degli anticorpi anti Borrelia nel siero, nel liquido cefalorachidiano e nel liquido sinoviale di soggetti affetti, con test di primo livello (ELISA o test immunoenzimatico) e test di secondo livello (Western-Blot); la diagnosi diretta si avvale di metodiche istologiche, immunoistochimiche, colturali e di ibridazione genetica, effettuate su tessuti e liquidi biologici 15. I test laboratoristici di routine sono in genere normali, la VES è normale (questo è un importante elemento per la diagnosi differenziale con l’artrite reumatoide e il lupus eritematoso sistemico). La negatività dei test sierologici non esclude la BL 49 e sono riportate, come descritto precedentemente, segnalazioni di artrite di Lyme sieronegativa 41. Tuttavia, quando si sospetta che una manifestazione clinica sia espressione di una malattia di Lyme in fase tardiva, la positività sierologica è un elemento fondamentale 50. Più del 75% dei pazienti con malattia cronica di Lyme ha un test ELISA negativo e un Western blot positivo. In genere, i pazienti con artrite oligoarticolare possono presentare un’intensa e positiva risposta IgG sia con l’ELISA che con il Western blot. Da analisi di Western blot risulta che, nella malattia di Lyme, la prima reazione immunologica si ha nei confronti della proteina flagellare 41-Kd e la proteina OspC 23. In genere, nella fase dell’eritema cronico migrante, si ha un’intensa reazione IgM contro le proteine 23-Kd e 41-Kd e nessuna reazione di tipo IgG. Nelle settimane successive persiste la reazione IgM, a volte accompagnata da reazioni minori contro le proteine 60-Kd e la 66 Kd, e perciò segue un innalzamento delle IgG contro le proteine 23-Kd e 41-Kd. In presenza di un caratteristico quadro clinico, la reazione immu- Vol. 140 - N. 4 TREVISAN nitaria contro la 23-Kd e la 41-Kd è considerata diagnostica della malattia di Lyme 6, 29, 51. La proteina 41 Kd non è caratteristica solo delle Bb, a differenza della 23-Kd. Altre proteine caratteristiche sono 35Kd, 37-Kd, 39-Kd, 83-Kd, e 93-Kd. Reazioni immunitarie contro la 31-Kd non compaiono fino a un anno o più dopo aver contratto l’infezione. Con la risoluzione dei sintomi, scompare o si attenua la reazione di tipo IgM, mentre la risposta di tipo IgG può persistere dopo la scomparsa dei sintomi, ma, comunque, in genere, si attenua o scompare dopo un adeguato trattamento antibiotico 6, 29. Alcuni pazienti possono manifestare i sintomi nonostante il Western blot risulti negativo 52. Questo fatto si spiega considerando che, a volte, la Borrelia rimane all’interno delle cellule, senza avere una fase extracellulare e ciò impedisce che si generi la risposta immunitaria dell’organismo nei confronti della spirocheta 25, 53, 54. I test serologici di ultima generazione sono volti a semplificare la serologia, pur mantenendo elevato il valore predittivo in termini di sensibilità e specificità al punto da poter sostituire, da solo, la procedura a 2 test (ELISA e Western blot) finora ritenuta la più predittiva. Recentemente si è utilizzato, infatti, un nuovo antigene, in metodologia immuno enzimatica, rappresentato da un corto peptide, detto VlsE 55. È noto che la Bb presenta sulla membrana esterna una serie di lipoproteine, le Osps, che vengono espresse in maniera differenziale nell’ospite mammifero e nel vettore. Le proteine OspA e OspB sono espresse nel vettore ma non nel mammifero, le OspC e le Vlse sono espresse in vivo nel mammifero 56. VlsE (variable major protein-like sequence, expressed) è una proteina di superficie della Bb che riveste un importante ruolo nella diagnosi sierologica della malattia di Lyme in quanto gioca un ruolo fondamentale nella sopravvivenza della spirocheta nell’uomo. Dopo la penetrazione nell’organismo ospite la Bb subisce importanti modificazioni a livello della VlsE, che le permettono di sfuggire al riconoscimento e all’attivazione del sistema immunitario 57, 58. La VlsE è divisa in varie parti: regioni conservate, che formano il dominio transmembrana e ancorano la proteina alla membrana batterica, e regioni variabili, che subiscono costantemente delle ricombinazioni. Per la produzione della Vlse il DNA di Bb contiene da 15 a 20 sequenze (cosiddette variable major protein-like sequenze [vls]) che contengono un gran numero di informazioni genetiche 57, 58. Ognuna è composta da 12 sezioni: 6 costanti e 6 variabili. Dalla ricombinazione di elementi differenti nascono proteine di superficie che differiscono nella loro regione variabile. Il DNA che si assembla e che si usa per la sintesi delle proteine è detto vlsE (E = expressed). Esso contiene anche i domini transmembrana della VlsE. VlsE è esclusivamente espressa in vivo nei mammiferi, quindi non è presente nelle colture di Bb 58. Le regioni costanti sono mascherate da quelle variabili e sono GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 431 TREVISAN LATE LYME DISEASE, CRONIC LYME DISEASE AND POST LYME DISEASE SYNDROME protette dal diretto attacco del sistema immunitario. Quando la Bb viene processata dalle cellule che presentano l’antigene, l’intera VlsE viene presentata al sistema immune con la formazione di anticorpi sia contro le regioni conservate che contro quelle variabili. Questi anticorpi in vivo non possono legare la Bb a causa dell’effetto maschera, ma sono particolarmente importanti nella diagnosi in quanto agenti contro una regione fortemente conservata della proteina 58. Il limite maggiore dei test sierologici è che non consentono di distinguere tra infezioni recenti e passate, soprattutto perché, spesso, il titolo delle IgG, e talvolta delle IgM, può mantenersi elevato anche in seguito al trattamento antibiotico. Un recente studio ha dimostrato che il titolo degli anticorpi della classe IgG anti- VlsE cala rapidamente dopo la terapia antibiotica; ciò suggerisce che una diminuzione di tale titolo anticorpale entro 6 mesi dall’infezione e dopo terapia antibiotica possa essere considerato come un marker indicativo dell’eradicazione della spirocheta (con una funzione assai simile al VDRL nella sifilide) 59. Altri studi che valutano la risposta anticorpale al peptide VlsE dopo terapia antibiotica, sia 6 mesi che anni dopo, nel siero di pazienti con manifestazioni cliniche di Lyme precoce o cronica, mostrano che la persistenza di titoli anticorpali anti Vlse per mesi o anni dopo la terapia antibiotica non può essere considerata una persistenza dell’infezione da Bb 60. Le metodiche dirette si rivelano particolarmente utili nelle fasi iniziali della malattia di Lyme, quando il movimento anticorpale non è ancora rilevante, o nei casi in cui la sierologia è negativa. L’isolamento colturale resta il gold standard e, in alcuni casi, la Bb si può coltivare da lesioni cutanee, siero, liquido cerebrospinale e sinoviale 15. La PCR è una metodica diretta che ricerca la presenza del DNA di Bb nei tessuti e nei liquidi biologici, ed è un esame di grande importanza per porre una diagnosi differenziale tra le varie sindromi associate al Lyme 15. Terapia Per ciò che riguarda il trattamento delle forme precoci di Lyme c’è un accordo generale tra i vari studi pubblicati; la terapia è essenzialmente di tipo antibiotico e altri farmaci sintomatici possono essere di volta in volta usati a seconda dei quadri clinici associati. La scelta deve essere effettuata in base a quadro clinico, stadio, sintomatologia, età del paziente, sesso e fattori concomitanti, quali una gravidanza. La BL è un’infezione multisistemica e tutti i distretti dell’organismo sono interessati, perciò è necessario tenere conto della capacità del farmaco di raggiungere le spirochete per svolgere l’effetto terapeutico. Inoltre, il farmaco deve essere in grado di diffondersi nei tessuti e nei liquidi biologici, di attraversare la barriera ematoencefalica, di penetrare all’interno delle cellule, di legarsi in maniera stabile alle strutture vitali del germe ed esplicare in questa sede l’attività antimicrobica 15. L’amoxicillina e la doxiciclina costituiscono i farmaci di prima scelta nella malattia di Lyme3 recente, mentre non 432 sono di utilità nelle forme croniche il cui trattamento è ancora dibattuto. Alcuni individui non rispondono alla terapia, cioè, dopo la terapia, hanno una fase di remissione completa dei sintomi generali, che riappaiono dopo un periodo più o meno lungo, talora con maggiore intensità. Altri, nonostante la terapia antibiotica, continuano a manifestare la sintomatologia; perciò si preferisce, in tal caso, solitamente somministrare terapie antibiotiche prolungate 36. In generale, l’artrite di Lyme viene trattata con terapia antibiotica sistemica orale o parenterale 4, 61. Doxiciclina o amoxicillina, entrambe somministrate anche fino a 28 giorni di trattamento, hanno mostrato buoni risultati e sono raccomandate se non vi sono segni clinici di un coinvolgimento di tipo neurologico, eventualità in cui sarà necessaria una terapia per via parenterale con cefotaxime o penicillina G. Per i pazienti pediatrici più piccoli si utilizza l’amoxicillina, per quelli di età superiore agli 8 anni la doxiciclina 3, 4. Per pazienti che manifestano disturbi articolari persistenti dopo un ciclo completo di terapia antibiotica, si preferisce ripetere il trattamento per altre 4 settimane con antibiotici per via orale o, alternativamente, con 2 settimane di cefotaxime endovena. Alcuni Autori sono concordi nel ritenere di attendere qualche mese prima di iniziare il nuovo ciclo di terapia antibiotica, essendo il processo di risoluzione dell’infiammazione a livello articolare estremamente lento. Se, nonostante i ripetuti cicli di terapia antibiotica non si evidenzia alcun beneficio, si può ricorrere alla sinoviectomia in artroscopia che riduce il periodo di infiammazione dell’articolazione 4. Nei pazienti che presentano disturbi di tipo neurologico con interessamento del sistema nervoso centrale o periferico, dopo un’attenta valutazione neurologica e l’esecuzione di una puntura lombare, la raccomandazione è somministrare il cefotaxime per via parenterale (2 g/die per 2-4 settimane) o la penicillina G 3, 4. Il trattamento della forma cronica non è a tutt’oggi ben definito. L’obiettivo principale sarebbe utilizzare antibiotici capaci di penetrare all’interno delle cellule, quali i macrolidi e le tetracicline 6, 39. In realtà, macrolidi non sono di norma utilizzati nel trattamento della malattia di Lyme, ma alcuni studi confermano la loro efficacia nel trattamento delle forme croniche se associati a un agente lisosomotropico che alcalinizza l’acidità intracellulare dei lisosomi, l’idrossiclorochina, appunto 62. L’attività della clorochina incrementerebbe di molto l’attività dei macrolidi che sono inattivati normalmente dal pH acido 6, 39. Il razionale di tale terapia si basa sul fatto che i sintomi della malattia di Lyme cronica sarebbero provocati da una persistenza intracellulare della Bb e, in questo caso, i macrolidi agirebbero nel distretto intracellulare 39, dove penicilline e cefalosporine non arrivano. Si è concordi nel ritenere che il trattamento di tali forme deve essere piuttosto prolungato, da 12 a 18 mesi. Una pic- GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 LATE LYME DISEASE, CRONIC LYME DISEASE AND POST LYME DISEASE SYNDROME cola percentuale di pazienti risulta, comunque, non rispondere nemmeno dopo terapie prolungate. Sebbene in parte definite e confermate, le varie sindromi correlate alla malattia di Lyme richiedono ancora un maggiore inquadramento nell’ambito di uno spettro clinico associato al Lyme e una diagnosi di certezza, che, relativamente semplice nelle forme precoci, spesso è difficile se non improbabile; sta al clinico, quindi, cercare di cogliere quegli elementi di diagnosi differenziale, sia clinici sia laboratoristici, tuttora oggetto di discussione tra vari Autori, per riconoscere e diagnosticare le varie forme cliniche, escludere sindromi simili e poter scegliere efficienti strategie terapeutiche. Riassunto La borreliosi di Lyme è un’infezione multisistemica causata da Borrelia burgdorferi che interessa principal- Vol. 140 - N. 4 TREVISAN mente la cute, le articolazioni, il sistema nervoso, il cuore e l’occhio. La valutazione clinica, supportata da dati epidemiologici, indagini sierologiche, istologiche e colturali, costituisce il fondamento per porre una diagnosi certa della malattia di Lyme. Le manifestazioni cutanee, nervose, osteoarticolari si possono manifestare, in alcuni casi, anche a distanza di tempo dalla puntura della zecca o, addirittura, in seguito a un’apparente eradicazione della stessa. In questo lavoro vengono descritte alcune sindromi cliniche correlate alla malattia di Lyme che presentano varie similitudini e sulla cui patogenesi sono state formulate e vagliate varie ipotesi; l’individuazione dei principali elementi di diagnosi differenziale, clinici e laboratoristici, costituisce il primum movens per riconoscere e diagnosticare le varie forme cliniche e scegliere un opportuno trattamento. PAROLE CHIAVE: Borreliosi di Lyme - Malattia di Lyme tardiva - Malattia di Lyme cronica - Artrite di Lyme resistente al trattamento - Post-malattia di Lyme. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 433 G ITAL DERMATOL VENEREOL 2005;140:435-43 Chronic urticaria A review B. WEDI, A. KAPP Chronic urticaria remains a major problem in terms of pathogenesis, diagnostic work-up and management. During the last years several new concepts regarding the disease have been developed. Some of these aspects resulted in a different management of patients with chronic urticaria whereas others still need research activities for confirmation or clarification of details. Symptoms are the result of the degranulation of mast cells and basophils. Possible mechanisms include autoimmune mechanisms, infectious diseases, pseudoallergic mechanisms and others such as internal diseases/malignancies. A detailed history plays a main role in the diagnostic program. Further diagnostic procedures depend on the urticaria subtype. Whereas in acute urticaria routine diagnostic is not recommended, in chronic urticaria a diagnostic programm considering associated infections (particularly with Helicobacter pylori, staphylococci, streptococci, yersinia), autoreactivity and non-allergic hypersensitivity reactions is reliable and successful. Special considerations are indicated in the case of recurrent angioedema without whealing and in childhood urticaria. In most cases a targeted diagnostic program leads to the identification of potential triggering factors and after their adequate treatment long-lasting and life quality impairing urticaria disappears or improves within several weeks. With regard to treatment non-sedating H1 antihistamines should be given regularly and daily, most often increased dosage is needed. Data on alternatives are insufficient but in selected cases cyclosporin A, leukotriene receptor antagonists, or hydroxychloroquine may be useful. Several questions have to be addressed in the future and there is hope that during the next years new therapeutic strategies will be developed to facilitate the management of this long-lasting and life-quality restricting disease. KEY WORDS: Chronic urticaria - Autoreactivity - Infections - Helicobacter pylori. Address reprint requests to: B. Wedi, MD, Associate Professor, Department of Dermatology and Allergology, Hannover Medical University, Ricklinger Str. 5, D-30449 Hannover, Germany. E-mail: [email protected]. Vol. 140 - N. 4 Department of Dermatology and Allergology Hannover Medical University, Hannover Germany C hronic urticaria remains a major problem in terms of pathogenesis, diagnostic work-up and management. During the last years several new concepts regarding the disease have been developed. Some of these aspects resulted in a different management of patients with chronic urticaria whereas others still need research activities for confirmation or clarification of details. This review summarizes the current concepts of classification and definition, pathogenesis and management of chronic urticaria. Classification and definition The 4 main subtypes of urticaria should be clearly differentiated: acute, chronic, physical urticaria and a heterogeneous group of other types that do not fit in these scheme such as urticaria pigmentosa/mastocytosis, urticaria vasculitis, familiar cold urticaria and hereditary or acquired angioedema with C1-INH deficiency.1-3 The cardinal clinical feature of urticaria is the occurrence of itchy wheals anywhere on the skin (Figure 1). Wheals are short-lived elevated erythematous lesions ranging from a few millimeters to several centimeters in diameter and can become confluent. Sometimes the GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 435 WEDI CHRONIC URTICARIA and there is no increased frequency of atopy. Nevertheless, a lot of direct and indirect releasing factors may be involved. Possible mechanisms include autoimmune mechanisms, infectious diseases (viral, bacterial, fungal, parasites), pseudoallergic mechanisms and others such as internal diseases/malignancies. Laboratory findings Figure 1.—Typical wheal and flare reaction in chronic urticaria. wheals are paler than the surrounding skin because of the compressing effect of the edema on the postcapillary venules. The itching can be pricking or burning and is usually worse in the evening or nighttime. Typically the lesions are rubbed and not scratched, therefore, excoriated skin is usually not a consequence of urticaria. Acute urticaria generally disappears within 2 to 4 weeks. By definition urticaria is chronic if wheal and flare reactions persist daily or nearly daily for at least 6 weeks. At least half of the patients suffer from concomitant and sometimes life threatening angioedema most typically involving the face, lips, tongue, pharynx, genitalia, and extremities.1 Systemic symptoms such as fatigue, respiratory, gastrointestinal and arthralgic symptoms are rare. Quality of life is significantly impaired like in patients with severe atopic dermatitis, psoriasis or coronary artery disease.2-4 Chronic urticaria usually persists for long time since not even 50% of patients that consulted a university dermatology department were symptomfree within 10 years.5 It is important to know that 2 or rarely more subtypes of urticaria can occur in the same patient such as chronic urticaria and dermographism or delayed pressure urticaria.1 In these cases urticaria is more worst.6 In the last years some interesting laboratory findings showed differences between patients with chronic urticaria and healthy controls although a specific marker is still not available. It has been shown that the number of basophil granulocytes is significantly decreased in chronic urticaria 7, 8 and is negatively correlated with disease activity.9 This led to the hypothesis that basophils may be actively recruited, for example by chemokine induced adhesion molecules on endothelial cells, from the circulation to the wheals. Interestingly, not only basophils, but also eosinophils and lymphocytes are decreased in chronic urticaria.9 Moreover, in autoimmune urticaria basophil counts are significantly lower compare to non-autoimmune urticaria.7 Additional data demonstrated that basophils and mast cells appear to be activated. In this aspect, Ferrer et al. have shown that serum IL-4 levels are significantly increased in chronic urticaria compared to healthy controls.10 In addition, intracellular Il-4 and IFN-γ were significantly increased in peripheral CD4+ lymphocytes, but not in CD8+-lymphocytes. Moreover, if urticaria was associated with angioedema, leukotriene levels appeared to be increased.10 Others showed that serum IL-2 receptor levels and tryptase are significantly higher in chronic urticaria pointing to T-cell and mast cell activation.11 In the subgroup with positive autologous serum skin test (ASST) and FcεRIα autoantibodies tryptase levels were highest. Moreover, CD40L expression on activated T-cells and bcl2 expression in activated T- and B-lymphocytes were increased in severe chronic urticaria.12 In contrast, ASST positive patients with chronic urticaria did not demonstrate increased stem cell factor (SCF) serum levels compared to controls.13 Pathogenesis In chronic urticaria symptoms are the result of the degranulation of mast cells and basophils. IgE-mediated hypersensitivity due to exogenous allergens is generally very rarely the cause of symptoms in chronic urticaria 436 Diagnosis The diagnosis of chronic urticaria is based upon a detailed history considering potential triggering factors, GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 CHRONIC URTICARIA WEDI a physical examination including a test for dermographism, laboratory investigations and, if needed, additional specific procedures. According to an international consensus 14 patient history should include at least the following items to define the subtype of urticaria and to identify potential related causes: 1) first manifestation, frequency, duration and daily variability; 2) border, size and distribution of wheals; 3) association with angioedema; 4) subjective symptoms like pruritus, pain; 5) family history for urticaria, atopy; 6) prior or current allergies, infections, internal diseases or other related causes; 7) exacerbation by physical factors or exercise; 8) drug intake (particularly NSAID and angiotensin converting enzyme inhibitors); 9) relation to food/food additives; 10) nicotine; 11) occupation, hobbies; 12) relation to weekends, holidays, holidays abroad; 13) surgical implants; 14) reactions to insect stings; 15) association with menstruation; 16) response to prior treatment; 17) stress and 18) quality of life impairment (Zuberbier T, Bindslev-Jensen C, Canonica W, Grattan CEH, Greaves MW, Henz BM et al. EAACI/GA_LEN guideline: definition, classification, and diagnosis of urticaria. Submitted to Allergy). Patient diaries are very helpful to become aware of the fluctuating intensity of the disease. Physical examination should determine number and size of wheals, angioedema and should include dermographism. If physical triggering is suspected, appropriate and standardized physical tests should be performed such as pressure test with defined weights,15-18 but physical urticaria will not be discussed in this review. Activity of the disease should be evaluated using a standardized score (Table I).19 This is of particular importance for treatment trials. In addition, impairment of daily life, occupational and psychosocial aspects should be considered. Standardized instruments for dermatologic diseases have been used,3, 4 however, instruments adapted for chronic urticaria may be more useful.2 If single wheals persist for longer than 24 h biopsies should be taken to exclude vasculitis by histology and immunofluorescence that may indicate systemic disease like lupus erythematodes.20 Routinely only differential count and general inflammatory parameters such as C-reactive protein (CRP) should be determined. An international consensus established by hand voting in the year 2001 Vol. 140 - N. 4 TABLE I.—Urticaria-Score according to Zuberbier T, Bindslev-Jensen C, Canonica W, Grattan CEH, Greaves MW, Henz BM et al. EAACI/GA_LEN guideline: definition, classification, and diagnosis of urticaria. Submitted to Allergy and Zuberbier et al.19 Score Wheals Pruritus 0 1 None Mild (<20 wheals/24 h) Moderate (21- 50 wheals/24 h) Severe (>50 wheals/24 h or large confluent areas of wheals) None Mild 2 3 Moderate Severe Sum of score: (0-6). numbered the following parameters as useful in chronic urticaria: serology for helicobacter, gastroscopy, anti-streptolysin titre, serology for hepatitis, pseudoallergen-low diet for 3 weeks, ASST, antinucleare antibodies, thyroid antibodies, oral provocation tests, specific IgE, ova/parasites and other investigations.19, 21 However, it should be noted, that this consens was established by simple hand voting and regionally, the optimal diagnostic schedule may differ, e.g. in Germany investigation for hepatitis and parasites is not very useful. Our research results during the last years revealed a reliable and successful diagnostic program based on the following 3 parameters:1, 22-27 1) infections; 2) autoreactivity; 3) non-allergic hypersensivity reactions (pseudoallergies). Therefore, the determination of the parameters presented is used routinely in our department (Figure 2). Infections Recently, we reviewed the published literature regarding “chronic urticaria and infections.” 23 It was found that studies investigating this topic were difficult to compare, evidence-based criteria were not applicable and a meta-analysis was impossible. Most studies did not consider the multiplicity of triggering factors. Further complicating factors are for example geographic variations in the frequency of infections (e.g. for parasitosis). GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 437 WEDI CHRONIC URTICARIA Infections Investigation of: - Helicobacter 1 - Streptococci 2 - Staphylococci 3 - Yersinia4 Autoreactivity - ASST5 - cellular activation (BHR, BasoAT, CAST)6 - thyroid autoAbs7 - (antinuclear Abs) Non-allergic hypersensitivity - Avoidance of aspirin and other NSAID - pseudoallergen-low diet8 Detailed urticaria history, dermographism (perhaps physical tests) differential function differentialcount, count,CRP9, CRP9, C1-INH Cl-INH function Figure 2.—Recommended diagnostic schedule in chronic urticaria. In children, serology for Epstein-Barr-virus and cytomegalovirus should be included; 1) urea breath test or gastroscopy with biopsies; 2) antiDNAseB-, antistreptolysin-titre; 3) antistaphylolysin-titre; 4) Yersinia-IgA, -IgG and immunoblot; 5) autologous serum skin test; 6) BHR = basophil histamine release, BasoAT = basophil activation test (CD63 expression), CAST = cellular antigen stimulation test (production of sulfidoleukotrienes); 7) basal TSH, microsomal Abs, thyroglobulin Abs, TSH receptor Abs; 8) in selected patients for at least 3 weeks; 9) general parameter for inflammation/malignancy. From the literature it can be seen that the prevalence of infections, either bacterial, viral, parasitic or fungal appears not to differ compared to the general population (for yersinia this has to be demonstrated). However, there is a very large amount of reports demonstrating benefit after eradication of infectious processes and it is hardly to believe that all these are due to spontaneous remissions. Nevertheless, available studies have common flaws and cannot be reviewed as a meta-analysis or according to evidencebased medicine rules. Best evidence exists for Helicobacter pylori infection.23 Systematically reviewing existing studies addressing the effect of antibiotic therapy for chronic urticaria patients infected with Helicobacter pylori revealed that resolution of urticaria was more likely when antibiotic therapy was successful in eradication of Helicobacter.28 Ten studies met the inclusion criteria and when data from these studies were combined, eradication of Helicobacter pylori was both quantitatively and statistically associated with remission of urticaria, with an odds ratio of 2.9 (95% confi- 438 dence interval 1.4-6.8; P= 0.005). The authors concluded that clinicians, after considering other causes of urticaria, should constitute 1) testing for Helicobacter pylori; 2) treating with appropriate antibiotics if Helicobacter pylori is present; and 3) confirming successful eradication of infection.28 We are recommending this approach for several years.22-24, 26, 27, 29, 30 However, Helicobacter is not the only triggering factor. Other persistent, chronic, in most cases subclinical infections, for example, with streptococci, staphylococci and yersinia can also be found.23 In the case of an identified infection, targeted eradication should be performed and it should be carefully looked whether eradication was successful. It should be borne in mind that often urticaria does not disappear until all triggering factors have been carefully addressed, e.g. Helicobacter pylori associated gastritis, persistent yersiniosis, elevated antistreptococcal titres with chronic sinusitis plus positive ASST plus exacerbation through regular aspirin intake. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 CHRONIC URTICARIA WEDI Autoreactivity Details of the autoimmune pathogenesis of chronic urticaria have been recently reviewed.31, 32 Evidence for autoreactive mechanisms in chronic urticaria is provided by a positive ASST (Figure 3). However, the clinical relevance is far from being clear because the presence of functional autoantibodies against FcεRI and/or IgE itself does not correlate with a positive ASST. Moreover, ASST can be still positive although urticarial symptomatology disappeared. Therefore, several authors recommed a functional cellular activity assay such as basophil histamine release for confirmation. In addition, the determination of sulfidoleukotriene de novo production in leukocyte suspensions (cellular antigen stimulation test, CAST) and flow cytometric CD63 surface expression on basophils are possible 33-35 although histamine release appears to be more specific. Nevertheless, Figure 3.—Positive autologous serum skin test in chronic urticaria, reading after 30 min. these functional assays are difficult to perform and to interpret due to their dependence on the releasability of the donor cells used. Up to the present, the direct chronic urticaria.37 After exclusion of other causes, in measurement of the autoantibodies is not available single cases a standardized pseudoallergen-low diet for routine purposes since a satisfactory ELISA has not might be indicated for at least 3 weeks.38 However, been developed. controlled provocation test often are not able to idenPatients with positive ASST more often have thyroid tify causal substances.39 autoantibodies. Thus, their determination is recommended at least in women with chronic urticaria. It is debated whether therapeutic implications are given SPECIAL DIAGNOSTIC ISSUE: CHILDHOOD CHRONIC URTICARIA when—as it is most often the case—thyroid function is normal. In selected cases assessment of antinuclear Recently, it has been shown that 20-30% of chilantibodies may be advisable to exclude systemic lupus dren with acute urticaria progressed into chronic erythematosus. urticaria. In almost all (91%) acute urticaria was considered to be induced by acute infection.40 Similarly, Non-allergic hypersensitivity (pseudoallergic reac- assocation with infections also plays a major role in chronic urticaria.23 Persistent chronic often bacterial tions) infections (e.g. with streptococci, staphylococci, but NSAID, particularly aspirin, are a common exacalso with Helicobacter pylori and yersinia) are most erbating factor in chronic urticaria and should be avoidcommon in chronic urticaria.40, 41 Furthermore, posied. However, as single causal factor they are rare. If sustive ASST indicating autoreactivity can also be found pected a placebo-controlled provocation test should in about 30% of children with chronic urticaria.41, 42 In be envisaged. children and young adults serology for Epstein-Barr In this aspect, recently an interesting publication virus and cytomegalovirus should be included in the demonstrated that 33% of patients with acute urticaria diagnostic program. due to NSAID intake developed chronic urticaria 1 to 10 years later in contrast to 1% of an atopic control popRECURRENT ANGIOEDEMA ulation.36 The authors concluded that NSAID intolerRecurrent angioedema without wheals may be ance may predispose to later development of chronic caused by hereditary or acquired C1-esterase inhibitor urticaria. In addition, food additives are suggested to trigger deficiency or dysfunction, drugs (particularly Vol. 140 - N. 4 GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 439 WEDI CHRONIC URTICARIA angiotensin converting enzyme inhibitiors, angiotensin II receptor antagonists),43, 44 and perhaps also persistent infections (e.g. Helicobacter pylori-associated gastritis, yersiniosis).45-47 If angioedema are described principally at least the functional activity of the C1-esterase inhibitor should be assessed to exclude deficiency or dysfunction.48 ACE-inhibitors and perhaps also AT II receptor antagonists should be avoided. It has to be considered that angioedema which often are located in the oropharynx occur within several weeks after initiation of treatment but can also occur first after several years of intake.49 Treatment Henderson et al.50 addressed the question what agents are commonly used to treat urticaria in 9.2 million visits in office-based practices between 1990 and 1997 in the USA. They found that H1-antihistamines were prescribed in 56% of consultations, systemic corticosteroids in 14% and other drugs in 12%. Interestingly, allergists were the least likely to prescribe corticosteroids whereas internists were the most likely.51 Looking at alternatives, H2-antihistamines, βagonists, doxepin, nifedipine and, interestingly enough methotrexate were prescribed. These drugs are not prescribed in our clinic. An italian study described prescription of antihistamines in 351 patients with chronic urticaria, with a good response in only 40%.51 In this study 221 patients were treated with other drugs such as steroids, cyclosporine A, cromolyn, LT receptor antagonists and adrenaline. It is undeniable that we do not have a standard treatment of chronic urticaria. To make preparations for the recent international urticaria consensus conference in Berlin 2004 we performed a systematic review of randomised controlled trials (RCTs) in chronic urticaria (Zuberbier T, Bindslev-Jensen C, Canonica W, Grattan CEH, Greaves MW, Henz BM et al. EAACI/GA_LEN guideline: definition, classification, and diagnosis of urticaria. Submitted to Allergy). A literature search using MEDLINE and EMBASE, in part also by hand-searching was done. In chronic urticaria RCTs demonstrated ineffective treatment with sedating H1-antihistamines plus cimetidine or terbutaline and also with tranexamic acid or cromolyn. However, these were single 440 studies with several flaws, so that their level of evidence is very low. In MEDLINE and EMBASE there is no study addressing the efficacy of corticosteroids in chronic urticaria although they are widely used. Sometimes corticosteroids are needed for example to achieve rapid control to cover social or occupational events but it is consent that prolonged daily treatment should be clearly avoided due to severe side effects. In the treatment of chronic urticaria best evidence exists for non-sedating H1-antihistamines such as azelastine, cetirizine, desloratadine, ebastine, fexofenadine, levocetirizine, loratadine and mizolastine (alphabetical order). The quality of these RCTs is high and taken together non-sedating H1 antihistamines can be recommended with highest Grade A. However, we should bear in mind that most of these studies included only patients with mild to moderate urticaria. High quality RCTs that compared non-sedating H1-antihistamines are not available and from the existing evidence it appears that differences are rather small if ever existing. However, from clinical experience it is well known that non-responders with one non-sedating antihistamines may respond favourably to another. Urticaria experts often use increased dosage and they are clearly needed in most patients with chronic urticaria. However, RCTs addressing this point in chronic urticaria are missing. Several drugs, such as cyclosporin A, LT receptor antagonists and stanazolol have been combined with non-sedating H1 antihistamines. However, from an evidence based view the grade of recommendation is low. The situation is even more bad looking at other alternatives such as doxepin, oxatomide, nifedipin, montelukast, and warfarin. For several drugs such as corticosteroids, dapsone, sulfasalazine, methotrexate, interferon, for plasmapheresis and intravenous immunoglobulins (IVIGs) we do not have RCTs. Evidence is based on case series, uncontrolled trials or expert opinion. Nevertheless, cyclosporin A may be a valuable alternative in severely affected patients although potential side effects have to be considered. Grattan et al. 52 investigated a dose of 4 mg/kg per day in combination with a doubled dose of cetirizine in ASST+ patients. An unblinded study by Baskan et al. 53 also included ASST+ patients and used the same dose but their aim was to compare 4 weeks versus 12 weeks treatment. The unblinded and uncontrolled study by Toubi 54 GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 CHRONIC URTICARIA WEDI investigated the effect of low-dose cyclosporine. They stated that efficacy of cyclosporine was independent of ASST reactivity. However, all these studies included very few patients. Available data for leukotriene receptor antagonists are inconsistent and difficult to compare.55-60 These drugs may be of benefit in subgroups of patients with chronic urticaria that have a positive ASST and/or suffer from ASA and/or food intolerance. It may be also worthwhile to compare patients with and without angioedema. In our hands hydroxychloroquine is effective in patients with autoimmune urticaria. Several years ago in vitro we found significant inhibitory effects of chloroquine on serum activity of ASST positive patients.35 Chloroquine inhibited histamine release, basophil CD63 expression and leukotriene de novo production that was induced by ASST positive chronic urticaria sera. This benefit may be also confirmed by an australian open label study 61 and a recent randomised placebocontrolled controlled trial 62 although both studies have several flaws. Taken together non-sedating H1 antihistamines remain the key treatment of chronic urticaria although these drugs are insufficient in several patients even in increased dosage. Thus, more and well designed RCTs are clearly needed to recommend or refuse potential alternative drugs. Although difficult to perform these RCTs should include best characterised patients and should try to minimise bias by carefully addressing critical points of internal validity. Several questions have to be addressed in the future and there is hope that during the next years new therapeutic strategies will be developed to facilitate the management of this long-lasting and life-quality restricting disease. Riassunto Orticaria cronica: una review L’orticaria cronica rimane una delle patologie maggiormente problematiche in tema di eziopatogenesi, work-up diagnostico e trattamento. Negli ultimi anni sono stati portati avanti nuovi concetti riguardanti questa patologia. Alcuni di questi aspetti hanno portato a modificare il management della malattia, mentre i risultati di altri studi di ricerca devono ancora essere confermati e validati. I sintomi dell’orticaria derivano, da un punto di vista fisiopatologico, dalla degranulazione Vol. 140 - N. 4 delle mast cell e dei basofili attraverso possibili meccanismi di tipo autoimmune, infettivo, pseudoallergico, e altri (connessi a malattie internistiche e a neoplasie). Un’accurata anamnesi della malattia gioca un ruolo fondamentale nel programma diagnostico. Ulteriori indagini sono indicate in base al sottotipo di patologia. Nell’orticaria acuta, il classico iter diagnostico non viene generalmente raccomandato, mentre nella forma cronica si consiglia vivamente di eseguire un programma mirato alla valutazione di infezioni (in particolar modo da Helicobacter pilori, stafilococchi, streptococchi, Yersinia), test di autoreattività e test di ipersensibilità non allergologici. Particolare considerazione deve essere posta all’angioedema e all’orticaria dell’infanzia. Nella maggior parte dei casi l’iter diagnostico porta alla identificazione dei fattori trigger della malattia; è stato visto che il trattamento a lungo termine dei fattori trigger migliora la qualità di vita dei pazienti e le manifestazioni cliniche della malattia possono scomparire o manifestarsi nuovamente, ma dopo parecchi anni. Vengono consigliati gli antistaminici H1 (pochi effetti collaterali) che devono essere somministrati quotidianamente e regolarmente; a volte è necessario aumentare la posologia. Altri trattamenti sono risultati non soddisfacenti anche se la ciclosprina A, gli antagonisti leucotrienici e l’idroclorochina possono essere efficaci in casi selezionati. Ci sono ancora molte domande a cui rispondere in futuro con la speranza che nei prossimi anni le strategie terapeutiche possano facilitare il management e la qualità di vita di questa patologia cronica e limitante le attività quotidiane. Parole chiave: Orticaria cronica - Autoreattività - Infezioni - Helicobacter pylori. References 1. Wedi B. Acute, chronic or physical urticaria. What causes the hives? MMW Fortschr Med 2002;144:28-32. German. 2. Baiardini I, Giardini A, Pasquali M, Dignetti P, Guerra L, Specchia C et al. Quality of life and patients’ satisfaction in chronic urticaria and respiratory allergy. Allergy 2003;58:621-3. 3. Poon E, Seed PT, Greaves MW, Kobza-Black A. The extent and nature of disability in different urticarial conditions. Br J Dermatol 1999;140:667-71. 4. O’Donnell BF, Lawlor F, Simpson J, Morgan M, Greaves MW. The impact of chronic urticaria on the quality of life. Br J Dermatol 1997;136:197-201. 5. Van der Valk PG, Moret G, Kiemeney LA. The natural history of chronic urticaria and angioedema in patients visiting a tertiary referral centre. Br J Dermatol 2002;146:110-3. 6. Kozel MMA, Mekkes JR, Bossuyt PM, Bos JD. Natural course of physical and chronic urticaria and angioedema in 220 patients. J Am Acad Dermatol 2001;45:387-91. 7. Bakos N, Hillander M. Comparison of chronic autoimmune urticaria with chronic idiopathic urticaria. Int J Dermatol 2003;42:613-5. 8. Grattan CE, Walpole D, Francis DM, Niimi N, Dootson G, Edler S et al. Flow cytometric analysis of basophil numbers in chronic urticaria: basopenia is related to serum histamine releasing activity. Clin Exp Allergy 1997;27:1417-24. 9. Grattan CE, Dawn G, Gibbs S, Francis DM. 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AntiFc(epsilon)RI auto antibodies and basophil histamine releasability in chronic idiopathic urticaria. J Allergy Clin Immunol 1998;102 (4 Pt 1):651-8. Wedi B, Novacovic V, Koerner M, Kapp A. Chronic urticaria serum 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. induces histamine release, leukotriene production and basophil CD63 surface expression – inhibitory effects of anti-inflammatory drugs. J Allergy Clin Immunol 2000;105:552-60. Asero R. Intolerance to nonsteroidal anti-inflammatory drugs might precede by years the onset of chronic urticaria. J Allergy Clin Immunol 2003;111:1095-18. Zuberbier T. Pseudoallergens and chronic urticaria. Allergologie 2001;24:457-62. German. Zuberbier T, Chantraine-Hess S, Hartmann K, Czarnetzki BM. Pseudoallergen-free diet in the treatment of chronic urticaria. A prospective study. Acta Derm Venereol 1995;75:484-7. Werfel T, Wedi B, Kleine-Tebbe J, Niggemann B, Saloga J, Sennekamp J et al. Vorgehen bei Verdacht auf eine pseudo-allergische Reaktion durch Nahrungsmittelinhaltsstoffe. Allergo J 1999;8: 135-141. Sackesen C, Sekerel BE, Orhan F, Kocabas CN, Tuncer A, Adalioglu G. The etiology of different forms of urticaria in childhood. Pediatr Dermatol 2004;21:102-8. Wieczorek D, Raap U, Liekenbrocker T, Kapp A, Wedi B. Chronic urticaria in childhood. Hautarzt 2004;55:357-60. German. Brunetti L, Francavilla R, Miniello VL, Platzer MH, Rizzi D, Lospalluti ML et al. High prevalence of autoimmune urticaria in children with chronic urticaria. J Allergy Clin Immunol 2004;114:922-7. Bowen T, Cicardi M, Farkas H, Bork K, Kreuz W, Zingale L et al. Canadian 2003 International Consensus Algorithm for the Diagnosis, Therapy, and Management of Hereditary Angioedema. J Allergy Clin Immunol 2004;114:629-37. Bork K, Hardt J, Schicketanz KH, Ressel N. Clinical studies of sudden upper airway obstruction in patients with hereditary angioedema due to C1 esterase inhibitor deficiency. Arch Intern Med 2003; 163:1229-35. Heymann WR. Acquired angioedema. J Am Acad Dermatol 1997;36:611-5. Sabroe RA, Kobza Black A. Angiotensin-converting enzyme (ACE) inhibitors and angio-oedema. Br J Dermatol 1997;136:153-8. Varvarovska J, Sykora J, Stozicky F, Chytra I. Acquired angioedema and Helicobacter pylori infection in a child. Eur J Pediatr 2003;162: 707-9. Charlesworth EN. Differential diagnosis of angioedema. Allergy Asthma Proc 2002;23:337-9. Wedi B, Raap U, Wieczorek D, Kapp A. Urticaria - an update. Allergologie 2004;27:435-43. German. Henderson RL, Fleischer AB, Feldman SR. Allergists and dermatologists have far more expertise in caring for patients with urticaria than other specialists. J Am Acad Dermatol 2000;43:1084-91. Nettis E, Pannofino A, D’Aprile C, Ferrannini A, Tursi A. Clinical and aetiological aspects in urticaria and angio-oedema. Br J Dermatol 2003;148:501-6. Grattan CE, O’Donnell BF, Francis DM, Niimi N, Barlow RJ, Seed PT et al. Randomized double-blind study of cyclosporin in chronic ‘idiopathic’ urticaria. Br J Dermatol 2000;143:365-72. Baskan EB, Tunali S, Turker T, Saricaoglu H. Comparison of shortand long-term cyclosporine A therapy in chronic idiopathic urticaria. J Dermatolog Treat 2004;15:164-8. Toubi E, Blant A, Kessel A, Golan TD. Low-dose cyclosporin A in the treatment of severe chronic idiopathic urticaria. Allergy 1997;52: 312-6. Bagenstose SE, Levin L, Bernstein JA. The addition of zafirlukast to cetirizine improves the treatment of chronic urticaria in patients with positive autologous serum skin test results. J Allergy Clin Immunol 2004;113:134-40. Di Lorenzo G, Pacor ML, Mansueto P, Pellitteri ME, Lo Bianco C, Ditta V et al. Randomized placebo-controlled trial comparing desloratadine and montelukast in monotherapy and desloratadine plus montelukast in combined therapy for chronic idiopathic urticaria. J Allergy Clin Immunol 2004;114:619-25. Nettis E, Colanardi MC, Paradiso MT, Ferrannini A. Desloratadine in GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 CHRONIC URTICARIA WEDI combination with montelukast in the treatment of chronic urticaria: a randomized, double-blind, placebo-controlled study. Clin Exp Allergy 2004;34:1401-7. 58. Erbagci Z. The leukotriene receptor antagonist montelukast in the treatment of chronic idiopathic urticaria: a single-blind, placebo-controlled, crossover clinical study. J Allergy Clin Immunol 2002;110: 484-8. 59. Reimers A, Pichler C, Helbling A, Pichler WJ, Yawalkar N. Zafirlukast has no beneficial effects in the treatment of chronic urticaria. Clin Exp Allergy 2002;32:1763-8. Vol. 140 - N. 4 60. Pacor ML, Di Lorenzo G, Corrocher R. Efficacy of leukotriene receptor antagonist in chronic urticaria. A double-blind, placebo-controlled comparison of treatment with montelukast and cetirizine in patients with chronic urticaria with intolerance to food additive and/or acetylsalicylic acid. Clin Exp Allergy 2001;31:1607-14. 61. Baumgart KW, Mullins R. Use of hydroxychloroquine in refractory urticaria. J Allergy Clin Immunol 2000;105:795-6. 62. Reeves GEM, Boyle MJ, Bonfield J, Dobson P. Impact of hydroxychloroquine therapy on chronic urticaria: chronic autoimmune urticaria study and evaluation. Int Med J 2004;34:182-6. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 443 CLINICAL CASES G ITAL DERMATOL VENEREOL 2005;140:445-9 Granular parakeratosis C. TOMASINI, Z. SEIA Granular parakeratosis is an acquired disorder of keratinization of unknown origin affecting mainly flexures of adults. A 48year-old woman presented a 1-year history of keratotic papules on the infra- and submammary regions. A skin biopsy revealed granular parakeratosis confined to epidermal foci. The patient's eruption resolved completely with topical steroid therapy. Etiopathogenesis and differential diagnosis are discussed. KEY WORDS: Granular parakeratosis - Keratinization disorder Skin. Division of Dermatology, University of Turin, Turin, Italy Herein, we report and discuss a case of GP in interand submammary regions. Case report G ranular parakeratosis (GP) is an histologic phenomenon with distinct clinical characteristics. It is normally localized in intertriginous areas and it was first described in 1991 by Northcutt et al. as axillary GP. 1 Since then, the disorder has been described in other intertriginous areas such as the inguinal region, inter- and submammary region, the vulva and perianal region, the trunk and the knee.27 Clinically, the GP presents with unilateral or bilateral erythematous hyperkeratotic papules and plaques that are often pruritic. Women beyond the 5th decade are mainly affected,3 although the disorder may also occur in children.8-11 The course is chronic with poor response to therapy and tendency to spontaneous resolution.1, 4, 6 Received: September 29, 2004. Accepted for publication: July 1, 2005. Address reprint requests to: Dott. C. Tomasini, Clinica Dermatologica II, Via Cherasco 23, 10126 Torino. E-mail: [email protected] Vol. 140 - N. 4 We present a case of a 48-year-old healthy woman with a 1-year history of dermatosis in the infra- and submammary regions. Symptoms included itching and burning sensation. On clinical examination there were numerous, small, erythematous, keratotic papules disseminated in the submammary region and in the inframammary folds (Figure 1). The lesions were friable and many of them could be removed by scraping. The patient referred that similar lesions had happened in the axillae and inguinal folds, and subsequently had spontaneously regressed. Family history was negative for blistering or keratinization diseases, psoriasis, or other cutaneous disorders. She was using no new personal hygiene products. A punch biopsy specimen was obtained from the right submammary fold. The histological examination revealed discrete foci of psoriasiform epidermal hyperplasia with minimal spongiosis, preservation of granular layer, and a thick parakeratotic cornified layer with retention of keratohyaline granules (Figures 2, 3). A focal collection of neutrophils GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 445 TOMASINI GRANULAR PARAKERATOSIS Figure 1.—Reddish hyperkeratotic papules in the inter-and submammary regions. Figure 2.—The horny layer is parakeratotic with granular appearance. in the cornified layer was detected. In the superficial dermis there were ectatic vessels and a superficial perivascular infiltrate of lymphocytes. Periodic acidSchiff stain was negative for fungi. A diagnosis of GP was established. A swab was also taken for microbiologic examination: cultures were negative for bacteria, dermatophytes and Candida. Treatment with topical fluconazole cream was ineffective. Topical betamethasone dipropionate ointment was then introduced twice daily with resolution of the rash within 30 days. Discussion and conclusions GP is an acquired disorder of keratinization that affects flexures, initially described in 1991 in 4 patients who had an erythematous eruption in the axillae.1 Since then, to the best of our knowledge, 42 additional cases - including the present case - have been reported. Thirteen of these cases occurred in sites other than the axillae, including the groin, infra- e submammary areas, perianal area, trunk and the knee.2-7 Eight cases occurred in infants with ages at presentation ranging from 9 to 22 months.8-11 Interestingly, in this small series of cases, 2 clinical patterns could be identified: bilateral linear plaques in the inguinal folds and erythematous, geometric plaques underlying pressure points from the diaper.8 The etiology of GP was initially thought to be an unusual contact reaction to a deodorant or antiper- 446 Figure 3.—The corneocytes are nucleated and replete with keratohyaline granules. spirant,1, 4, 12, 13 but this could not explain the frequent unilateral involvement, 4 the inconsistent response to discontinuation of the suspected irritant, the negative patch tests, and the localization in nonaxillary intertriginous folds.1 The absence of spongiosis on histology also militates against contact dermatitis as an etiologic factor. Conversely, the tendency to localize to intertriginous or occluded regions would implicate that heat, moisture, friction, and obesity are contributing factors.1, 4, 12, 13 Furthermore, the well-demarcated, geometric distribution underlying pressure points of disposable diapers would also suggest that diapers play a role in GP of the infancy.8-11 GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 GRANULAR PARAKERATOSIS TOMASINI A possible role of fungal/yeast infections in the development of GP has been suggested by finding of positive cultures for Candida albicans from lesions of GP and histologic observation of hyphal elements within granular parakeratotic corneocytes.12-14 In adults, the putative pathophysiological mechanism of GP involves a defective pathway of keratinization in which profilaggrin is unable to form filaggrin, the protein component of keratohyaline granule. 12 The function of filaggrin has not been completely elucidated, but in the cornified cells it is thought to serve as the matrix protein that embeds and promotes the aggregation and disulphide binding of keratin filaments producing the keratin pattern structure of the lower cornified cells. The conversion of profilaggrin into its monomeric subunits must be carefully controlled to prevent premature aggregation. Patients with GP exhibit a lack of degradation of keratohyaline granules and aggregation of keratin filaments.2 The clinical differential diagnosis of GP involving the flexures includes a wide spectrum of disorders characterized by erythemato-keratotic papules, such as Hailey-Hailey disease, Darier disease, pemphigus vegetans, plane warts, acanthosis nigricans, psoriasis, tinea infection, seborrheic dermatitis, intertrigo, candidiasis, Langerhans cell histiocytosis, nummular eczema and seborrheic keratosis in early stage. The diagnosis is confirmed by examination of a biopsy specimen showing characteristic findings of parakeratotic corneocytes containing keratohyaline granules situated usually over a hyperplastic epidermis, although occasionally granular parakeratotic changes are confined only to follicular infundibulum.7 Interestingly, in newborns GP shows striking clinical and pathological resemblance to a disorder firstly described in German literature in 1975 by Gartmann et al. as pomaden Kruste 15 and later reported in English literature under the name "pigmented and hyperkeratotic napkin dermatitis",16 attributed to overtreatment of the groins of babies with ointments and oils. It is likely that these diseases actually represent examples of GP. In conclusion, GP represents a pattern of altered keratinization in which multiple etiologies may be operative, one of them is microbic, and this might also explain why GA has a propensity for intertriginous sites. Vol. 140 - N. 4 Approaches to treatment of GP have been largely empirical. Although many cases have a self resolution,4-12, 17 a variable response has been observed with oral and topical corticosteroid, antimicrobials, antifungals, and keratolytics. 18-21 Whilst topical retinoids do not appear to be effective, the use of vitamin D analogues was noted to be effective in some cases, supporting the theory of abnormal differentiation.1, 6, 17-19 References 1. Northcutt AD, Nelson DM, Tschen JA. Axillary granular parakeratosis. J Am Acad Dermatol 1991;24:541-4. 2. Wallace CA, Pichardo RO, Yosipovitch G, Hancox J, Sangueza OP. Granular parakeratosis: a case report and literature review. J Cutan Pathol 2003;30:332-5. 3. English JC, Derdeyn AS, Wilson WM, Patterson JW. Axillary granular parakeratosis. J Cutan Med Surg 2003;7:330-2. 4. Meheregan DA, Thomas JE, Meheregan DR. Intertriginous granular parakeratosis. J Am Acad Dermatol 1998;29:495-6. 5. Meheregan DA, Vandersteen P, Sikorski L, Meheregan DR. Axillary granular parakeratosis. J Am Acad Dermatol 1995;33:373-5. 6. Wohlrab J, Juftul M, Wolter M, Marsch WC. Submammary granular parakeratosis: an acquired punctate hyperkeratosis of exogenic origin. J Am Acad Dermatol 1999;40:813-4. 7. Resnik KS, DiLeonardo M. Follicular granular parakeratosis. Am J Dermatopathol 2003;25: 428-9. 8. Chang MW, Kaufmann JM, Orlow SJ, Cohen DE, Mobini N, Kamino H. Infantile granular parakeratosis: recognition of two clinical patterns. J Am Acad Dermatol 2004;50 (5 Suppl):S93-6. 9. Trowers AB, Assaf R, Jaworsky C. Granular parakeratosis in a child. Pediatr Dermatol 2002;19:146-7. 10. Patrizi A, Neri I, Misciali C, Fanti PA. Granular parakeratosis: four pediatric cases. Br J Dermatol 2002;147:1003-6. 11. Pimentel DR, Michalany N, Morgado de Abreu MA, Petlik B, Mota de Avelar Alchorne M. Granular parakeratosis in children: case report and review of the literature. Pediatr Dermatol 2003;20:215-20. 12. Metze D, Rutten A. Granular parakeratosis:a unique acquired disorder of keratinization. J Cutan Pathol 1999;26:339-52. 13. Barnes CJ, Lesher JL, Sangueza OP. Axillary granular parakeratosis. Int J Dermatol 2001;40:439-41. 14. Resnik KS, Kantor GR, DiLeonardo M. Dermatophyte-related granular parakeratosis. Am J Dermatopathol 2004;26:70-1. 15. Gartmann H, Steigleder GK. [Inguinal "pomade" crust of infants] Z Hautkr 1975;50:667-9. German. 16. Patrizi A, Neri I, Marzaduri S, Fiorillo L. Pigmented and hyperkeratotic napkin dermatitis: a liquid detergent irritant dermatitis. Dermatology 1996;193:36-40. 17. Sceppa J, Mowad C, Elenitsas R. Crusted plaques in the axillae. Arch Dermatol 2001;137:1241-6. 18. Brown SK, Heilman ER. Granular parakeratosis: resolution with topical tretinoin. J Am Acad Dermatol 2002;47(5 Suppl):S279-80 19. Webster CG, Resnik KS, Webster GF. Axillary granular parakeratosis: response to isotretinoin. J Am Acad Dermatol 1997;37: 789-90. 20. Chamberlain AJ, Tam MM. Intertriginous parakeratosis responsive to potent topical corticosteroids. Clin Exper Dermatol 2003; 28:50-2. 21. Contreras ME, Gottfried LC, Bang RH, Palmer CH. Axillary intertriginous granular parakeratosis responsive to topical calcipotriene and ammonium lactate. Int J Dermatol 2003;42:382-3. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 447 TOMASINI GRANULAR PARAKERATOSIS Paracheratosi granulare L a paracheratosi granulare (granular parakeratosis, GP), descritta per la prima volta nel 1991 da Northcutt et al. con il nome di GP ascellare 1, è un pattern istologico a cui corrisponde un quadro clinico peculiare che si osserva alle pieghe corporee. Da allora sono stati riportati casi di GP a interessamento inguinale, intra e sottomammario, genitale, perianale, al tronco e, persino alle ginocchia 2-7. Dal punto di vista clinico si apprezzano papule e placche eritematose ipercheratosiche uni o bilaterali, spesso pruriginose. La dermatosi colpisce prevalentemente le pazienti di sesso femminile di età inferiore ai 50 anni 3, sebbene siano stati descritti anche casi pediatrici 8-11. Il decorso è, in genere, cronico con scarsa risposta alla terapia e tendenza alla risoluzione spontanea 1, 4, 6. In questo lavoro viene segnalato un caso di GP della regione intra e sottomammaria con particolare attenzione ai problemi interpretativi che esso ha suscitato. Caso clinico Una paziente di 48 anni, in buone condizioni di salute, giungeva all’ osservazione degli Autori per una dermatosi localizzata nella regione infra e sottomammaria da un anno. La paziente lamentava intenso prurito e sensazione di bruciore. All’ anamnesi non risultava l’ uso di nuovi prodotti per l’ igiene personale. Dal punto di vista clinico si osservavano numerose piccole papule eritematose e cheratosiche disseminate in zona sottomammaria e tra le pieghe mammarie (Figura 1). La componente cheratosica appariva friabile e facilmente asportabile con il grattamento. La paziente riferiva che lesioni simili erano apparse dapprima ai cavi ascellari e alle pieghe inguinali e successivamente erano regredite spontaneamente. Negativa risultava la familiarità per malattie bollose, disordini della cheratinizzazione, psoriasi o altre dermatosi. Veniva effettuato un prelievo bioptico di una lesione localizzata alla piega sottomammaria destra. L’ esame istologico rivelava la presenza di aree di iperplasia epidermica psoriasiforme con minima spongiosi. Lo strato granuloso appariva conservato, mentre lo strato corneo era ispessito e paracheratosico con ritenzione di granuli di cheratoialina e accumuli di neutrofili (Figure 2, 3). Nel derma superficiale si potevano osservare vasi ectasici e un infiltrato linfocitario superficiale perivascolare. La colorazione PAS risultava negativa. Sulla base di queste caratteristiche istologiche veniva formulata la diagnosi di GP. Un tampone per esame microbiologico non evidenziava presenza di batteri, dermatofiti o Candida. Veniva inizialmente intrapresa una terapia con fluconazolo topico con scarsi risultati e, successivamente, veniva utilizzato un composto a base di betametasone dipropionato in 2 applicazioni al dì con remissione della dermatosi a distanza di 4 settimane. 448 Discussione e conclusioni La GP è un disordine acquisito della cheratinizzazione che colpisce le pieghe, descritto per la prima volta nel 1991 in 4 pazienti affetti da un’ eruzione eritemato-cheratosica localizzata alle ascelle 1. Da allora, per quanto ne sappiamo, sono stati riportati 42 casi: di questi 13 coinvolgevano le ascelle, le pieghe inguinali, le aree infra e sottomammarie, perianali e le ginocchia 2-7 e 8 riguardavano bambini di età compresa tra i 9 e i 22 mesi 8-11. In questa serie limitata venivano individuati 2 diversi pattern clinici: placche lineari, bilaterali in sede inguinale e placche eritematose geometriche localizzate nei punti di pressione delle zone del pannolino 8. L’ eziologia della GP non è al momento nota. Inizialmente si era ipotizzato che la GP fosse dovuta a un’ anomala reazione da contatto a un deodorante o a un prodotto antiperspirante 1, 4, 12, 13, ma quest’ ipotesi non rendeva ragione del frequente coinvolgimento monolaterale 4, la mancanza di regressione con la sospensione del prodotto topico, la negatività dei patch-test e la localizzazione in altre aree intertriginose 1. Inoltre, l’ assenza di spongiosi nei preparati istologici non supportava tale ipotesi. Secondo alcuni Autori la particolare disposizione delle lesioni nelle aree intertriginose suggeriva che il calore, l’ umidità la frizione e/o l’ obesità fossero fattori predisponenti di tale dermatosi 1, 4, 12, 13. La netta delimitazione nei punti di pressione al di sotto dei pannolini potrebbe implicare un ruolo favorente degli assorbenti nella GP dei neonati 8-11. La possibile eziologia micotica è stata suggerita dalla positività per Candida Albicans e dal riscontro istologico di ife tra i corneociti in alcuni casi di GP 12-14. Negli adulti, il meccanismo patofisiologico della GP potrebbe consistere in un difetto di cheratinizzazione con mancata sintesi di profilaggrina in filaggrina, una componente proteica dei granuli di cheratoialina 12. La funzione della filaggrina non è ancora del tutto chiara, ma si presume che, all’ interno delle cellule cornee, funga da matrice di adesione essenziale per la produzione di ponti disolfuro tra i filamenti di cheratina, producendo, così, il pattern strutturale della cheratina tipico delle cellule cornee. La conversione della profillaggrina nelle due subunità monometriche deve essere attentamente controllata per prevenire un’ aggregazione prematura. I pazienti affetti da GP presentano un difetto di degradazione dei granuli di cheratoialina e di aggregazione dei filamenti di cheratina 2. La diagnosi differenziale clinica di una dermatosi papulo-cheratosica localizzata alle pieghe comprende un ampio spettro di disordini quali la malattia di Hailey-Hailey, la malattia di Darier, il pemfigo vegetante, le verruche piane, l’ acanthosis nigricans, la psoriasi, la tinea, la dermatite seborroica, l’ intertrigo, la candidosi, l’ istiocitosi a cellule di Langerhans, l’ eczema nummulare e le cheratosi seborroiche in fase iniziale 1, 4-6, 9, 12. L’ esame istologico è dirimente per la GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 GRANULAR PARAKERATOSIS TOMASINI corretta diagnosi evidenziando la caratteristica paracheratosi granulare a livello dell’ epidermide, sebbene in qualche caso tale fenomeno sia limitato all’ infundibulo follicolare 7. Nei neonati, la GP presenta notevoli similarità a una particolare dermatosi descritta per la prima volta da Gartmann et al. nel 1975 con il nome di “Pomaden Kruste” 15 nella letteratura tedesca e poi riportata da quella anglosassone come “pigmented and hyperkeratotic napkin dermatitis” 16, attribuita a un trattamento eccessivo della cute genitale dei neonati con creme e olii. È verosimile che questi quadri, in realtà, rappresentino esempi di GP. In conclusione, la GP rappresenta un disordine della cheratinizzazione a eziologia verosimilmente multifattoriale. Ciò potrebbe spiegare la particolare predisposizione per le aree intertriginose. Il trattamento della GP non è codificato. Sebbene molti casi mostrino una risoluzione spontanea 4,12, 17, si è notata una risposta variabile agli steroidi topici e per os, agli antimicrobici, agli antifungini e ai cheratolitici 18-21. Sebbene i retinoidi topici non appaiano efficaci, l’ uso di retinoidi ana- Vol. 140 - N. 4 loghi della vitamina D ha sortito risultati incoraggianti, avvalonando l’ ipotesi di un’ anomalia della differenziazione cheratinocitaria alla base di questa dermatosi 1, 6, 17-19. Riassunto La paracheratosi granulare (GP) è un disordine acquisito della cheratinizzazione a eziologia sconosciuta che colpisce prevalentemente le pieghe di soggetti adulti. In questo lavoro è riportato il caso di una paziente di 48 anni che presentava da circa un anno papule cheratosiche alle pieghe infra e sottomammarie. Una biopsia cutanea evidenziava il quadro della paracheratosi granulare. L’ eruzione regrediva rapidamente con terapia steroidea topica. Vengono, inoltre, discusse le problematiche eziopatogenetiche e di diagnosi differenziale. PAROLE CHIAVE: Paracheratosi granulare - Disordini della cheratinizzazione - Cute. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 449 LETTERS TO THE EDITOR G ITAL DERMATOL VENEREOL 2005;140:451-8 Non recurrent eosinophilic cellulitis: report of an atypical case M. L. BERNARDINI 1, G. BRANDOZZI 1, A. CAMPANATI 1, L. GIORNETTA 1, M. GIANGIACOMI 2, A. OFFIDANI 1 1Division of Dermatology Università Politecnica delle Marche Ospedali Riuniti Hospital, Ancona, Italy 2Division of Anatomy and Istology Università Politecnica delle Marche Ospedali Riuniti Hospital, Ancona, Italy Dear Sir, Figure 1.—Large oedematous and erythematous lesion on the right knee. E osinophilic cellulitis (EC) was described for the first time by Wells in 1971 1 as a distinctive pathologic entity of unknown etiology, although it was given prominence by many authors because of its association with skin viral and bacterial infections, arthropod bites, cutaneous parasitic infestations (onchocerciasis, ascariasis and toxocariasis), hypereosinophilic syndromes, myeloproliferative disorders, leukaemias, anal carcinoma, drug intake or surgical intervention. Further associations were a history of Reynaud positive phenomenon and urticaria, genetic inheritance.2 Patients affected by Wells’ syndrome (WS) usually present with one or more cutaneous plaques resembling an acute bacteric cellulitis while cultural examination for bacteria is always negative and oral antibiotics never seemed to improve clinical or histologic lesions, except for a single case report in which there was a good response to minocycline.3 Lesions resolve spontaneously after weeks or months without residual scarring. We report the case of a 23 year-old female presenting with a 2 weeks history of an itchy annular-erythematous and oedematous plaque, surrounded by a wide violaceous and swelling border first localized on the anterior surface of the right knee and then also on the anterior surface of the left knee (Figures 1, 2). Our patient had already been administered an antihistaminic drug per os (cetirizine 10 mg/die for 10 days) which barely influenced her clinical manifestations. Fever and general malaise were absent. At her first dermatologic visit plaques appeared hot on palpation and the examination revealed the presence of a slight regression of the erythema in the central area and of an enlarging red-violaceous and swelling border. According to a first diagnosis of The case has been presented as a communication at the 79th SIDEV National Congress of the Società Italiana di Dermatologia e Venereologia 2004 and at the 1st Congresso Marchigiano di Dermatologia e Venereologia (25 September, 2004). Vol. 140 - N. 4 Figure 2.—Smaller lesion involving the left knee. acute bacteric cellulitis she was prescribed antibiotics per os (amoxicillin/clavulanic acid, 2 g/die) for 10 days but her clinical manifestations did not improve in any way. Laboratory analysis disclosed: the absence of peripheral eosinophilia; erythrocyte sedimentation rate: 40 mm in the first hour; normal urinalysis, immunoglobulin (IgE included) levels, immunologic serology (C3 and C4; anti-nuclear antibodies); cultural examination for bacteria and fungi was negative; stool examinations were negative for parasites and ova. Ultrasonography showed an absence of joint involvement. Haematoxylin-eosine stained histologic section, on bilater- GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 451 LETTERS TO THE EDITOR Figure 3.—EE-10×. Acanthosis and focal hyperkeratosis. Presence of dermal perivascular lymphocytic infiltration. Figure 4.—EE-20×. Magnification of the previous image. Infiltration of the dermis with eosinophilic granulocytes, accompanied by slight perivascular lymphocytic infiltration. Oedema is absent in the upper dermis. al biopsy specimens, showed oedematous dissociation of dermal collagen fibers and moderate perivascular lymphoplasmacellular infiltrate associated with the presence of eosinophilic infiltration. Both diffuse infiltration of dermal fibers or small clusters of eosinophilic granulocytes (E) were present. Vasculitis was absent. Histiocytes palisade surrounding focal deposition of eosinophilic material and nuclear debris (Figures 3-5), the so called “flame figure” (FF) was detected. Because of both clinical and histologic findings 452 Figure 5.—EE-40×. Flame figure. Histiocytes surrounding focal deposition of eosinophilic material. we concluded that our patient fulfilled the criteria for the diagnosis of WS. We explained the poor presence of flame figures in our patient’s histologic sections as a probable consequence of the first antihistaminic treatment she was administered. We prescribed betametasone per os, 2 m/die, progressively tapered over 10 days and antihistamines per os (cetirizine 10 m/die) for 15 days. Skin lesions resolved within 2 weeks and she has experienced no manifestation recurrence since, after 2 years follow-up. In WS a predominant eosinophilic infiltrate was observed early in the upper and deep dermis. Distinctive FF become apparent in the subacute phase when degranulating E coat basophilic collagen bundles with eosinophilic major basic protein (MBP). The last phase showed a histiocyte palisade around FF. Typical FF were detected but scantely in the patient’s histologic sections: this was probably due to the antihistaminic treatment she was already administered before she was referred to our Dermatologic Clinic. The excessive dermal infiltration of E with FF appears to be a peculiar response to a variety of potential triggering events. Clonally expanded type-2 helper T cells overproducing interleukin-5 (IL-5), the main cytokine in the development and differentiation of E, could be the underlying mechanism in eosinophilic diseases. Yagi et al.4 have observed a high proportion of circulating T CD4+CD7- cells in patients with WS before treatment and their findings suggest that such cells could play a pivotal role in the pathogenesis of EC by producing IL-5. IL-5 not only mobilizes eosinophils from bone marrow to blood but also IL-5 seems to promote skin homing by presumably altering the expression of adhesion molecules. Espana et al.5 found a good correlation between the clinical activity of the disease, the levels of E in the blood and bone marrow and the IL-5 and eosinophilic cationic protein levels in the peripheral blood. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 LETTERS TO THE EDITOR The FF associated with EC are not pathognomonic, as they can be seen in other unrelated dermatoses. FF may be seen incidentally in bullous pemphigoid, pemphigoid gestationis, tinea, spider and insect bite reactions and other inflammatory conditions in which numerous eosinophils are present.6 Diagnosis is, therefore, the result of the matching of all anamnestic, clinical and histologic findings. References 1. Wells GC. Recurrent granulomatous dermatitis with eosinophilia. Trans St Johns Hosp Dermatol Soc 1971;57:46-56. 2. Weiss G, Shemer A, Confino Y, Kaplan B, Trau H. Wells’ syndrome: report of a case and review of the literature. Int J Dermatol 2001;40: 148-52. 3. Stam-Westerveld EB, Daenen S, Van der Meer Jb, Jonkman MF. Eosinophilic cellulitis (Wells’ syndrome): treatment with minocycline. Acta Derm Venereol 1998;78:157. 4. Yagi H, Tokura Y, Matsushita K, Hanaoka K, Furukawa F, Takigawa M. Wells’ syndrome: a pathogenic role for circulating CD4+ CD7- cells expressing interleukin-5 mRNA. Br J Dermatol 1997;136:918-23. 5. Espana A, Sanz ML, Sola J, Gil P. Well’s sindrome (eosinophilic cellulitis): correlation between clinical activity, eosinophil levels, eosinophil cation protein and interleukin-5. Br J Dermatol 1999;140:127-30. 6. Moosavi M, Mehregan DR. Wells’ syndrome: a clinical and histopathologic review of seven cases. Int J Dermatol 2003;42:62-7. Address reprint requests to: Dr. M. L. Bernardini, Via V. Veneto 24, 60122 Ancona. E-mail: [email protected] Un caso di cellulite eosinofilica (Sindrome di Wells) non recidivante con quadro istologico atipico Egregio Direttore, L a cellulite eosinofilica (CE) è stata descritta per la prima volta da Wells nel 1971 come un’entità patologica distinta a eziologia sconosciuta 1. Viene, comunque, segnalata in letteratura una concomitanza della malattia con infezioni cutanee batteriche o virali, morsi di artropodi, infestazioni (oncocerchiasi, ascariasi e toxocariasi), sindromi ipereosinofile, disordini mieloproliferativi, leucemie, carcinoma anale, assunzione di farmaci o interventi chirurgici; più raramente è stata descritta un’anamnesi positiva per il fenomeno di Reynaud, orticaria ed ereditarietà genetica 2. I pazienti con sindrome di Wells (SW) in genere presentano una o poche placche cutanee che ricordano una cellulite acuta, tuttavia l’esame colturale per batteri non risulta mai positivo e gli antimicrobici somministrati per via sistemica non sembrano migliorare il quadro clinico o istologico delle lesioni, a eccezione di un singolo caso di risposta alla minociclina riportato in letteratura 3. Le lesioni iniziali evolvono velo- Vol. 140 - N. 4 cemente in placche che tendono alla risoluzione spontanea, dopo settimane o mesi, senza residuare cicatrici. Segnaliamo il caso di una paziente di 23 anni che si presentava alla nostra attenzione per la comparsa, da circa 15 giorni, di una placca anulare eritemato-edematosa, con ampio bordo periferico edematoso e violaceo, localizzata dapprima sulla superficie estensoria del ginocchio destro e, in seguito, anche alla superficie estensoria del ginocchio sinistro (Figure 1, 2). La paziente veniva inizialmente trattata con un antiistaminico per os (cetirizina 10 mg/die per 10 giorni) che sortiva solo modesti effetti clinici. Al momento della prima visita dermatologica la paziente lamentava una sintomatologia locale fortemente pruriginosa. Risultavano assenti febbre e malessere generale. Le placche, calde al tatto, apparivano di grandi dimensioni, di colore rosso-rosa, con area centrale color camoscio e bordo periferico rosso-violaceo ed edematoso in espansione. Nell’ipotesi di una forma di cellulite batterica acuta veniva prescritto un farmaco antibiotico (amoxicillina/acido clavulanico 1 g, 2 compresse/die) per 10 giorni. Il quadro sintomatologico e obiettivo, tuttavia, non traeva beneficio dalla terapia e la paziente mostrava una lieve progressione delle manifestazioni. Venivano, pertanto, programmati specifici esami di laboratorio e una biopsia incisionale bilaterale per la diagnosi istologica delle lesioni. Le indagini di laboratorio mostravano: emocromo con formula leucocitaria nella norma e assenza di eosinofilia periferica. Gli indici di flogosi erano positivi: VES=40 mm 1° ora. Nella norma risultavano anche le analisi urinarie, il dosaggio delle immunoglobuline, incluse le IgE, la sierologia immunologica (complemento, anticorpi anti-nucleo); l’esame colturale per batteri e funghi era negativo; gli esami delle feci risultavano negativi per uova e parassiti. All’esame ecografico risultava assente il coinvolgimento delle articolazioni del ginocchio sottostanti. La sezione istologica, colorata in ematossilina-eosina, mostrava una discreta dissociazione edematosa delle fibre collagene del derma e un moderato infiltrato linfoplasmacellulare perivascolare, associato alla presenza di granulociti eosinofili (E). Questi ultimi apparivano sia dispersi tra le fibre che in piccoli aggregati. Non vi era alcuna evidenza di vasculite. Era, inoltre, presente un deposito focale di materiale eosinofilico con detriti nucleari, circondato da alcuni istiociti (Figure 3-5). Tale reperto era interpretabile come una classica «figura a fiamma» (FF), caratteristica, sia pure non patognomonica, delle CE. L’insieme dei reperti unitamente ai dati clinico-anamnestici orientavano verso una diagnosi di SW. Veniva prescritto, pertanto, cortisone per via generale (betametasone per os, 2 mg/die a scalare) per un totale di 10 giorni di terapia e, in associazione, un anti-istaminico per os (cetirizina compresse 10 mg/die) per 15 giorni. Il quadro clinico tendeva a mostrare netti segni di miglioramento già dopo la prima settimana di terapia e si aveva una completa risoluzione entro 2 settimane. La paziente non ha mostrato a tutt’oggi recidive, a un follow-up di circa 2 anni. Nella SW i reperti istologici sono generalmente caratterizzati da un iniziale infiltrato di E nel derma superficiale e profondo. Le FF si formano durante la fase subacuta quando gli E, degranulanti, rivestono i fasci di fibre col- GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 453 LETTERS TO THE EDITOR lagene di proteina eosinofilica basica maggiore. Nella fase di risoluzione gli istiociti fagocitici si dispongono a palizzata circondando le FF. Le classiche FF, nel nostro caso, appaiono poco evidenti: con tutta probabilità ciò è dovuto al fatto che una terapia anti-istaminica era già stata instaurata prima che la paziente si rivolgesse alla nostra attenzione. La massiva infiltrazione dermica di E, con la comparsa di FF, sembra essere una risposta peculiare a una varietà di potenziali eventi trigger. L’iperproduzione di interleuchina 5 (IL-5), la citochina più importante per lo sviluppo e la differenziazione degli E, da parte di cellule T helper 2 in espansione clonale, potrebbe rappresentare il meccanismo che sottende le patologie caratterizzate dalla presenza di infiltrato di E. Yagi et al. 4 hanno osservato che i linfociti T CD4+CD7- risultano aumentati nel siero di pazienti con SW non in trattamento e suggeriscono che queste cellule svolgano un ruolo fondamentale nella patogenesi della CE grazie alla produzione di IL-5. IL-5 non solo mobilita gli E dal midollo osseo al sangue periferico, ma sembra promuovere anche il processo di homing cutaneo degli stessi, presumibilmente alterando l’espressione delle specifiche molecole di adesione. Espana et al. 5 hanno mostrato l’esistenza di una buona correlazione tra attività clinica di malattia, livelli di E nel sangue periferico e midollo osseo e livelli di IL-5 e proteina cationica eosinofilica nel sangue periferico. Una CE con FF non è patognomonica di questa sindrome poiché rappresenta una reazione istologicamente ben definita, osservabile anche in altre patologie. Può essere, infatti, occasionalmente presente in corso di pemfigoide bolloso, herpes gestationis, tigna, reazione da puntura di insetti o di aracnidi e altre condizioni infiammatorie in cui risultino presenti numerosi eosinofili 6. La diagnosi, pertanto, deve essere il risultato della combinazione di reperti anamnestici, clinici e istologici. Topical tacrolimus in the tretment of localized bullous pemphigoid E. FRIGERIO, C. FRANCHI G. CAINELLI,G. F. ALTOMARE Department of Dermatology University of Milan, Galeazzi Hospital, Milan, Italy Dear Sir, T acrolimus ointment is a new topical immunomodulator that by specifically inhibiting the activity of calcineurin blocks the early phases of T cell activation and the production of various cytokines (IL2, IL3, IL4, G-CSF, TNF…).1 A number of clinical studies have so far been carried out in order to investigate the use of topical tacrolimus in the treatment of atopic dermatitis, since it proved to be effecti- 454 Figura 1. — At the time of our observation: a large bulla with a serous content at the level of the internal malleolus and some erosions partially covered by squamous scabs on the medial surface of the left leg. ve in reducing the symptoms and severity of the disease in both adults and children. However, although limited to individual cases, an increasing number of reports have described its efficacy in treating other immunomediated dermatoses, including lichen ruber planus, lichen sclerosus et atrophicus, gangrenous pyoderma and alopecia areata.2 The case of a woman with localized bullous pemphigoid that rapidly resolved after topical treatment with 0.1% tacrolimus ointment is reported. A 45-year-old woman in good general conditions attended our outpatient clinic about 1 month after the appearance of erythematovesicular-bullous lesions associated with intense pruritus on the lower third of her left leg (Figure 1). These lesions arose in the area of a large scar that was the sequela of 2 previous surgical operations undergone because of a torn Achilles tendon (the first in 1997 and the second in 1999). No lesions were found in other skin areas nor any mucosal involvement. Histological and direct immunofluorescence examinations of the perilesional skin confirmed the clinical diagnosis of localized bullous pemphigoid. The results of routine blood chemistry tests were normal, and indirect immunofluorescence was negative. Given the marked atrophy of the cicatricial tissue, we decided to avoid the use of highly potent topical steroids and administer twice-daily a local therapy with 0.1% tacrolimus ointment with an occlusive bandage and systemic antihistamine treatment to control the pruritic symptoms (levocetirizine 5 mg/day). At the control examination after 2 weeks, the manifestation had improved with the re-epithelialisation of the previous bulla and the absence of new lesions (Figure 2); moderate pruritus persisted. One month therapy led to complete GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 LETTERS TO THE EDITOR does not show the atrophising effect which is typical of topical corticosteroids. Its only side effect is a sensation of burning and transient pruritus at the application site, which our patient did not experience despite the use of an occlusive bandage. Although large-scale studies are necessary in order to demonstrate the real efficacy of this new immunomodulator, our experience suggests that tacrolimus is a possible alternative to topical corticosteroids also in the treatment of autoimmune bullous dermatoses. Bibliografia Figura 2. — At the two-week control examination: re-epithelialisation of the previous bulla and the absence of new lesions. remission of the dermatosis, and so maintenance treatment with a single daily administration of 0.1% tacrolimus ointment was recommended. No relapse occurred after 2 months. In the localized bullous pemphigoid the same desmosomal antigens as those of generalized bullous pemphigoid have been recognized, and T lymphocytes play a determining role in the pathogenesis of the lesions. It has been demonstrated that T lymphocytes are important for the induction and regulation of both cell- and antibody-mediated immune responses in various autoimmune diseases. It has been found that the serum of patients with bullous pemphigoid contains self-reactive T lymphocytes that produce both Th2 (IL4 and IL13) and Th1 cytokines (gammaIFN), which respectively regulate the secretion of IgG4 and IgG1 by B lymphocytes. In particular, self-reactive T cells that recognized the bullous pemphigoid antigen (BP 180) and give rise to a Th2-mediated response have only been found in the serum of bullous pemphigoid patients.3 Given the limited extension of the lesions, the majority of cases of localized bullous pemphigoid do not require sytemic immunosuppressive treatment, and the therapy of choice is based on the use of highly potent topical corticosteroids. The authors of 2 case reports published in 2003 (1 of dyshidrosis-form localized bullous pemphigoid and 1 of generalized bullous pemphigoid) described the good results obtained using this new topical immunomodulator inhibiting T lymphocytes.4, 5 In our case, we preferred using this new immunomodulator rather than conventional topical corticosteroid therapy in order to avoid worsening the already considerably atrophied cicatricial tissue at the site of the bullous lesions because, unlike corticosteroids, tacrolimus does not give rise to local side effects even after long term treatment; in particular, it Vol. 140 - N. 4 1. Dumont FJ. FK 506, an immunosuppressant targeting calcineurin function. Curr Med Chem 2000;7:731-48. 2. Gupta AK, Adamiak A, Chow M. Tacrolimus: a review of its use for the management of dermatoses. J Eur Acad Dermatol Venereol 2002;16:100-14. 3. Büdinger L, Borradori L, Yee C, Eming R, Ferencik S, Grosse-Wilde H et al. Identification and characterization of autoreactive T cell responses to bullous pemphigoid antigen 2 in patients and healthy controls. J Clin Invest 1998;102:2082-9. 4. Ko M-J, Chu C-Y. Topical tacrolimus therapy for localized bullous pemphigoid. Br J Dermatol 2003;149:1079-80. 5. Chu J, Bradley M, Marinkovich MP. Topical tacrolimus is a useful adjunctive therapy for bullous pemphigoid. Arch Dermatol 2003;139:813-5. Address reprint requests to: Prof. G. Altomare, Via Riccardo Galeazzi 4, 20161 Milano. E-mail: [email protected] Tacrolimus topico nella terapia del pemfigoide bolloso localizzato Egregio Direttore, I l tacrolimus è un nuovo immunomodulatore che inibendo in modo specifico l’attività della calcineurina blocca le fasi precoci di attivazione delle cellule T e la produzione di svariate citochine (IL2, IL3, IL4, G-CSF, TNF …)1. A tutt’oggi sono stati effettuati vari studi clinici riguardanti l’utilizzo del tacrolimus topico nella terapia della dermatite atopica dove si è rivelato efficace nel ridurre i sintomi e la gravità della malattia sia nell’adulto che in età pediatrica. Sono, comunque, in continuo aumento le segnalazioni, seppur spesso limitate a singoli casi, della sua efficacia terapeutica anche in altre dermatosi immunomediate, tra cui, per citarne alcune, il lichen ruber planus, il lichen scleroatrofico, il pioderma gangrenoso e l’alopecia areata 2. Riportiamo il caso di una paziente affetta da pemfigoide bolloso localizzato in cui il trattamento topico con tacrolimus 0,1% unguento ha determinato la rapida risoluzione della dermatosi. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 455 LETTERS TO THE EDITOR La paziente, di 45 anni, in buone condizioni generali, si è presentata presso il nostro ambulatorio per la comparsa da circa 1 mese al III inferiore della gamba sinistra di alcuni elementi eritemato-vescico-bollosi associati a intenso prurito (Figura 1). Tali lesioni insorgevano nel contesto di un’ampia cicatrice esito di 2 pregressi interventi chirurgici per la rottura del tendine d’Achille (il primo nel 1997 e il secondo nel 1999). Non si evidenziavano lesioni in altre zone cutanee né interessamento delle mucose. L’esame istologico e l’immunofluorescenza diretta effettuati su cute perilesionale confermavano la diagnosi clinica di pemfigoide bolloso localizzato. Gli esami ematochimici di routine risultavano nella norma e l’immunofluorescenza indiretta era negativa. Considerata la spiccata atrofia del tessuto cicatriziale, abbiamo ritenuto opportuno evitare l’utilizzo di topici steroidei a elevata potenza e abbiamo deciso di impostare una terapia locale con tacrolimus 0,1% unguento 2 volte/die con bendaggio occlusivo e anti-istaminici sistemici per controllare la sintomatologia pruriginosa (levocetirizina 5 mg/die). Al controllo dopo 2 settimane la manifestazione era migliorata con riepitelizzazione della pregressa bolla e assenza di nuove lesioni (Figura 2), permaneva modesto prurito. A 1 mese dall’inizio della terapia la dermatosi andava in completa remissione per cui, come mantenimento, veniva consigliata un’unica applicazione quotidiana di tacrolimus 0,1% unguento. Non si sono evidenziate recidive a distanza di 2 mesi. Nel pemfigoide bolloso localizzato, vengono riconosciuti gli stessi antigeni desmosomiali del pemfigoide bolloso generalizzato e nella patogenesi delle lesioni rivestono un ruolo determinante i linfociti T. È stato dimostrato che i linfociti T sono importanti sia nell’induzione sia nella regolazione di entrambe le risposte immuni, cellulo-mediate e anticorpo-mediate, in diverse patologie autoimmuni. Nel siero di pazienti affetti da pemfigoide bolloso sono stati riscontrati linfociti T autoreattivi producenti sia citochine Th2 (IL4 e IL13) che Th1 (IFN gamma) le quali regolano rispettivamente la secrezione di IgG4 e IgG1 da parte dei linfociti B. In particolare, solo nel siero di questi pazienti sono state ritrovate cellule T autoreattive che riconoscono l’antigene del pemfigoide bolloso (BP180) dando origine a una risposta autoimmune Th2 mediata 3. Il pemfigoide bolloso localizzato, nella maggior parte dei casi, data la limitata estensione delle lesioni, non necessita del ricorso a un trattamento immunosoppressivo sistemico, infatti la terapia d’elezione si basa sull’utilizzo di corticosteroidi topici a elevata potenza. Nel 2003 sono stati riportati 2 casi, un pemfigoide bolloso localizzato disidrosiforme e un pemfigoide bolloso generalizzato, in cui gli Autori si sono avvalsi, con buoni risultati, di questo nuovo immunomodulatore topico che inibisce i linfociti T 4, 5. Nel nostro caso abbiamo preferito ricorrere a tacrolimus unguento piuttosto che alla terapia convenzionale con corticosteroidi topici per evitare il peggioramento della già 456 importante atrofia del tessuto cicatriziale sede delle lesioni bollose. Il tacrolimus, infatti, a differenza dei corticosteroidi non determina effetti collaterali locali anche nell’utilizzo per lunghi periodi, in particolare non presenta quell’effetto atrofogenico proprio dei corticosteroidi topici. L’unico effetto collaterale consiste nell’insorgenza di bruciore e prurito transitorio nella sede di applicazione, effetto tra l’altro non verificatosi nella nostra paziente nonostante il ricorso alla terapia in occlusiva. Sulla base della nostra esperienza, in attesa di studi su ampie casistiche che permettano di dimostrare l’effettiva efficacia di questo nuovo immunomodulatore, riteniamo il tacrolimus una possibile alternativa ai corticosteroidi topici anche nelle dermatosi bollose autoimmuni. The safety profile of topical pimecrolimus in the treatment of atopic dermatitis G. GIROLOMONI 1, C. GELMETTI 2, A. VIERUCCI 3 of Biomedical and Surgical Sciences Section of Dermatology, University of Verona, Verona, Italy 2Institute of Dermatological Sciences IRCCS Ospedale Maggiore, University of Milan, Milan, Italy 3Department of Pediatrics, University of Florence A. Meyer Pediatric Hospital, Florence, Italy 1Department Dear Editor T he United States Food & Drug Administration (FDA) has recently issued a Public Health Advisory about the safety profile of topical calcineurin inhibitors (better known as topical immunomodulators or TIMs: pimecrolimus and tacrolimus). The FDA also announced of its intention to add a “black-box” warning (a special warning on the drug package) to the labeling for the 2 drugs, and that current indications for which these drugs have been granted approval might be subject to change. This action was based on a recommendation from the FDA Pediatric Advisory Committee because of concerns of potential safety risks (especially skin cancer and lymphoma) in pediatric patients affected with atopic dermatitis (AD) and receiving therapy with a TIM. We feel deeply troubled by the FDA’s actions, because there is no evidence that topical use of pimecroliums and tacrolimus is harmful. The American Academy of Dermatology, the American Academy of Allergy, Asthma and Immunology, the Society for Pediatric Dermatology, The British Association of Der- GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005 LETTERS TO THE EDITOR matologists, the European Society for Pediatric Dermatology, the German Society of Dermatology, the Austrian Society of Dermatology, the European Dermatology Forum, the European Academy of Dermatology and Venereology, and other scientific societies as well as patient associations (National Society for Atopic Eczema) have unanimously expressed similar concern for the FDA’s actions. Over the last decade, both agents have been extensively studied in clinical trials and their efficacy and safety in the treatment of AD have been demonstrated.1-5 In particular, pimecrolimus has been investigated in more than 19 000 patients (of which 2 600 were infants and 7 300 were children). In addition, more than 5 million patients, over half of them children, have been treated with pimecrolimus since its approval, and post marketing surveillance studies (PMS) show that there is no clinical evidence suggesting an increased risk of malignancies in patients treated with pimecrolimus. In particular: a) the incidence of malignancies in patients treated with pimecrolimus in the course of clinical studies was lower than that in patients in the control group treated with placebo or topical corticosteroids (2 cases out of 19 000 versus 5 cases out of 4 000); b) PMS data from clinical use in over 5 million patients treated with pimecrolimus, as of March 2005, show very few cases of malignancies. The number is well below the expected background incidence in the population treated and, in addition, is lower than the rate expected in the general population. Moreover, no causal relationship with pimecrolimus was established for any of the cases reported; c) those lymphomas identified by spontaneous adverse event reporting systems do not have the clinical presentation and histology that characterize lymphomas occurring in the setting of immunosuppressive therapy. Systemic absorption of both drugs is very limited,6 and even though in some patients blood concentrations have been detected, the values are usually very low and insufficient to cause the sustained systemic immunosuppression that would be responsible for lymphomas. This is true even in young children with moderate-to-severe dermatitis affecting a large body surface area. There is no evidence of photocarcinogenic or mutagenic potential in animals treated with pimecrolimus. Lymphomas that have the characteristics of diseases related to systemic immunosuppression have only been observed in animals exposed to high systemic levels of calcineurin inhibitors. These animals experienced prolonged systemic exposure that is much greater than that achieved with topical application in humans. There is no increased incidence of systemic or cutaneous infections in patients treated with topical pimecrolimus. There is no evidence of systemic immunosuppression due to topical pimecrolimus as showed by antibody response to vaccination and by tests on delayed-type hypersensitivity.7 The European Medicines Agency (EMEA) did not take any immediate action but decided to start a referral process. This Vol. 140 - N. 4 is a process consisting of a review of the full body of the clinical data that will allow a full evidence-based evaluation of the risk /benefit profile of these drugs.8 The EMEA feels that such a comprehensive review of the clinical data is the most effective way to achieve an objective assessment and provide guidance for patients and physicians regarding the use of TIMs. In the meantime, drug labels remain unchanged. AD is a chronic, recurring and frustrating condition. Many patients suffer from AD on the face and sensitive skin sites where long-term application of topical corticosteroids is not indicated. Patients need alternatives to topical steroids due to side effects. The health and safety of our patients are of paramount importance to physicians. We are concerned that the aforementioned warnings confuse and unnecessarily worry our patients and their families as well as health care providers. It is the responsibility of health authorities to present a balanced and fair review of the evidence. Current labeling sufficiently describes the appropriate use and safety of these medications. We strongly believe that the recent recommendations of the Pediatric Advisory Committee and the FDA Health Alert are not justified on the basis of scientific evidence and should be revised. In order to provide further evidence confirming the safety of pimecrolimus, long-term clinical studies have been started, with a registry including 4 000 pediatric patients who will be followed for a period of 10 years. References 1. Ashcroft DM, Dimmock P, Garside R, Stein K, Williams HC. Efficacy and tolerability of topical pimecrolimus and tacrolimus in the treatment of atopic dermatitis: meta-analysis of randomised controlled trials. Br Med J 2005;330:516-24. 2. Meurer M, Fartasch M, Albrecht G, Vogt T, Worm M, Ruzicka T et al. Long-term efficacy and safety of pimecrolimus cream 1% in adults with moderate atopic dermatitis. Dermatology 2004;208:365-72. 3. Kempers S, Boguniewicz M, Carter E, Jarratt M, Parisier D, Stewart D et al. A randomized investigator-blinded study comparing pimecrolimus cream 1% with tacrolimus ointment 0.03% in the treatment of pediatric patients with moderate atopic dermatitis. J Am Acad Dermatol 2004;51:515-25. 4. Papp KA, Werfel T, Folster-Holst R, Ortonne JP, Potter PC, de Prost Y et al. Long-term control of atopic dermatitis with pimecrolimus cream 1% in infants and young children: a two-year study. J Am Acad Dermatol 2005;52:240-6. 5. Luger TA, Lahfa M, Folster-Holst R, Gulliver WP, Allen R, Molloy S et al. Long-term safety and tolerability of pimecrolimus cream 1% and topical corticosteroids in adults with moderate to severe atopic dermatitis. J Dermatol Treat 2004;15:169-78. 6. Billich A, Aschauer H, Aszòdi A, Stuetz A. Percutaneous absorption of drugs used in atopic eczema: pimecrolimus permeates less through skin than corticosteroids and tacrolimus. Int J Pharmacol 2004;269: 29-35. 7. Papp KA, Breuer K, Meurer M, Ortonne JP, Potter PC, de Prost Y et al. Long-term treatment of atopic dermatitis with pimecrolimus cream 1% in infants does not interfere with the development of protective antibodies after vaccination. J Am Acad Dermatol 2005;52:247-53. 8. European Medicines Agency: Committee for Medical Products for Human Use. Press release. April 18-21, 2005. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA 457 LETTERS TO THE EDITOR Il profilo di sicurezza del pimecrolimus topico nella terapia della dermatite atopica Egregio Direttore, L a Food & Drug Administration (FDA) americana ha recentemente richiamato l’attenzione sul profilo di sicurezza degli inibitori della calcineurina a uso topico, meglio noti come immunomodulatori topici (o topical immunomodulators, TIM): pimecrolimus e tacrolimus. In questo avviso, inoltre, la FDA informa la classe medica e il pubblico della propria intenzione di apporre una speciale avvertenza («black-box» warning) sulla confezione dei farmaci riguardo il potenziale rischio di sviluppo di neoplasie (specialmente linfomi e tumori cutanei) nei pazienti pediatrici affetti da dermatite atopica (DA) sottoposti a terapia con TIM, e l’eventualità di apportare una modifica alle attuali indicazioni per cui tali farmaci sono registrati. Questa eventuale azione dell’FDA ci preoccupa, in quanto non esiste alcuna evidenza che l’uso topico di questi farmaci sia pericoloso. Preoccupazione riguardo a quest’azione dell’FDA è stata manifestata dalla American Academy of Dermatology, American Academy of Allergy, Asthma and Immunology, Society for Pediatric Dermatology, British Association of Dermatologists, European Society for Pediatric Dermatology, German Society of Dermatology, Austrian Society of Dermatology, European Dermatology Forum, European Academy of Dermatology and Venereology, e altre società scientifiche, nonché da associazioni di pazienti (National Society for Atopic Eczema). Durante gli ultimi 10 anni, entrambi i farmaci sono stati studiati in maniera approfondita in numerosi trials clinici, e la loro efficacia e sicurezza nella terapia della DA è stata ampiamente dimostrata 1-5. In particolare, gli studi clinici con pimecrolimus hanno coinvolto più di 19 000 pazienti (di cui 7 600 bambini e 2 600 con meno di 2 anni di età). Inoltre, più di 5 milioni di pazienti, più della metà dei quali bambini, sono stati trattati con pimecrolimus dopo la sua approvazione, e studi di farmacovigilanza postmarketing hanno dimostrato che non esiste alcuna evidenza clinica che suggerisca un rischio aumentato di tumori nei pazienti trattati con pimecrolimus. In particolare si è evidenziato che: a) l’incidenza di neoplasie nei pazienti trattati con pimecrolimus nel corso degli studi clinici è inferiore a quella dei pazienti del gruppo di controllo trattati con placebo o corticosteroidi ( 2 casi su 19 000 contro 5 casi su 4 000); b) i dati di sorveglianza postmarketing, aggiornati a Marzo 2005, riportano un numero esiguo di neoplasie, inferiore a quello atteso nella popolazione generale. Inoltre, in nessuno dei casi riportati 458 è stata stabilita una relazione causale con pimecrolimus; c) i rari casi di linfoma osservati presentano un pattern istologico differente da quello che tipicamente caratterizza i linfomi insorgenti nei pazienti sottoposti a terapie immunosoppressive. L’assorbimento sistemico di entrambi i farmaci è assai limitato 6, e anche in quei rari casi in cui le concentrazioni ematiche dei farmaci siano rilevabili, sono molto basse e assolutamente insufficienti a causare un’immunosoppressione protratta. Questo si verifica anche nei bambini con dermatite moderata/severa estesa a vaste aree corporee. Non esiste alcuna evidenza di un potenziale fotocarcinogenico o mutagenico del pimecrolimus negli animali. I linfomi che tipicamente si associano alla immunosoppressione sono stati descritti solo in animali esposti per tempi prolungati a elevati livelli sistemici di inibitori della calcineurina, livelli decine di volte più alti di quelli che sono mai stati misurati nell’uomo dopo applicazione topica. Non esiste alcuna evidenza che i pazienti trattati con pimecrolimus abbiano un’incidenza più alta di infezioni sistemiche o cutanee, e non c’è alcuna evidenza che terapie anche prolungate con pimecrolimus causino immunosoppressione sistemica, come dimostrato dalle risposte anticorpali alle vaccinazioni anti-infettive o dalle riposte ai test che misurano l’ipersensibilità ritardata 7. L’agenzia europea per i farmaci (European Medicines Agency, EMEA) non ha preso alcun provvedimento immediato, ma ha deciso di aprire una procedura di «referral» che consiste in una revisione dettagliata dei dati clinici che consentirà una valutazione accurata, basata su prove scientifiche convincenti, del rapporto rischio/beneficio di questi farmaci 8. L’EMEA ritiene che tale procedura rappresenti il mezzo migliore e più efficace per consentire una valutazione obiettiva in grado di fornire a medici e pazienti le regole per un corretto utilizzo di questi farmaci. Nel frattempo le indicazioni resteranno invariate. La DA è una patologia cronica, ricorrente e a suo modo frustrante. In molti pazienti la malattia colpisce il volto, il collo e altre sedi «sensibili» dove l’applicazione prolungata di corticosteroidi non è appropriata. Questi pazienti necessitano, quindi, di alternative agli steroidi topici. Noi siamo preoccupati che l’allarme menzionato sia causa di confusione e di preoccupazione impropria nei pazienti, nelle loro famiglie e nel personale sanitario. Dal momento che è responsabilità delle autorità sanitarie presentare le evidenze in maniera giusta e corretta, riteniamo che le informazioni attualmente presenti nei farmaci descrivono in maniera appropriata le loro modalità d’impiego e il profilo di sicurezza. Pertanto, reputiamo che le recenti raccomandazioni dell’FDA non siano giustificate dall’evidenza scientifica e dovrebbero essere riconsiderate. Al fine di fornire ulteriore conferma della sicurezza di pimecrolimus, sono stati iniziati studi a lungo termine e un registro che include 4 000 pazienti pediatrici che saranno seguiti per un periodo di 10 anni. GIORNALE ITALIANO DI DERMATOLOGIA E VENEREOLOGIA Agosto 2005