Colorectal tumors: The histology report
Transcript
Colorectal tumors: The histology report
Digestive and Liver Disease 43S (2011) S344–S355 Colorectal tumors: The histology report Giovanni Lanza a, *, Luca Messerini b , Roberta Gafà c , Mauro Risio d On behalf of the “Gruppo Italiano Patologi Apparato Digerente (GIPAD)” and of the “Società Italiana di Anatomia Patologica e Citopatologia Diagnostica”/International Academy of Pathology, Italian division (SIAPEC/IAP) a Department of Experimental and Diagnostic Medicine, Section of Anatomic Pathology, University of Ferrara, Italy of Medical and Surgical Critical Care, Section of Pathological Anatomy, University of Florence, Italy c Department of Imaging Diagnostics and Laboratory Medicine, Anatomic Pathology, Azienda Ospedaliero-Universitaria of Ferrara, Italy d Unit of Pathology, Institute for Cancer Research and Treatment, Candiolo-Torino, Italy b Department Abstract Epithelial colorectal tumors are common pathologic entities. Their histology report should be comprehensive of a series of pathologic parameters essential for the correct clinical management of the patients. Diagnostic histologic criteria of adenomatous, serrated, inflammatory, and hamartomatous polyps and of polyposis syndromes are discussed. In addition, the pathologic features of early and advanced colorectal cancer are described and a checklist is given. Finally, molecular prognostic and predictive factors currently employed in the treatment of colorectal cancer are discussed. © 2011 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. Keywords: Adenocarcinoma; Adenoma; Colorectal; GIPAD report; KRAS; Microsatellite instability; Polyposis syndromes; Serrated polyps 1. Introduction 2. Polypoid and nonpolypoid colorectal adenomas [1–3] Epithelial tumors of the colon and rectum are frequent pathologic entities and deserve to be histologically reported with accuracy and completeness. Colorectal cancer is one of the commonest malignant tumors worldwide and represents in Italy the second cause of cancer-related death. Epithelial polyps of the large intestine are often sampled or removed endoscopically. Adenomas are the precursors of most colorectal cancers, whereas about 15% of colorectal carcinomas develop through an alternative morphogenetic pathway from serrated polyps. Polypoid adenoma is a circumscribed lesion projecting into the bowel lumen composed of mucosal neoplasia, encompassing categories 3–4 of the revised Vienna classification (i.e.: low and high grade dysplasia/intraepithelial neoplasia, non invasive carcinoma/carcinoma in situ, intramucosal carcinoma) [2] (Table 1). Polypoid adenoma consists of the peduncolated and sessile (broad base) morphology, whereas nonpolypoid adenomas include flat and depressed adenomas, that are histologically defined as those in which the thickness of the lesion is less than twice that of the height of the adjacent normal colonic mucosa, or even thinner than the surrounding mucosa. The histological identification of nonpolypoid lesions needs the complete endoscopical removal and the well-oriented paraffin embedding of tissue samples. The endoscopy-based Paris classification compares the height of the lesion to that of closed cups of a biopsy forceps (2.5 mm) and allows to Correspondence to: Prof. Giovanni Lanza, Department of Experimental and Diagnostic Medicine, Section of Anatomic Pathology, University of Ferrara, Via Fossato di Mortara 64/b, 44100 Ferrara, Italy. 1590-8658/$ – see front matter © 2011 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. G. Lanza et al. / Digestive and Liver Disease 43S (2011) S344–S355 S345 Table 1 Grading of superficial colorectal neoplasia WHO 1989 WHO 2000 Revised Vienna Classification Category 1: Negative for neoplasia Category 2: Indefinite for neoplasia Low grade dysplasia (mild and moderate dysplasia) Low grade intraepithelial neoplasia (mild and moderate dysplasia) Category 3: Mucosal low grade neoplasia Low grade adenoma Low grade dysplasia High grade dysplasia (severe dysplasia) High grade intraepithelial neoplasia (severe dysplasia, adenocarcinoma in situ) Carcinoma in situ (carcinoma in situ, intramucosal carcinoma) Intramucosal neoplasia (intramucosal adenocarcinoma) Category 4: Mucosal high grade neoplasia 4.1. High grade adenoma/dysplasia 4.2. Non-invasive carcinoma (carcinoma in situ) 4.3. Suspicious for invasive carcinoma 4.4. Intramucosal carcinoma Invasive carcinoma (spreading into the submucosa) Invasive carcinoma (invasion into the submucosa) distinguish sessile polypoid lesions (Type 0-Is, protruding above 2.5 mm) from the nonpolypoid ones (Type 0-IIa protruding below 2.5 mm), the distinction being crucial given the highly significative differences in cancerization rates between nonpolypoid and sessile polypoid (adenomatous and serrated) lesions [4]. The histological report for nonpolypoid lesions should therefore confirm the adenomatous nature of the lesion, determine the degree of neoplasia, exclude invasion and, when possible, state the likelihood of consistency with the endoscopical typing according to the Paris classification. Category 5: Submucosal invasion by carcinoma 2.4. Villous histology [Diagnostic strength: Level 3] Villous structures form at least 80% of villous adenomas, and more than 20% of tubulovillous adenomas. Villi can assume different morphologies, all of them equally contributing to villous histology of an adenoma: classical villi (long, slender, finger-like processes), palmate villi (leaf-like, broad, branched processes), foreshortened villi (isolated, slender outgrowths). The presence of at least one identifiable villus in polyp biopsies or fragments deserves to be reported as “predominantly tubular histology”. 2.1. Gross specimens and handling 2.5. Grade of neoplasia [Diagnostic strength: Level 3] Size should be carefully measured identifying the maximum diameter of the adenomatous component, length and diameter of the stalk, if present. The resection margin should be identified, described, and sliced in a way that allows complete assessment. Polyps need to be sliced and totally embedded. At least three levels should be cut and H&E stained through each block. 2.2. Pathology report Since size, villous histology, and grade of neoplasia represent the most important determinants for the malignant progression of colorectal adenomas, as well as for the risk of development of synchronous and/or metachronous large bowel neoplasia, histological report should focus on all of them. 2.3. Size [Diagnostic strength: Level 2] The size of adenomas influences surveillance intervals, and an accurate measurement by the pathologist to the nearest millimeter is required. Grossly, measurement of the size can be done from the formalin-fixed specimen, excluding the stalk, if present. Microscopy allows the precise measurement of the adenomatous sectors of polyps, excluding non neoplastic tissues. When the two measurements significantly differ, microscopic size should be made explicit in the report and auditable. Severity of both architectural (crypt branching and budding, complex glandular crowding, intraluminal papillary tufting) and cytological features (loss of cell polarity and differentiation, nuclear stratification, enlarged and rounded nuclei with nucleoli) distinguish mucosal low grade neoplasia (Category 3, equating low grade dysplasia) from mucosal high grade neoplasia (Category 4.1, equating high grade dysplasia in older systems) (Table 1). Only one or two glands with high grade dysplasia and identifiable at low power examination are needed to classify an adenoma as high grade. Trauma, erosion, prolapse and bleeding, mainly occurring in the superficial portions of polyps, elicit reparative changes in the adenomatous epithelium (loss of polarity and nuclear stratification), mimicking high grade neoplasia, but these are not to be overinterpreted as such. Cribriform or solid pattern of epithelial growth consistent with carcinoma in situ and invasion by the neoplastic epithelium into the lamina propria or muscularis mucosae consistent with intramucosal carcinoma are included (categories 4.2 and 4.4, respectively) within the spectrum of mucosal high grade neoplasia and as such reported. S346 G. Lanza et al. / Digestive and Liver Disease 43S (2011) S344–S355 3. Cancerized adenoma, malignant polyp (pT1 colorectal cancer) [3] Neoplastic invasion into the submucosa through the muscularis mucosae (Category 5 in accordance with the revised Vienna Classification) is diagnostic of cancerized adenoma [Diagnostic strength: Level 3]. Colonic intramucosal carcinoma behaves like a benign adenoma and for this reason polyps harbouring “in situ” or “intramucosal” cancer (Categories 4.2 and 4.4, respectively) are not generally regarded as “malignant” polyps and classified as high grade dysplasia, high grade intraepithelial neoplasia or mucosal high grade neoplasia (Table 1). Cancerized adenomas represent the earliest form of clinically relevant colon cancer in most patients. Invasion of the submucosa, in fact, opens the way to metastasis via the haematogenous and lymphatic routes, and the choice between surveillance and major surgery when an endoscopically removed polyp is found to be an ACIC will hinge on its metastatic potential, taking into account that post operative mortality (within 30 days) ranges from 0.6 to 4.4% in T1 cancers. At present, histopathologic parameters alone determine whether a high (35%) or low (7%) risk of nodal metastases exists [5–8]. Histologic risk factors – namely positive resection margin, poor differentiation of cancer, vascular invasion and tumor budding – do not only predict lymph node disease rate but also distant metastasis and mortality rates. 3.3. Margin involvement [Diagnostic strength: Level 3] A negative deep (basal) margin is reported in the absence of cancer within the diathermy and one high-power field from diathermy, or more than 1 mm from the actual margin of resection The presence of adenomatous tissue in the lateral mucosal resection margin should also be independently reported as an indication for further endoscopic treatment. 3.4. Tumor budding [Diagnostic strength: Level 2] An isolated single cancer cell and a cluster composed of fewer than 5 cancer cells are defined as “budding” foci. Although several studies have shown that tumor budding is independently associated with lymph node and distant metastasis as well as shorter disease free and overall survival in colorectal cancer, there is no consensus with regard to the assessment methods and cut-off values. Due to the usually small dimension of the submucosal invasive front in cancerized adenomas, tumor budding should be assessed in the area where the highest activity is identified on H&E, reserving immunohistochemistry for the better assessment of this feature. Awaiting the results from reproducibility studies, a quantitative, two-grade system (low vs. high grade) is advisable, counting tumor buds (< or ≥10) within a 0.385 mm2 area using a ×25 lens. 3.1. Carcinoma grading [Diagnostic strength: Level 3] 3.5. Microstaging [Diagnostic strength: Level 2] Histologic grade should be in accordance with the system used in advanced colorectal cancer and distinguish well/ moderately (low grade) from poorly differentiated (high grade) tumors. It is based on the worst area, independently from its extension. An anaplastic component (even in the form of small, single or scattered foci) should be identified, as its occurrence strongly correlates with the risk of lymph node metastases. Signet ring cell carcinoma should be graded as poorly differentiated. Tumor budding at the front of invasion should not influence carcinoma grading and should be scored separately (see below). The level of submucosal invasion, assessed as Haggitt levels I–IV in peduncolated polyps and as sm1–3 Kikuchi levels in sessile polyps, is important in predicting the outcome of cancerized adenomas. On the other hand, cancerized adenomas with slight submucosal invasion to the depth of 200–300 micrometers are very unlikely to have metastatic lymph nodes and the quantitative measurements of both depth (< or >2000 micrometers) and width (< or >4000 micrometers) of the cancerous submucosal invasion are strongly indicative of the nodal involvement and worthy reporting. In this setting the rough estimate of the ratio of adenomatous to carcinomatous tissue is meaningful, indicating the prevalence of the potentially metastatic cancerous tissue within the polyp, the risk of lymph node metastasis being likely high (even in the absence of other risk parameters) in pT1 polypoid adenocarcinoma, consisting entirely of cancer without adenoma. 3.2. Vascular invasion [Diagnostic strength: Level 2] Lymphatic invasion requires the presence of cancer cells within endothelial-lined channels or spaces, distinguishing such features from retraction artefacts. Venous invasion is defined as tumor emboli within endothelial-lined channels surrounded by a smooth muscle wall. Uncertainty in the assessment (presence vs absence) of neoplastic vascular invasion, even after the examination of serial and deeper routinely H&E stained sections, should be reported. Evidence is lacking for the additional use of immunohistochemistry in detecting vascular invasion. 4. Serrated polyps [3,9,10] Epithelial serration, namely the saw-toothed outline derived from infolded epithelial tufts in the crypt and in the luminal surface can be found in several histotypes of colorectal polyps. Transient serration associated with hyperproliferation can be seen in inflammatory polyps, above all of myoglandular type. Steady serration is displayed in the common hyperplastic polyps of the large bowel and, coupled with focal or diffuse epithelial neoplastic changes, in serrated G. Lanza et al. / Digestive and Liver Disease 43S (2011) S344–S355 adenomas and mixed polyps. Furthermore, cell maturation and differentiation are altered in serrated epithelium, particularly in the mucus-producing process, so that abnormal goblet cell, microvesicular, and mucus-poor cell components can be found allowing the identification of different cytotypes of serrated polyps: these, however, seem unrelated to different risks of tumor progression. S347 4.4. Mixed polyps [Diagnostic strength: Level 2] In these polyps one or more serrated components (hyperplastic, SSA, TSA) are associated and/or intermingled with classical adenomatous tissue (tubular, tubulovillous and villous). Extension and grading of neoplastic sectors should be exhaustively described in the diagnosis, in order to support clinical decision making. 4.1. Hyperplastic (metaplastic) polyps [Diagnostic strength: Level 3] 5. Inflammatory polyps [13] Epithelial serration together with minimal architectural changes, and without cytological atypia are the diagnostic features of hyperplastic polyps of the large bowel. They are small-sized (0.2–0.5 cm) mucosal bumps of the sigmoid colon and rectum, consisting of straight, parallel, slightly elongated crypts lined by serrated epithelium in the intermediate and upper third, and by undifferentiated cells in the lower third. The nuclei are round or oval and located at the base with little or no stratification. 4.2. Sessile serrated adenomas (SSA) /sessile serrated polyps [Diagnostic strength: Level 2] SSA are mainly right-sited, large-sized (>1 cm) sessile serrated lesions displaying patchy or diffuse distorsions of tissue organization: branching of crypts, serration of foveolar cell phenotype at the base of the crypt, dilatation at the base of the crypt, horizontal crypt growth [11]. Most findings are only suggestive for SSA, and are localized in the deeper sectors of the mucosa and require well-oriented samples for identification. Subtle nuclear changes, when present, are focal and include small prominent nucleoli, open chromatin, and irregular contours. Frankly dysplastic nuclear abnormalities, indicative of the initial transition toward traditional serrated adenomas should be reported, in terms of extension and neoplasia grade, in order to manage patient surveillance. Intermingling and/or intermediate features of SSA and hyperplastic polyps can often be seen in the same histolgical section, impairing diagnostic reproducibility. The term “sessile serrated polyp” has therefore been suggested for equivocal lesions and even as a synonym of SSA, considering the improper use of “adenoma” for lesions lacking true neoplastic changes. Inflammatory polyps are defined as mucosal elevations resulting from an inflammatory process. They may occur in patients with inflammatory bowel disease (IBD), as well as in patients with other forms of colitis, such as infectious colitis, ischemic colitis, or diverticulitis. They may be also induced by a mucosal trauma due to an inappropriate peristalsis and in such cases are defined as mucosal prolapse-related inflammatory polyps. Different entities such as inflammatory cap polyp, colitis cistica profunda and inflammatory myoglandular polyp are considered to belong to the mucosal prolapse syndrome. Inflammatory polyps do not show an increased risk of neoplastic transformation. Inflammatory polyps may be sessile or pedunculated, and their gross appearance varies from rounded lesions to fingerlike projections (filiform polyps, usually associated with IBD). They may be single or multiple and their size generally ranges from few mm to 2 cm; sometimes, they may be extremely large (“giant” inflammatory polyps) and can cause intestinal obstruction. 5.1. Histological features [Diagnostic strength: Level 3] The lamina propria shows an intense inflammatory cell infiltrate and contains dilated and branched colonic crypts; neutrophilic cryptitis and crypt abscess may be prominent. The colonic surface epithelium and the colonic crypts may show both epithelial damage and regeneration. Intense vascular congestion and hemorrhage may also be present. Mucosal prolapse-related inflammatory polyps show additional histological features such as thickening and vertical extension of the muscularis mucosae toward the lamina propria. 5.2. Differential diagnosis [Diagnostic strength: Level 2] 4.3. Traditional serrated adenomas (TSA) [Diagnostic strength: Level 2] Nuclear (elongated, hyperchromatic, stratified nuclei) and cytological frankly dysplastic features are seen within the serrated epithelium, besides architectural changes and are diagnostic for TSA [12]. Rapid and protuberant growth parallels nuclear dysplasia and, grossly, TSA can be indistinguishable from villous or tubulovillous colorectal adenomas. The differential diagnosis between reactive epithelial changes and epithelial dysplasia or neoplasia is the main potential pitfall that can occur in evaluating inflammatory polyps. The regenerating epithelium usually shows maturation toward the surface while dysplastic or neoplastic epithelium do not show surface maturation. Moreover, the presence of an inflammatory background should be carefully considered to avoid an overdiagnosis of dysplasia or neoplasia, particularly in patients with IBD. Inflammatory polyps may contain bizarre stromal cells, that may be mistaken for a sarcoma (“pseudosarcomatous” S348 G. Lanza et al. / Digestive and Liver Disease 43S (2011) S344–S355 change). The location of these cells underneath areas of ulceration, the presence of an inflammatory infiltrate together with the absence of atypical mitoses can help in the differential diagnosis. Inflammatory polyps may also mimic juvenile polyps, since the histological features of both are very similar; the clinical data can help in the distinction between these two types of polyps. 6. Hamartomatous polyps and polyposes Hamartomatous polyps are characterized by a disorganized overgrowth of tissues native to the anatomic area in which they arise. They may occur as solitary polyps or in the context of polyposis syndromes, and are defined by their histology. 6.1. Solitary juvenile polyp (JP) Most JPs are located in the distal large intestine and their size is generally less than 1–2 cm; their surface is smooth and lobulated and they are usually pedunculated. Rectal bleeding is the most frequent symptom; sometimes autoamputation of the polyp can occur. Solitary JPs have no increased risk of malignancy. JP is composed of an expanded lamina propria with irregular glands that are often cistically dilated. The lamina propria contains a variable amount of inflammatory cells but there is no proliferation of smooth muscle. JP is the most frequent hamartomatous colorectal polyp, and is especially common before 10 years of age; however, it may occur also in adults. 6.2. Juvenile polyposis [14] The polyps are histologically similar to solitary JPs. Juvenile polyposis should be suspected when three or more JPs are found; however the diagnostic criteria for Juvenile polyposis are the following: five or more juvenile polyps of the colorectum and/or juvenile polyps throughout the gastrointestinal tract and/or any number of juvenile polyps with a family history of Juvenile polyposis. There are three types of Juvenile polyposis: (1) Juvenile polyposis of infancy, that is usually fatal from non-neoplastic conditions; (2) Juvenile polyposis coli, that shows an increased risk of colorectal cancer; (3) Generalized Juvenile polyposis, which shows an increased risk of development of gastrointestinal carcinomas. Most patients with Juvenile polyposis show mutations of the SMAD4/DPC4 gene. Polyps closely resembling juvenile polyps are found in other conditions such as Cowden disease, Bannayan-Riley-Ruvalcaba syndrome and Cronkhite-Canada syndrome. The first two syndromes are related to inherited mutation of the PTEN gene, whereas Cronkhite-Canada syndrome is a non-hereditary gastrointestinal polyposis of unknown etiology. Patients with these syndromes show gastrointestinal multiple polyps, but they may also have extragastrointestinal clinical manifestations as well as an increased risk of malignancy. 6.3. Peutz-Jeghers syndrome [15] Peutz-Jeghers polyps mainly occur in the small bowel, and less frequently in the large bowel and the stomach. They are variable in size, being sessile or pedunculated. They are generally multiple, but solitary Peutz-Jeghers type polyps have been described. Mucocutaneous hyperpigmentation around the mouth, eyes, perianal area, and on the fingers often precede gastrointestinal manifestations. The diagnosis of Peutz-Jeghers syndrome is based upon clinical findings and the histological appearance of the polyps [Diagnostic strength: Level 3]. The incidence of gastrointestinal cancer as well as extra-gastrointestinal neoplasms in Peutz-Jeghers syndrome is greater than in the normal population [16]. Mutations of the LKB1/STK11 gene have been found in nearly 60% of affected patients. The main histological feature is the presence of arborizing bands of smooth muscle, derived from muscularis mucosae, that are covered by normal or hyperplastic glandular epithelium. Epithelial misplacement into the intestinal wall may be present and may mimic invasive adenocarcinoma. It can be recognized by normal differentiation of the epithelium, lack of stromal reaction and haemosiderin deposition. 7. Adenomatous polyposis [17] Familial adenomatous polyposis (FAP) and its variant syndromes (Gardner syndrome and Turcot syndrome) are different phenotypic manifestations caused by germline mutations of the adenomatous polyposis coli gene. FAP is characterized by hundreds of adenomatous polyps in the large bowel, that generally begin to emerge in the second decade of life. If adenomas are not removed, colorectal cancer is inevitable, since the penetrance of the disease is very high, reaching nearly 100%. [Diagnostic strength: Level 3] Extracolonic manifestations associated with FAP are: upper gastrointestinal adenomas and adenocarcinomas, adenocarcinoma of the ampulla of Vater, gastric fundic glands polyposis, desmoid tumors, central nervous system tumors, cancer of the thyroid, hepatoblastoma, hypertrophy of the retinal pigment epithelium, osteomas, epidermoid cysts, and dental abnormalities. Duodenal cancer and desmoid tumors are the main causes of death in FAP patients treated with prophylactic colectomy. Attenuated familial adenomatous polyposis (AFAP) [18] is characterized by multiple adenomas of the colon. In this syndrome the number of adenomas is lower than in classical FAP and it ranges from 2 to 100, averaging around 20–30 adenomas, with prominent variations among family members. [Diagnostic strength: Level 3] AFAP is associated with germline mutations of the APC gene in only a small proportion of cases (approximately 10%). Furthermore, adenomas are frequently flat and often localized in the proximal colon. Extra-colonic manifestations may or not be present, but upper gastrointestinal lesions are generally G. Lanza et al. / Digestive and Liver Disease 43S (2011) S344–S355 present. AFAP patients have a high risk of colorectal cancer. These patients develop colorectal cancer 10 years earlier than individuals with sporadic colorectal cancer, but 15–20 years later than patients with classical FAP. MUTYH-associated adenomatous polyposis (MAP) [19]. Multiple adenomas can also occur in a subset of APC mutationnegative patients. These patients harbor germline mutations in the repair gene MUTYH (MYH). The number of adenomas ranges from 15 to more than 100. These patients present a very high risk of developing colorectal cancer. Furthermore, the incidence of extra-colonic tumors, especially duodenal cancer, is higher than that of the general population. [Diagnostic strength: Level 3] 8. Hyperplastic polyposis (HP) [20] The following criteria have been proposed to diagnose HP: 1) at least five histologically diagnosed hyperplastic polyps proximal to the sigmoid colon of which two are greater than 10 mm in diameter, or 2) any number of hyperplastic polyps occurring proximal to the sigmoid colon in an individual who has a first degree relative with HP, or 3) more than 30 hyperplastic polyps of any size, but distributed throughout the colon. [Diagnostic strength: Level 2] Individuals with HP develop colorectal cancer on a background of multiple polyps that are mainly hyperplastic polyps. However, SSAs, TSAs and mixed polyps can also be found in these patients; therefore it the suggestion is to consider all types of serrated polyps in diagnosing HP. 9. Surgically resected colorectal cancer Careful pathologic reporting of colorectal cancer resection specimens is of paramount importance to determine the prognosis and to plan the treatment of individual patients. In addition, pathologists have an increasing role in the identification of hereditary tumors and in prediction of tumor response to specific type of therapy. There is clear evidence that the use of checklists improves the standard of colorectal cancer reporting and therefore their use is strongly recommended (Table 2). Pathologic parameters that need to be absolutely reported on the basis of their prognostic value or that are required for therapy will be discussed. In addition, molecular prognostic and predictive factors recently introduced in the clinical setting will be examined. 9.1. Histologic type [1,21] [Diagnostic strength: Level 2] Histologic type should be evaluated according to the WHO classification as follows [1]. • Adenocarcinoma • Mucinous adenocarcinoma • Signet-ring cell carcinoma • Small cell carcinoma • S349 Squamous cell carcinoma Adenosquamous carcinoma Medullary carcinoma • Undifferentiated carcinoma Most colorectal carcinomas (nearly 85%) are usual adenocarcinomas, and 10 to 15% are classified as mucinous adenocarcinomas (mucinous component >50%). The other tumor types are rare. Tumor typing does not have major prognostic relevance, but signet-ring cell carcinoma and small cell carcinoma have been demonstrated to have adverse prognostic significance independent of stage. Notwithstanding, the various subtypes of colorectal cancer are characterized by different genetic alterations and probably more detailed prognostic information will be obtained in the future by an integrated histologic and molecular classification [22]. With respect to usual adenocarcinomas, mucinous tumors have a greater tendency to develop peritoneal metastases, to invade adjacent organs and to show extensive lymph node involvement [21]. Mucinous carcinomas are evenly distributed in the right and left colon and more frequently than common adenocarcinomas harbour defects in the DNA mismatch repair (MMR) system [23]. Mucinous adenocarcinomas differ from common adenocarcinomas also on the basis of other molecular features, such as higher frequency of CIMP phenotype and rare p53 mutations, suggesting that these tumors really represent a distinct pathologic entity [22]. A less favourable clinical outcome for patients with mucinous cancers has been demonstrated in many studies but not in others and the prognostic significance of the mucinous phenotype remains at present undetermined. In addition, the prognosis of mucinous carcinomas with microsatellite instability (MSI) has been reported to be better than that of microsatellite stable mucinous carcinomas. Signet-ring cell carcinoma is composed of at least 50% signet-ring cells. It represents less than 1% of all colorectal carcinomas and occurs more frequently in individuals younger than 50 years of age and in patients with ulcerative colitis. Histologically, colorectal signet-ring cell carcinomas differ from gastric ones for the common presence of abundant extracellular mucin and infrequent diffuse tissue infiltration. Signet-ring cell carcinomas are often at an advanced stage at diagnosis and are associated with a worse outcome than common adenocarcinomas. Peritoneal carcinomatosis develops in the majority of patients who die of the disease. About 30% of signet-ring cell carcinomas display defect of MMR function; however, MSI status does not appear to be a significant predictor of survival in this tumor type. Small cell carcinomas are histologically identical to those arising in the lungs. They often appear to develop within an adenoma and usually express neuroendocrine markers. Part of the cases show a distinct adenocarcinomatous component. Most patients have distant metastases at diagnosis and the prognosis is very poor. Large cell poorly differentiated neuroendocrine carcinomas may also occur in the colorectum and their clinical behaviour is similar to that of small cell carcinomas. Medullary carcinomas are generally described as tumors • • S350 G. Lanza et al. / Digestive and Liver Disease 43S (2011) S344–S355 Table 2 Surgically resected specimens of colorectal cancer – Checklist Table 2 (continued) Extramural venous invasion Not identified Present Tumor site Cecum Ascending colon Hepatic flexure Transverse colon Splenic flexure Descending colon Sigmoid colon Rectosigmoid junction Rectum Tumor size Maximum tumor diameter: cm Histologic type Adenocarcinoma Mucinous adenocarcinoma Signet-ring cell carcinoma Small cell carcinoma Squamous cell carcinoma Adenosquamous carcinoma Medullary carcinoma Undifferentiated carcinoma Other (specify): Grade of differentiation Low grade (well or moderately differentiated) High grade (poorly differentiated or undifferentiated) High grade component (%): Depth of tumor invasion No evidence of primary tumor Tumor invades submucosa (pT1) Tumor invades muscularis propria (pT2) Tumor invades through the muscularis propria into the subserosal adipose tissue or the nonperitonealized pericolic or perirectal soft tissues (pT3) Tumor penetrates to the surface of the visceral peritoneum (serosa) (pT4a) Tumor directly invades other organs or structures ) (pT4b) (specify: Tumor penetrates to the surface of the visceral peritoneum (serosa) and directly invades other organs or structures ) (pT4b) (specify: Margins of resection Proximal/distal margin Cannot be assessed Invasive carcinoma present Invasive carcinoma absent mm Distance of invasive carcinoma from closest margin: Circumferential (radial) margin Not applicable Cannot be assessed Invasive carcinoma present Invasive carcinoma absent Distance of invasive carcinoma from non-peritonealised margin: Regional lymph nodes Number examined: Number involved: Tumor deposits Not identified Present (number: ) Response to neoadjuvant therapy Not applicable (no prior treatment) Complete regression Minimal residual tumor No marked regression Pathologic staging (pTNM) TNM descriptors (required only if applicable) m (multiple primary tumors) r (recurrent) y (posttreatment) Primary tumor (pT) pTX: Cannot be assessed pT0: No evidence of primary tumor pTis: Carcinoma in situ, intraepithelial or invasion of lamina propria pT1: Tumor invades submucosa pT2: Tumor invades muscularis propria pT3: Tumor invades through the muscularis propria into pericolorectal tissues pT4a: Tumor penetrates the visceral peritoneum pT4b: Tumor directly invades other organs or structures Regional lymph nodes (pN) pNX: Cannot be assessed pN0: No regional lymph node metastasis pN1a: Metastasis in 1 regional lymph node pN1b: Metastasis in 2 to 3 regional lymph nodes pN1c: Tumor deposit(s) in the subserosa, or nonperitonealized pericolic or perirectal tissues without regional lymph node metastasis pN2a: Metastasis in 4 to 6 regional lymph nodes pN2b: Metastasis in 7 or more regional lymph nodes Distant metastasis (pM) Not applicable pM1: Distant metastasis Specify site(s): pM1a: Metastasis to single organ or site (e.g., liver, lung, ovary, nonregional lymph node) pM1b: Metastasis to more than one organ/site or to the peritoneum Additional pathologic findings None identified Diverticular disease, ulcerative colitis, Crohn’s disease, familial adenomatous polyposis, other forms of polyposis, synchronous carcinoma(s) (complete a separate form for each cancer), etc. Specify: Polyps present (specify type and number): Comment(s): mm characterized by solid growth pattern and marked intratumoral and peritumoral lymphocytic infiltration, and composed by cells with vesicular nuclei, prominent nucleoli, and abundant eosinophilic cytoplasm [1,24]. Tumor cells form sheets or may have an organoid or trabecular architecture; focal mucin production or glandular differentiation are often present. In our experience, medullary carcinomas often display a clearly evident glandular and/or mucinous component and are generally formed by quite uniform cells with mild or moderate nuclear atypia and variable amount of cytoplasm [25]. Moreover, intense intratumoral and peritumoral lymphocytic infiltration is not always present. At the molecular level, the great majority of medullary carcinomas show MSI, which probably explains their better prognosis with respect to common poorly differentiated adenocarcinomas [25]. Undifferentiated carcinomas are tumors lacking morphological evidence of differentiation beyond that of an epithelial tumor [1]. Some authors include in this category tumors G. Lanza et al. / Digestive and Liver Disease 43S (2011) S344–S355 showing a minimal component of gland formation (<5%). Undifferentiated carcinomas may form sheets of cells, cords, or trabecular structures. Grade of anaplasia is variable, some tumors displaying marked nuclear atypia and pleomorphism, and others having relatively uniform and less atypical cytologic features. The clinical outcome of undifferentiated carcinomas is quite unpredictable and seems to be mainly related to the MMR status of the tumor. Other subtypes of large bowel carcinoma (such as sarcomatoid carcinoma, choriocarcinoma, and clear cell carcinoma) are extremely rare. Furthermore, in the recent years new subtypes of large bowel adenocarcinoma – named serrated, micropapillary, and villous adenocarcinoma – have been described, but their clinico-pathologic and molecular features need to be more precisely defined. 9.2. Grade of differentiation [1,24,26] [Diagnostic strength: Level 2] Diverse grading systems for colorectal cancer are currently employed. We recommend the use of a 2-tiered grading system based on the WHO classification: • Low grade: well differentiated and moderately differentiated • High grade: poorly differentiated and undifferentiated A 2-tiered stratification demonstrated greater reproducibility and prognostic relevance than stratification into 3 or 4 categories. Specifically, high grade tumors have repeatedly been shown to behave more aggressively than low grade carcinomas in multivariate analysis. According to the WHO criteria, grading should be based on the evaluation of the worst area, excluding areas of focal dedifferentiation present at the invasive margin of the tumor. However, the grading systems proposed by the College of American Pathologists [26] and the Royal College of Pathologists [27] are both based on the assessment of the predominant grade of differentiation. We suggest that, using the WHO system, the percentage of high grade tumor area when present should be also reported. 9.3. Depth of tumor invasion [28,29] [Diagnostic strength: Level 3] This is evaluated as the pT component of the TNM classification [28,29]: • Tumor invades submucosa (pT1) • Tumor invades muscularis propria (pT2) • Tumor invades through the muscularis propria into pericolorectal tissues (pT3) • Tumor penetrates to the surface of the visceral peritoneum (pT4a) • Tumor directly invades other organs or structures (pT4b) The TNM classification includes a pTis “carcinoma in situ” level comprising intraepithelial carcinoma (confined within the glandular basement membrane) and carcinoma showing invasion of the lamina propria (intramucosal). As colorectal neoplasia does not have metastatic potential until it has invaded the submucosa, it is preferable to avoid the term S351 pTis and use the term high grade dysplasia to indicate these lesions. The outer edge of the muscolaris propria represents the line of demarcation between pT2 and pT3. pT3 indicates spread in continuity beyond the bowel wall and does not apply to lymphatic or venous invasion. At variance with the sixth edition, according to the new TNM and AJCC classifications tumor deposits in the mesenteric fat are no longer classified in the pT category as discontinuous extramural extensions. If a tumor spreads to the outer edge of the muscolaris propria but not beyond and no muscle separates cancer cells from the perivisceral tissues, then the tumor should be classified as pT3. Tumors that have penetrated the visceral peritoneum as a result of direct extension through the wall and subserosa are assigned to the pT4 category, as tumors that directly invade other organs or structures, whether or not they penetrate the serosal surface. Identification of visceral peritoneal involvement needs adequate tumor sampling and eventually multiple levels sectioning and microscopic examination. The histologic findings considered to represent carcinomatous serosal involvement are [26,30]. • Tumor present at the serosal surface with inflammatory reaction, mesothelial hyperplasia, and/or erosion or ulceration • Free tumor cells on the serosal surface with underlying ulceration of the visceral peritoneum Previous studies indicated that serosal involvement was associated with worse outcome than invasion of adjacent organs. However, recent data obtained from a very large cohort of colorectal cancer patients indicate that penetration of the visceral peritoneum carries a 10% to 20% better 5-year survival rate than invasion of adjacent organs for each category of N [31,32]. Therefore, designation of the T4 subsets was changed in the seventh edition of the TNM classification. 9.4. Margins of resection [24,26,27] [Diagnostic strength: Level 3] Proximal, distal, circumferential, and mesocolic margins of resection need to be evaluated in colorectal cancer surgical specimens. It is very useful to mark the margin(s) closest to the tumor with ink after careful examination of the serosal surface. Proximal and distal resection margins are rarely involved unless close (<2 cm) to the tumor or the tumor shows histologically poor differentiation or a diffusely infiltrating pattern of growth. Sections to assess proximal and distal margins can be obtained either by longitudinal sections perpendicular to the margin or by en face sections parallel to the margin. The distance from the tumor edge to the distal resection margin is very important for low anterior resection and a clearance of 2 cm (1 cm for T1 and T2 tumors) is considered adequate. The circumferential (radial) margin represents the adventitial soft tissue margin closest to the deepest penetration of tumor and is created surgically. Tumor involvement of S352 G. Lanza et al. / Digestive and Liver Disease 43S (2011) S344–S355 the radial margin is the most critical factor in predicting local recurrence in rectal cancer and is also associated with an increased risk of death from the disease [33]. The circumferential margin is considered positive if the tumor is located 1 mm or less from the nonperitonealized surface. This assessment includes tumor within veins, lymphatics or lymph nodes and tumor deposits, as well as direct tumor extension. However, if circumferential margin involvement is based only on intranodal tumor, this should be stated. In rectal cancer, the distance of the tumor from the radial margin should be always reported. The circumferential margin should be also assessed in colonic segments incompletely encased by peritoneum (e.g., cecum, ascending colon, and descending colon). The clinical relevance of pathologic examination of the surgical mesocolic plane of dissection has been recently recognized [34]. 9.5. Regional lymph nodes [28,29] [Diagnostic strength: Level 3] The number of metastatic lymph nodes and the total number of lymph nodes examined must always be reported. Regional lymph node status should be assessed according to the new TNM classification [28,29], as follows. pN0 No regional lymph node metastasis pN1 Metastasis in 1–3 regional lymph nodes pN1a Metastasis in 1 regional lymph node pN1b Metastasis in 2–3 regional lymph nodes pN1c Tumor deposit(s), i.e., satellites, in the subserosa, or in non-peritonealized pericolic or perirectal soft tissue without regional lymph node metastasis pN2 Metastasis in 4 or more regional lymph nodes pN2a Metastasis in 4–6 regional lymph nodes pN2b Metastasis in 7 or more regional lymph nodes With respect to the previous edition a subdivision of pN1 and pN2 categories has been introduced, reflecting differences in clinical outcome observed within each category on the basis of the number of affected lymph nodes [31,32]. Histological examination of a regional lymphadenectomy specimen ordinarily includes 12 or more lymph nodes. If the lymph nodes are negative, but the number ordinarily examined is not met, the tumor will be classified as pN0. All macroscopically evident lymph nodes in the surgical specimen should be dissected and examined histologically. Many factors influence lymph node recovery and evaluation, such as extent of surgical resection, quality of pathologic examination, patient factors, and tumor characteristics [35,36]. The mean number of nodes detected in a series of dissections is now considered to be indicative of colon cancer quality care [37] and should be comprised between 12 and 15 [24]. As nodal metastases in colorectal cancer are often found in small lymph nodes (<5 mm in diameter), meticulous search is required on gross examination by the pathologist. If less than 12 lymph nodes are retrieved, re-examining the specimen could be useful. Use of visual enhancement techniques and inclusion of pericolic fat are not generally recommended. In rectal cancer specimens from patients treated with neoadjuvant therapy the number of recovered lymph nodes is often lower than 12 despite accurate search. Several studies reported that the total number of lymph nodes evaluated after surgical resection is an important prognostic factor in colorectal cancer. In particular, most studies showed that increased survival of patients with stage II colon cancer is associated with increased number of lymph nodes examined [35]. A similar positive association was also demonstrated among patients with stage III colon cancer [35]. In addition, recent investigations indicated that the ratio of metastatic to examined lymph nodes is related to clinical outcome in patients with colon and rectal cancer [38]. The biologic bases of these findings are not fully understood and factors other than staging accuracy are probably involved. Some studies suggested that identification of micrometastases in lymph nodes of patients with colorectal cancer staged as pT3N0 by conventional pathologic examination could be of prognostic value [39]. However, at present data are insufficient to recommend use of special measures to detect micrometastases or isolated tumor cells in the routine assessment of regional lymph node status [26]. Peritumoral deposits (satellite nodules) are macroscopic or microscopic carcinomatous nests or nodules in the pericolorectal adipose tissue lymph drainage area of a primary carcinoma without histologic evidence of residual lymph node tissue [28,29]. They may represent discontinuous spread of the tumor, venous invasion with extravascular spread or totally replaced lymph nodes. According to the latest edition of the TNM classification, if a nodule is considered by the pathologist to be a totally replaced lymph node (generally having a smooth contour), it should be recorded as a positive lymph node and not as a satellite. In addition, pT1–2 lesions that lack regional lymph node metastasis but have tumor deposit(s) will be classified as pN1c whereas the pT category is unchanged. There is no general agreement on the classification of pericolic tumor nodules [24,40] and probably these new statements of the TNM classification will not be accepted by part of the pathologists [41]. Tumor deposits are associated with unfavourable disease outcome [40] and therefore their presence and number should be separately recorded [29]. [Diagnostic strength: Level 2] 9.6. Response to neoadjuvant therapy [27,42] [Diagnostic strength: Level 2] Preoperative (neoadjuvant) chemo- and radiotherapy for rectal cancer induces several secondary changes including tumor regression and downstaging [42,43]. In the case of rectal cancers treated with neoadjuvant therapy pathologic staging should be performed according to the ypTNM system and based on evaluation of viable cancer cells. Marked tumor regression and especially complete tumor eradication are associated with better clinical outcome. Therefore, specimens from patients treated with neoadjuvant therapy should be carefully examined and extensively sampled in particular to demonstrate complete tumor regression. Several systems G. Lanza et al. / Digestive and Liver Disease 43S (2011) S344–S355 for grading tumor regression have been proposed and there is no general consensus on the histological classification of this parameter. However, 3-tiered grading systems have been generally recommended by National and International Associations [24,26,27,29]. We suggest the use of the grading system proposed by the Royal College of Pathologists, which includes the following categories: • No residual tumor cells and/or mucus lakes only (complete regression) • Minimal residual tumor, i.e. only occasional microscopic tumor foci are identified with difficulty • No marked regression According to the TNM classification, cases with complete regression are recorded as ypT0. S353 10.1. KRAS mutation [48,49] [Diagnostic strength: Level 3] Presence of KRAS gene mutations (codons 12 and 13) is associated with lack of clinical response to therapy with monoclonal antibodies (cetuximab and panitumumab) directed against the epidermal growth factor receptor (EGFR) in metastatic colorectal cancer. Actually, tumors of patients with metastatic disease who are candidates for anti-EGFR antibody therapy have to be tested for KRAS mutations and only patients whose tumors show absence of KRAS mutations at codon 12 or 13 can be treated with anti-EGFR antibodies. However, only a fraction of patients with KRAS wild type carcinomas will respond to anti-EGFR antibody treatment and further predictive molecular markers are actively investigated nowadays [49]. 9.7. Vascular invasion [26,27] [Diagnostic strength: Level 2] Venous invasion has been shown in several studies to be an independent negative prognostic factor in colorectal cancer. In particular, invasion of large extramural veins has been repeatedly associated with increased risk of cancer-related death. The prognostic value of intramural venous invasion and of invasion of lymphatics or thin-walled vessels is less clear. We believe that extramural vein invasion should be always reported, whereas reporting of intramural and thin-walled structures is optional. Elastic fiber stains could help in the recognition of venous invasion, when suspected, on haematoxylin and eosin stained sections; however routine use of elastic stains, as recently suggested, is not at present recommended. 9.8. Additional histologic prognostic factors Several other histopathologic variables, including perineural invasion, pattern of growth, lymphocytic infiltration at the tumor margin, tumor infiltrating lymphocytes (TILs), Crohn-like reaction, and tumor budding have been proposed as prognostic factors in colorectal cancer. These parameters are not commonly employed in the clinical setting and their reporting is optional. Nowadays, tumor budding [44] and grade of intratumoral lymphocytic infiltration [45] represent the most promising prognostic factors to be introduced in the pathologic evaluation of these tumors, provided that their assessment will be standardized and their prognostic value clearly defined. 10. Molecular prognostic and predictive factors [46,47] Much effort has been produced to identify immunohistochemical, biologic and molecular prognostic and predictive factors to be utilized in the management of patients with colorectal cancer [46,47]. Actually, only KRAS mutational status analysis and evaluation of proficiency of the DNA mismatch repair (MMR) system by immunohistochemistry and microsatellite instability (MSI) analysis are employed in clinical practice. 10.2. DNA mismatch repair status [50] [Diagnostic strength: Level 2] About 15% of colorectal cancers are characterized by MSI (or MSI-H, high frequency MSI), which is determined by impaired function of the MMR system. Most MSI-H carcinomas (about 70%) are sporadic and produced by somatic biallelic promoter methylation of the MLH1 gene. It is believed that sporadic MSI colorectal carcinomas develop through the CpG island methylator pathway. The other MSI-H tumors are hereditary and caused by germline mutation of a MMR gene (MLH1, MSH2, and less frequently MSH6 and PMS2) with somatic inactivation of the remaining wild type allele (Lynch syndrome). Genetic or epigenetic inactivation of MMR genes is nearly always associated with immunohistochemical loss of expression of the corresponding protein. Furthermore, as MMR proteins work as heterodimers (MSH2 dimerizes with MSH6, and MLH1 with PMS2) abnormalities of the obligatory partners (MSH2 and MLH1) will result in proteolytic degradation of their dimer and concurrent loss of both the obligatory and secondary partner proteins (MSH2/MSH6 and MLH1/PMS2). Conversely, mutations in genes of the secondary partner proteins (MSH6 and PMS2) will be characterized by selective loss of MSH6 and PMS2 expression respectively, as their function may be compensated by other proteins [51]. Therefore, immunohistochemical pattern of MMR protein expression allows the identification of the gene that is most likely inactivated. Several studies demonstrated an excellent correlation of the results obtained by immunohistochemistry and MSI analysis and both methods can be used confidently for the identification of MMR-deficient colorectal carcinomas. However, a small fraction of hereditary cases having a missense mutation (generally of MLH1) that lead to a nonfunctional protein with intact antigenicity might result in MSI-H with intact expression of MMR proteins. Lynch syndrome accounts for 2–3% of the total burden of large bowel carcinomas. MSI testing and immunohistochemical analysis of MMR proteins expression are worldwide employed for the identification of colorectal cancer patients with presumptive Lynch syndrome, to be tested for MMR genes germline mutations after appropriate genetic coun- S354 G. Lanza et al. / Digestive and Liver Disease 43S (2011) S344–S355 selling. It is recommended that MSI testing should be carried out on tumors from patients clinically at high risk or selected on the basis of the revised Bethesda guidelines [52]. However, molecular screening investigations performed by MSI and/or immunohistochemical analysis on large series of unselected surgically removed colorectal cancers followed by MMR genes mutation analysis have indicated that a large fraction of Lynch syndrome cases would go unrecognized using common criteria of selection, and some Authors suggest screening all colorectal carcinomas for MSI or abnormal MMR protein expression. Recent studies have shown that sporadic MSI-H MLH1negative tumors frequently harbour BRAF V600E gene mutation. Conversely, BRAF mutations have not been detected in MSI-H MLH1-negative tumors from patients with Lynch syndrome. Therefore, BRAF gene mutation analysis could be employed as an aid for discriminating hereditary from sporadic MLH1-negative MMR-deficient carcinomas [53]. MMR status has been demonstrated to be an independent prognostic factor in colorectal cancer. In fact, several studies displayed higher survival rates for patients with stage II and III MSI carcinomas with respect to patients with non-MSI tumors [54,55]. In addition, emerging data suggest that patients with MSI tumors do not have significant benefit from adjuvant 5fluorouracil-based chemotherapy [54,56]. However, the use of MMR status assessment as a prognostic and predictive test has not yet been validated and incorporated into clinical practice. 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