Clinicopathologic Factors Identify Sporadic Mismatch Repair Defective Colon Cancers

Anatomic Pathology / MORPHOLOGY IN MMR-DEFECTIVE COLON CANCER Clinicopathologic Factors Identify Sporadic Mismatch Repair Defective Colon Cancers Britta Halvarsson, MD, PhD, 1 Harald Anderson, PhD, 2 Katarina Domanska, 3 Gudrun Lindmark, MD, PhD, 4 and Mef Nilbert, MD, PhD 3,5 Key Words: Mismatch repair; Microsatellite instability; Tumor-infiltrating lymphocyte; Morphology; Histopathology Abstract Identification of sporadic mismatch repair (MMR)- defective colon cancers is increasingly demanded for decisions on adjuvant therapies. We evaluated clinicopathologic factors for the identification of these prognostically favorable tumors. Histopathologic features in 238 consecutive colon cancers were linked to MMR status based on immunostaining and BRAF mutation status. MMR defects were identified in 22.7% of the tumors, with 46 classified as sporadic. When the clinical parameters of age, sex, and proximal tumor location were combined with the morphologic features with the highest relative risks (RRs), eg, mucinous differentiation (RR, 9.0), tumor-infiltrating lymphocytes (RR, 7.5), absence of necrosis (RR, 7.5), and expanding growth pattern (RR, 5.0) into a 7-factor index, the presence of at least 4 features identified the MMRdefective tumors with 92.3% sensitivity and 75.3% specificity and excluded 61.5% of the tumors from MMR testing. This clinicopathologic index thus successfully selects MMR-defective colon cancers. Chromosomal instability (CIN) and microsatellite instability (MSI) constitute the predominant tumorigenic pathways in colon cancer. 1 CIN is associated with high mutation rates in genes tightly linked to the development of colorectal cancer, eg, APC, K-ras, and TP53, and current estimates indicate that some 70 genetic alterations lead to the development of CIN tumors. 2 The MSI pathway is linked to germline mismatchrepair (MMR) gene mutations within the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome, which accounts for 2% to 3% of colorectal cancer. 3,4 However, defective MMR caused by hypermethylation of the MLH1 promoter characterizes 15% to 20% of sporadic tumors, particularly within the proximal colon. 5,6 The MMR-defective tumors can be identified with high sensitivity and specificity through immunohistochemical loss of MMR protein expression, and the presence of BRAF mutations may help distinguish hereditary from sporadic tumors with loss of MLH1/PMS2 staining. 7-9 Identification of HNPCC-associated colon cancer is important to prevent additional cancer cases in family members through surveillance programs for high-risk people. 10 Identification of sporadic MMR-defective tumors is central to improved prognostication because this subset of tumors has been suggested to have a favorable prognosis and questionable benefit from adjuvant 5-fluorouracil based chemotherapy. 11-15 Several histopathologic features, including proximal tumor location, expanding growth pattern, mucinous histologic features, poor differentiation, lack of dirty necrosis, and lymphocytic reactions, ie, peritumoral lymphocytes, Crohnlike reactions, and tumor-infiltrating lymphocytes (TILs), have been linked to MMR-defective tumors. However, there are no established guidelines for the identification of these tumors. 16-21 Routine inclusion of MSI analysis and/or MMR 238 Am J Clin Pathol 2008;129:238-244 Downloaded 238 from https://academic.oup.com/ajcp/article-abstract/129/2/238/1760174

Anatomic Pathology / ORIGINAL ARTICLE protein immunostaining is, therefore, performed by many laboratories, whereas others have not taken such action for reasons of cost and partly contradictory results regarding the prognostic impact of MSI. To assess whether clinicopathologic criteria can be used to select sporadic tumors for evaluation of MMR status, we characterized the morphologic features in a consecutive, unselected series of 238 colon cancers with correlations to immunohistochemical MMR protein expression with the aim of assessing the predictive strength of combined clinical and morphologic features. Materials and Methods Cases The study was approved by the Lund University Ethics Committee, Lund, Sweden, and comprised all colon cancers diagnosed from January 1999 through December 2001 at the regional Helsingborg Hospital, Helsingborg, Sweden. The 238 tumors developed in 118 women and 109 men with a mean age of 71 years (37-92 years). Synchronous colon cancers were diagnosed in 10 patients (1 of whom had 3 tumors), and 5 patients had previously been diagnosed with colorectal cancers. Tumor location was the proximal colon in 137 tumors, the distal colon in 99 tumors, and inadequately classified in 2 cases. Rectal cancers were excluded from the study. Tumor stage was pt1 in 2.9%, pt2 in 8.0%, pt3 in 73.9%, and pt4 in 15.1% of the tumors. Tumor differentiation was moderate in 177 (74.4%) and poor (including 2 undifferentiated tumors) in 61 (25.6%) tumors, with mucinous differentiation identified in 22 tumors and signet-ring cell cancer in 1. Histopathologic Evaluation All H&E-stained routine slides were morphologically reevaluated by one investigator (B.H.) according to a standardized protocol using previously described definitions and cutoff levels Table 1. 16,18-20,22-26 The clinical information available included that provided by the surgeon referring the specimen, but clinical files or family history of cancer were not obtained. However, information about other neoplasms operated on at the same hospital was available in the Department of Pathology, Helsingborg Hospital. Tumor stage was determined according to American Joint Committee on Cancer and International Union Against Cancer criteria and grade according to the World Health Organization classification. 22 Tumors with a mucinous or signet-ring cell component identified in more than 50% of the tumor area were classified as mucinous/signet-ring cell cancers and were considered poorly differentiated. Tumors with a trabecular, solid, or medullary growth pattern or a mucinous or signet-ring cell component encompassing 10% to 50% of the tumor area were classified as heterogeneous. 18,27 The tumor growth pattern was classified as expanding or infiltrative. 24 Dirty necrosis, defined as the presence of cell detritus and inflammatory cells within glandular lumina, was scored as present or absent. 19 Lymphocytic reactions were primarily determined in infiltrating tumor components and hot-spot areas. A Crohn-like infiltrate was defined as 3 or more nodular lymphoid aggregates deep to the advancing tumor margin within a single 4 field. 18,26 Peritumoral lymphocytes were considered to be present when a cap of chronic inflammatory cells was seen in the deep invasive tumor border. 18 The presence of TILs was defined as 7 or more TILs per 10 high-power fields ( 40) on H&E-stained slides (not including intramucosal carcinomas and early invasive tumor components). 16,20 Residual adenomas were classified as tubular, tubulovillous, villous, or serrated. 22 Immunostaining and BRAF Mutation Analysis Immunohistochemical analysis was used to assess MMR defects. Fresh, 4-µm sections were immunostained using antibodies against MLH1 (clone G168-15, dilution 1:200; BD PharMingen, San Diego, CA), PMS2 (clone A16-4, dilution 1:500; BD PharMingen), MSH2 (clone FE-11, dilution 1:100; Oncogene Sciences, San Diego, CA), and for MSH6 (clone 44, dilution 1:1,000; BD Transduction Laboratories, Lexington, Table 1 Morphologic Criteria Applied in the Evaluation of Sporadic Mismatch Repair Defective Colon Cancers Factor Definition Reference Tumor localization Proximal or distal to the splenic flexure Jass et al 23 Growth pattern Pushing with even, rounded infiltration; infiltrating when Jass et al 24 invading foci identified Differentiation Well, moderate, poor, or undifferentiated Hamilton and Aaltonen 22 Mucinous differentiation Mucin in >50% of the tumor area Hamilton and Aaltonen 22 Heterogeneous differentiation Poor; mucinous and/or signet-ring cell components in <50% of the tumor area Alexander et al 25 Necrosis Dirty necrosis within glandular lumina Greenson et al 19 Crohn-like reaction 3 nodular or Crohn-like lymphoid aggregates per 10 high-power fields Young et al 18 and Graham et al 26 Peritumoral lymphocytes A cap or mantle of lymphocytes at the infiltrating tumor border Young et al 18 Tumor-infiltrating lymphocytes 7/10 high-power fields Smyrk et al 16 and Jass 20 Downloaded from https://academic.oup.com/ajcp/article-abstract/129/2/238/1760174 Am J Clin Pathol 2008;129:238-244 239 239 239

Halvarsson et al / MORPHOLOGY IN MMR-DEFECTIVE COLON CANCER KY). The EnVision detection kit (DakoCytomation, Glostrup, Denmark) was used, and the staining procedure has previously been described. 28 Immunohistochemical MMR protein expression was classified as retained when nuclear staining in the tumor cells was identified and as lost when the tumor cells showed loss of staining with retained staining in stromal, epithelial, inflammatory, or infiltrating lymphoid cells. All immunostainings were evaluated without knowledge of the results of morphologic review, and consensus was reached (without discrepant findings) by 2 investigators (B.H. and M.N.). Tumors that showed loss of MLH1/PMS2 expression were sequenced for BRAF exons 11 and 15 (for primer sequences see Davies et al 29 ) to gain support for whether the underlying cause was a germline HNPCC-associated mutation or somatic hypermethylation of the MLH1 promoter. DNA was extracted from 1-µm cores from the paraffin-embedded tumor blocks by using a protocol available at http://cc.ucsf.edu/people/waldman/protocols/paraffin.html, and sequencing was performed on an ABI Prism 3100 (Applied Biosystems, Foster City, CA). Statistical Methods Associations between defective MMR and various clinical and histopathologic factors were analyzed by means of contingency tables and the Fisher exact test. The risk of an MMR defect was calculated conditionally on each factor value, and risk ratios (RRs), including approximate 95% confidence intervals, were determined. 30 An index for the identification of sporadic MMR-defective tumors was applied, and its sensitivity and specificity is illustrated by means of a receiver operating characteristic curve. Results Overall, MMR defects, defined as loss of at least 1 of the MMR proteins, were identified in 54 (22.7%) of 238 colon cancers. These tumors were diagnosed in patients at mean age of 72 years (range, 47-87 years) and 46 (85%) of 54 were localized in the proximal colon. Among these 54 MMR-defective tumors, 7 colon cancers from 5 patients were likely to be caused by HNPCC based on loss of MSH2 and/or MSH6 staining, and 1 tumor with loss of MLH1/PMS2 staining was classified as uncertain based on a heterogeneous staining pattern. These 8 colon cancers were excluded from further analysis because other means (eg, the Bethesda guidelines) of identifying HNPCC-associated tumors have been defined and the study was aimed at identification of sporadic (non-hnpcc) MMR-defective tumors. The study was thus based on the remaining 230 colon cancers, 46 of which were considered sporadic MMR-defective tumors. The BRAF V600E mutation was identified in 34 (74%) of these, which further supports sporadic tumor development. These colon cancers developed in 115 women and 106 men, 8 of whom had synchronous colon cancers. Tumor location was within the proximal colon in 57.0%. Morphologic review Table 2 and Image 1 showed poor differentiation in 24.8%, an expanding growth pattern in 24.0%, and necrosis in 47.8%. Mucinous/signet-ring cell differentiation was identified in 10.0% of the tumors, but tumor components (10%-50% of the tumor area) with, eg, mucinous or signet-ring cell differentiation were observed in 44.3% of the tumors. Lymphocytic infiltrates appeared as Crohn-like reactions in 44.7%, peritumoral lymphocytes in 14.7%, and TILs in 16.6%. Residual adenomatous components were identified in 67 cancers, 11 of which showed serration, 22 were tubular, 31 showed villous differentiation, and 3 had tubulovillous and serrated components. The 46 sporadic MMR-defective tumors developed in patients at a mean age of 74 years (range, 60-87 years) and 35 (76%) of these tumors occurred in women. Of these tumors, 8 were synchronous MMR-defective colon cancers with concordant loss of MLH1/PMS2 and the presence of the BRAF V600E mutation in all 4 patients. Features associated with MMR-defective tumors were, as expected, overrepresented among the 46 sporadic MMR-defective colon cancers; 40 (87%) were located within the proximal colon, 27 (61%) of 44 showed an expanding growth pattern, 25 (54%) were poorly differentiated, 39 (85%) had any degree of mucinous differentiation, and 41 (89%) lacked dirty necrosis. Lymphocytic reactions were common with, eg, TILs present in 59% of the tumors. The highest RR for MMR tumor development related to mucinous differentiation in more than 10% of the tumor (RR, 9.0), TILs (RR, 7.5), lack of dirty necrosis (RR, 7.5), and an expanding growth pattern (RR, 5.0) (Table 2 and Image 1). These morphologic factors were, together with female sex, age 60 years or older, and a proximal tumor location, combined to an index equal to the number of risk factors, and the predictive power of the index to identify sporadic MMRdefective tumors is illustrated as a receiver operating characteristic curve Figure 1. The presence of at least 3 of these factors allows identification of the MMR-defective tumors with 97.7% sensitivity and 48.3% specificity and the corresponding figures for tumors with at least 4 factors were 93.2% and 75.3%, respectively Table 3. If the index was applied with a 4-factor cutoff, 61.5% of the colon cancers could be excluded from MMR analysis. Discussion Recognition of a colon cancer phenotype linked to MMR defects is beneficial for the identification of hereditary cases and for prognostic estimates in sporadic colon cancers. 240 Am J Clin Pathol 2008;129:238-244 Downloaded 240 from https://academic.oup.com/ajcp/article-abstract/129/2/238/1760174

Anatomic Pathology / ORIGINAL ARTICLE Table 2 Tumor Morphologic Features in Relation to MMR Status in Sporadic MMR-Defective Colon Cancers * Factor Whole Material Fraction MMR Defective P Risk Ratio (95% CI) Tumor location (n = 228) Proximal 131 (57.5) 40/131 (30.5) <.001 4.9 (2.2-11.2) Distal 97 (42.5) 6/97 (6.2) Growth pattern (n = 221) Expanding 53 (24.0) 27/53 (50.9) <.001 5.0 (3.0-8.5) Infiltrating 168 (76.0) 17/168 (10.1) Differentiation (n = 230) Moderate 173 (75.2) 21/173 (12.1) Poor 57 (24.8) 25/57 (43.9) <.001 3.6 (2.2-5.9) Mucinous adenocarcinoma 23 (10.0) 11/23 (47.8) Any mucinous differentiation 88 (38.3) 39/88 (44.3) <.001 9.0 (4.2-19.2) No mucinous differentiation 142 (61.7) 7/142 (4.9) Necrosis (n = 230) Absent 120 (52.2) 41/120 (34.2) <.001 7.5 (3.1-18.3) Present 110 (47.8) 5/110 (4.5) Crohn-like reaction (n = 226) Present 101 (44.7) 29/101 (28.7).002 2.4 (1.4-4.2) Absent 125 (55.3) 15/125 (12.0) Peritumoral lymphocytes (n = 225) Present 33 (14.7) 12/33 (36.4).02 2.1 (1.2-3.5) Absent 192 (85.3) 34/192 (17.7) Tumor-infiltrating lymphocytes (n = 223) Present 37 (16.6) 27/37 (73.0) <.001 7.5 (4.7-12.2) Absent 186 (83.4) 18/186 (9.7) CI, confidence interval; MMR, mismatch repair. * Data are given as number (percentage) or number of MMR defective/total (percentage). The number possible to evaluate varied between 44 and 46. Fisher exact test. Includes 2 undifferentiated tumors. Includes 1 signet-ring cell cancer. Sensitivity 1.00 0.75 0.50 0.25 0.00 0.00 0.25 0.50 1 Specificity 0.75 1.00 Figure 1 Receiver operating characteristic (ROC) curve demonstrating sensitivity and specificity for predicting sporadic mismatch repair using a 7-factor index with age ( 60 years), female sex, proximal tumor location, and the morphologic variables tumor-infiltrating lymphocytes, lack of dirty necrosis, any mucinous differentiation (>10% mucinous areas), and expanding growth. Area under the ROC curve = 0.92. HNPCC-associated tumors were excluded from this study because Bethesda guidelines have been developed for the identification of this subset and because the clinical and morphologic features differ between sporadic and HNPCC-associated MMR-defective tumors. 18 Within our series, 36 tumors fulfilled the Bethesda guidelines, and all but 2 of 7 likely Table 3 MMR Defects in Relation to Number of Factors * in 218 Sporadic MMR-Defective Colon Cancers No. MMR Deficient/ Sensitivity Specificity No. of Factors Proficient (%) (%) 0 0/3 100 0 1 0/29 100 1.7 2 1/52 100 18.4 3 2/47 97.7 48.3 4 8/25 93.2 75.3 5 5/13 75.0 89.7 6 16/4 63.4 97.1 7 12/1 27.3 99.4 MMR, mismatch repair. * The factors were female sex, age 60 years, proximal location, expanding growth, lack of necrosis, mucinous differentiation, and tumor-infiltrating lymphocytes. HNPCC-associated tumors would have been identified using these guidelines (data not shown). The main aim of our study, however, was to evaluate the predictive strength of tumor morphologic features for the identification of sporadic MMRdefective colon cancers. Epigenetic alterations in several cancer-associated genes, including MLH1, are age-dependent, and the sporadic MMR-defective colon cancers predominantly develop in females and at older age, which is in agreement with 70% of the tumors occurring in females and at a mean age of 74 years. 17,18,31 MMR-defective tumors show a predilection for Downloaded from https://academic.oup.com/ajcp/article-abstract/129/2/238/1760174 Am J Clin Pathol 2008;129:238-244 241 241 241

Halvarsson et al / MORPHOLOGY IN MMR-DEFECTIVE COLON CANCER A B C D Image 1 Tumor morphologic features that best determined mismatch repair (MMR) status in colon cancer. A, Tumor-infiltrating lymphocytes (H&E, 40). B, Necrosis (a lack associated with MMR defects) (H&E, 4). C, Expanding growth pattern (H&E, 2). D, Mucinous differentiation (H&E, 2). the proximal colon, with 75% to 85% of the MMR-defective tumors being located here, which is in line with our finding of 87% of MMR-defective tumors being proximal.17,18 Concordant MMR status has been demonstrated in synchronous tumors from the same person, suggesting an individual predilection for tumors developing along a certain pathway. However, MMR-defective synchronous tumors do not necessarily signify HNPCC but may indicate other patientspecific factors such as exposure or yet unidentified genetic causes.23 MSI is identified in 1 of 3 of synchronous, as well as metachronous colon cancers, and, among these, 80% to 90% show concordant MSI status in the tumors.32 Differences apply, however, between these groups; hypermethylation of 242 Am J Clin Pathol 2008;129:238-244 Downloaded 242 from https://academic.oup.com/ajcp/article-abstract/129/2/238/1760174 the MLH1 promoter is identified in about 80% of synchronous tumors but in only about 40% of metachronous tumors.32,33 These results are supported also by the observation that 9 of 10 patients with synchronous tumors showed concordant MMR status. Several morphologic features have been associated with the development of sporadic MMR-defective tumors, including expanding growth, poor and mucinous differentiation, and lymphocytic infiltration. The identification of an expanding growth pattern varies between 15% and 93%, and in our series it was 61% (27/44).17,19,25,34 The presence of an expanding growth pattern was associated with a relative risk of 5.0 for an MMR-defective tumor. Poor and mucinous differentiation

Anatomic Pathology / ORIGINAL ARTICLE has, in most studies, been identified in one third to one half of the MSI tumors. 17,19,35,36 Mucinous tumors represent 10% to 15% of colon cancer, and between one third and one half of these are MMR-defective. Mucinous tumor components (although not reaching the amount requested for a diagnosis of a mucinous cancer) are frequent in MMR-defective tumors. 19,23,27,35 In our series, 54% of the MMR-defective tumors were poorly differentiated, and 85% showed any degree of mucinous differentiation; these factors were associated with RRs of 3.6 and 9.0 for MMR-defective tumors, respectively. Lymphocytic tumor infiltrates have been associated with MMR-defective tumors and include Crohn-like lymphoid reactions in 34% to 69% of the tumors, peritumoral lymphocytes in 30% to 64%, and TILs in 28% to 90% of the tumors. 17,19,21,23,25 We also identified correlations between these features and MMR-defective tumors (Table 2). In accordance with the findings in previous studies, 17,19,25,27 TILs showed a particularly high RR (7.5). Combinations of histopathologic features have been reported to improve the identification of MMR-defective tumors, and assessment of TILs in combination with mucinous areas has been reported to identify MMR-defective tumors with 70% to 80% sensitivity and 80% to 95% specificity. 25 When combining TILs with any amount of mucinous differentiation and absence of dirty necrosis, the sensitivity increased to about 100%, and it has been suggested that about half of the colon cancers thereby can be excluded from analysis of MMR status. 19,20,25 We combined age of 60 years or older, female sex, and a proximal tumor location with the features with the highest RRs, ie, mucinous differentiation, TILs, lack of necrosis, and an expanding growth pattern into an index aimed at identifying MMR-defective tumors (Figure 1). We thus demonstrated that sporadic MMR-defective tumors may successfully be identified and, at the same time, a substantial fraction of the tumors can be excluded from testing; the presence of 3 or more factors identified sporadic MMRdefective tumors with 97.7% sensitivity, excluding 39.0% of the tumors from MMR assessment, and if 4 or more factors were required, 93.2% sensitivity was reached, and 61.5% of the tumors could be removed from MMR testing. Our study demonstrated strong associations between several morphologic features and MMR-defective tumors and defined an index based on the clinical factors of sex, age, and tumor location and the morphologic factors of mucinous differentiation, an expanding growth pattern, TILs, and lack of dirty necrosis that successfully identifies MMR-defective tumors. We suggest that such an approach may be used to select tumors that should undergo evaluations of MMR status and should be further evaluated in independent materials. From the Departments of 1 Pathology and Cytology and 4 Surgery, Helsingborg Hospital, Helsingborg, Sweden; 2 Cancer Epidemiology and 3 Oncology, Lund University Hospital, Lund, Sweden; and 5 Clinical Sciences, Copenhagen University and Clinical Research Centre, Hvidovre University Hospital, Copenhagen, Denmark. Financial support was granted from the Swedish Cancer Society, the Swedish Research Council, the Nilsson Cancer Fund, the Zoega Fund, and the Region Skåne Research Funds, Sweden. Address reprint requests to Dr Halvarsson; Dept of Pathology, Helsingborg Hospital, 251 87 Helsingborg, Sweden. Acknowledgments: We acknowledge Eva Rambech and Anna Karlsson for technical help and excellent immunostaining. References 1. Lengauer C, Kinzler KW, Vogelstein B. Genetic instabilities in human cancers. Nature. 1998;396:643-649. 2. Sjoblom T, Jones S, Wood LD, et al. The consensus coding sequences of human breast and colorectal cancers. Science. 2006;314:268-274. 3. 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