Isolated loss of PMS2 staining is an uncommon immunophenotype

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1 Colorectal Carcinomas With Isolated Loss of PMS2 Staining by Immunohistochemistry Lindsay Alpert, MD; Reetesh K. Pai, MD; Amitabh Srivastava, MD; Wendy McKinnon, MS; Rebecca Wilcox, MD; Rhonda K. Yantiss, MD; Ramir Arcega, MD; Hanlin L. Wang, MD, PhD; Marie E. Robert, MD; Xiuli Liu, MD, PhD; Rish K. Pai, MD, PhD; Lei Zhao, MD, PhD; Maria Westerhoff, MD; Heather Hampel, MS, LGC; Sonia Kupfer, MD; Namrata Setia, MD; Shu-Yuan Xiao, MD; John Hart, MD; Wendy L. Frankel, MD Context. Isolated loss of PMS2 staining is an uncommon immunophenotype in colorectal carcinomas, accounting for approximately 4% of tumors with microsatellite instability. Limited information regarding these tumors is available in the literature. Objective. To compare the clinicopathologic features of colorectal carcinomas with isolated PMS2 loss by immunohistochemistry to those with other forms of mismatch repair deficiency. Design. Ninety-three colorectal carcinomas with isolated PMS2 loss by immunohistochemistry and 193 with other forms of mismatch repair deficiency were identified. Forty (43%) of the isolated PMS2 loss cases and 35 control cases (18%) had a known germline mutation or a clinical diagnosis of Lynch syndrome. Accepted for publication July 17, Published as an Early Online Release January 16, From the Departments of Pathology (Drs Alpert, Setia, Xiao, and Hart) and Medicine (Dr Kupfer), University of Chicago, Chicago, Illinois; the Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (Dr Reetesh Pai); the Department of Pathology, Brigham and Womens Hospital, Boston, Massachusetts (Dr Srivastava); the Departments of Medicine (Ms McKinnon) and Pathology and Laboratory Medicine (Dr Wilcox), University of Vermont Medical Center, Burlington; the Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, New York (Dr Yantiss); the Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles (Drs Arcega and Wang); the Department of Pathology, Yale-New Haven Hospital, New Haven, Connecticut (Dr Robert); the Department of Pathology, Cleveland Clinic, Cleveland, Ohio (Drs Liu and Rish Pai); the Department of Pathology, University of Alabama, Birmingham (Dr Zhao); the Department of Pathology, University of Washington, Seattle (Dr Westerhoff); and the Departments of Internal Medicine (Ms Hampel) and Pathology (Dr Frankel), The Ohio State University Wexner Medical Center, Columbus. Dr Rish Pai is now with the Department of Laboratory Medicine and Pathology, Mayo Clinic, Scottsdale, Arizona. Dr Liu is now with the Department of Pathology, University of Florida, Gainesville. Dr Zhao is now with the Department of Pathology, Brigham and Womens Hospital, Boston, Massachusetts. Dr Westerhoff is now with the Department of Pathology, University of Michigan, Ann Arbor. The authors have no relevant financial interest in the products or companies described in this article. Presented in part at the United States and Canadian Academy of Pathology; March 14, 2016; Seattle, Washington. Reprints: Lindsay Alpert, MD, Department of Pathology, University of Chicago, 5841 S Maryland Ave, MC 6101, Chicago, IL ( lindsay.alpert@uchospitals.edu). Results. Overall, isolated PMS2-loss tumors occurred in significantly younger patients (P,.001) and in fewer female patients (P ¼.006). These tumors were significantly less likely to be right-sided (P ¼.001), high-grade (P ¼.01), or display histologic features of microsatellite instability (P,.001). The isolated PMS2-loss group also exhibited increased odds of disease-specific death (odds ratio [OR], 3.09; 95% CI, ; P ¼.007). When the analysis was restricted to germline mutation/lynch syndrome cases and controls, no significant differences were detected for age, sex, tumor location, tumor grade, histologic features, or distant metastases, although a trend toward increased odds of disease-specific death in the isolated PMS2-loss group was evident (OR, 3.87; 95% CI, ; P ¼.10). Conclusions. Unusual clinicopathologic features observed in colorectal carcinomas with isolated PMS2 loss are likely related to the high proportion of cases caused by germline mutations. Isolated PMS2-loss tumors may demonstrate more aggressive behavior than other tumors with microsatellite instability, but larger studies are needed to investigate that possibility further. (Arch Pathol Lab Med. 2018;142: ; doi: / arpa oa) Isolated loss of PMS2 staining is an uncommon immunophenotype among colorectal carcinomas (CRCs), accounting for approximately 4% of tumors with microsatellite instability (MSI). 1,2 The most common underlying molecular alteration found in such cases is a germline mutation of the PMS2 gene. Lynch syndrome caused by PMS2 mutations is known to have some unusual clinical features 2 5 ; not only do such patients have a lower lifetime risk of CRC (approximately 10% 20% by age 70 years) than do patients with MLH1 or MSH2 mutations (approximately 40% 50% by age 70 years), but the risk of extracolonic tumors is also lower among PMS2 mutation carriers than it is among patients with mutations in othermismatchrepair(mmr)genes. 5 Because of the lower likelihood of tumor development in these patients, clinical detection of families with PMS2 mutations is difficult. 4,6,7 Because of this difficulty, immunohistochemical screening of CRCs has an important role in identifying families with PMS2 mutations. However, isolated loss of PMS2 staining is not entirely specific for the presence of a germline PMS2 mutation; a subset of patients with isolated loss of PMS2 Arch Pathol Lab Med Vol 142, April 2018 Isolated PMS2 Loss in Colorectal Cancer Alpert et al 523

2 Colonic adenocarcinoma exhibiting isolated loss of PMS2 immunohistochemical staining in tumor cell nuclei (a) with intact MLH1 (b), MSH2 (c), and MSH6 (d) staining (original magnification 3200 [a through d]). staining have germline mutations in MLH1 rather than PMS2 because germline point mutation in MLH1 produces a nonfunctional MLH1 protein that fails to bind properly with PMS2. 1,8 To complicate matters further, up to 30% of cases with isolated loss of PMS2 staining lack detectable germline mutations in PMS2 or MLH1, and several studies have confirmed that a subset of these CRCs result from somatic inactivation of the PMS2 gene. 1,4,8 13 Although CRCs with isolated loss of PMS2 staining represent a molecularly heterogeneous group, the underlying molecular alterations resulting in this phenotype are often unknown in day-to-day practice. In addition, limited information regarding the clinical and pathologic features of these tumors is available in the literature. In this multiinstitutional study, we investigated the clinicopathologic features and outcomes of patients with CRCs that showed isolated loss of PMS2 immunostaining and compared them to CRCs with other forms of MMR deficiency and/or MSI. MATERIALS AND METHODS Case Selection The study group consisted of 93 CRC cases with isolated loss of PMS2 immunohistochemical staining, identified between 2009 and 2015, from the pathology archives of 11 institutions. Immunohistochemistry for MMR proteins was performed using a variety of antibodies against MLH1 (including clone G , Cell Marque, Rocklin, California, 1:50 dilution; clone G , BD Biosciences, Franklin Lakes, New Jersey, 1:100 dilution; clone M1, Ventana Medical Systems, Tucson, Arizona, prediluted; and clone ES05, Leica Biosystems, Buffalo Grove, Illinois, 1:500 dilution), MSH2 (including clone G , Cell Marque, 1:500 or 1:200 dilutions; clone G , Ventana, prediluted; and clone FE11, Calbiochem, EMD Millipore, Billerica, Massachusetts, 1:100 or 1:3000 dilutions), MSH6 (including clone EP49, Cell Marque, 1:100 dilution; clone 44, Ventana, prediluted; clone 44, BD Biosciences, 1:800 dilution; clone 44, Biocare Medical, Pacheco, California, 1:50 dilution; and clone EP49, Epitomics/Abcam, Burlingame, California, 1:800 dilution), and PMS2 (including clone EP51, Cell Marque, 1:30 dilution; clone EPR3947, Ventana, prediluted; clone A16-4, BD Biosciences, 1:100 dilution; clone ERP3947, Cell Marque, 1:4 dilution; and clone A16-4, BD Biosciences, 1:300 dilution). Isolated loss of PMS2 staining was defined as no convincing nuclear staining for PMS2 in any tumor cells present (0% staining) with intact nuclear staining for MLH1, MSH2, and MSH6 in at least 5% of the tumor (Figure). The formalin-fixation time for specimens tested by immunohistochemistry was unknown, but intact staining of lymphocytes was used as a positive internal control in every case. Results of polymerase chain reaction testing for MSI, BRAF V600E mutational analysis, and MLH1 promoter hypermethylation testing 524 Arch Pathol Lab Med Vol 142, April 2018 Isolated PMS2 Loss in Colorectal Cancer Alpert et al

3 were recorded when available. The cases were compared with a control group consisting of 193 cases of CRC from 2 institutions (University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, and University of Chicago Medical Center, Chicago, Illinois) with known MMR deficiency/msi and no evidence of isolated PMS2 loss by immunohistochemistry. Clinicopathologic Evaluation Histologic sections were initially evaluated and/or reviewed by gastrointestinal pathologists. Tumors were assessed for histologic features associated with MSI according to the revised Bethesda guidelines (tumor-infiltrating lymphocytes [TILs], Crohn-like lymphocytic reaction, mucinous or signet ring cell component, and medullary growth pattern). 14 Tumors located in the cecum, ascending colon, hepatic flexure, and transverse colon were regarded as right-sided tumors. Tumors were classified histologically as low or high grade based on their degree of differentiation; well or moderately differentiated tumors were classified as low grade, and poorly or undifferentiated tumors were classified as high grade. For cases with multiple synchronous tumors, all tumors with clinicopathologic data available were included in the histologic and tumor location analyses, but only the tumor with the highest T stage was used for the analysis of stage at presentation. Clinical data were collected from the electronic medical records of the participating institutions in accordance with the guidelines of their institutional review boards. Data collected included personal and family cancer history, results of germline mutational analysis, development of recurrent CRC, survival status, and cause of death. Cases and controls were also subclassified into germline/ probable Lynch syndrome or likely sporadic, based on the information collected. Inclusion criteria for the germline/probable Lynch syndrome group were detection of a germline mutation in a relevant MMR gene/epcam or a clinical diagnosis of Lynch syndrome, based on family history when germline mutational analysis results were not available. Inclusion criteria for the likely sporadic group were absence of known germline mutations in relevant MMR genes or EPCAM or detection of a BRAF V600E mutation or somatic MLH1 inactivation (including MLH1 promoter hypermethylation), and no strong clinical suspicion for Lynch syndrome. Statistical Analysis Descriptive statistics for categorical variables were conducted by v 2 statistics; 2-tailed Pearson significance (P) values are reported, except for tables with any cell frequency less than 5, for which Fisher exact statistics are reported. Student t tests were conducted for continuous variables; 2-tailed P values, 95% CI, and P values assuming unequal variances are reported. Linear and logistic regressions were conducted for continuous and categorical outcomes, respectively. RESULTS Follow-up Testing and Case Classification Results of MSI testing by polymerase chain reaction were available for 70 of 93 (75.3%) of the study cases, 65 (92.9%) of which were found to be MSI-H. Data from germline analysis were available in 52 of 93 cases (55.9%), 32 (61.5%) of which were found to have PMS2 germline mutations and 7 (13.5%) had germline MLH1 mutations. Germline analysis revealed no PMS2 alterations in 13 patients (25.0%), including 7 with wild-type MLH1 and 1 patient with a clinical diagnosis of Lynch syndrome based on family history without additional germline testing of MLH1. Of the 7 cases with wild-type germline PMS2 and MLH1, 2 (28.6%) showed MLH1 promoter hypermethylation and 1 (14.3%) showed a somatic MLH1 mutation with loss of heterozygosity. One case with wild-type germline PMS2 and MLH1 was clinically concerning for Lynch syndrome, and the possibility of a cryptic PMS2 mutation was considered, but a firm clinical diagnosis of Lynch syndrome could not be established. Based on these findings, 40 CRCs (43.0%) that showed isolated PMS2 loss were categorized as germline/probable Lynch syndrome, 8 (8.6%) were categorized as likely sporadic, and the remaining 45 cases (48.4%) could not be further categorized based on available information. Of the 193 control cases, 123 (63.7%) were MSI-H by polymerase chain reaction and were found to have a BRAF V600E mutation, whereas the other 70 control cases (36.3%) demonstrated MMR deficiency by immunohistochemistry. Of the 70 controls with MMR deficiency by immunohistochemistry, 39 (55.7%) showed loss of MLH1 and PMS2, 21 (30.0%) showed loss of MSH2 and MSH6, 9 (12.9%) showed isolated loss of MSH6, and 1 case (1.4%) with 2 tumors demonstrated loss of MLH1 and PMS2 in one tumor and isolated loss of MSH6 in the other tumor. Results of germline analysis were available for 33 of the 193 controls (17.1%) and revealed an MMR gene or EPCAM mutation in 32 patients (97.0%). A clinical diagnosis of Lynch syndrome was established in 3 additional cases (1.9%) without germline mutational analysis. One patient (3.0%) underwent germline mutational analysis of all 4 MMR genes and EPCAM, but no mutations were detected. Mutational analysis identified BRAF V600E mutations in 128 of the 193 controls (66.3%), and 4 others (2.1%) showed MLH1 promoter hypermethylation. Based on these results, 35 controls (18.1%) were classified as germline/probable Lynch syndrome, 133 (68.9%) were classified as likely sporadic, and the remaining 25 cases (13.0%) could not be further classified based on available information. Clinicopathologic Features and Outcome The clinicopathologic features of the study cases and controls are summarized in Table 1. Patients in the study group were significantly younger than controls (mean, 59 years versus 69 years; P,.001) and were significantly less likely to be female (46 of 91 [50.5%] versus 130 of 193 [67.4%]; P ¼.006). Tumors in the study group were also significantly less likely to be right-sided (59 of 93 [63.4%] versus 162 of 202 [80.2%]; P ¼.002), high grade (29 of 93 [31.2%] versus 92 of 194 [47.4%]; P ¼.009), or to display histologic features of MSI (40 of 57 [70.2%] versus 165 of 189 [87.3%]; P ¼.002). There were no significant differences between study cases and controls with respect to T stage or lymph node metastases at presentation (Table 1). Distant metastases at presentation were rare in both groups, although they were significantly more common in patients with isolated PMS2 loss in their tumors compared with controls (11 of 87 [12.6%] versus 10 of 192 [5.2%]; P ¼.03). When the outcomes of disease recurrence and diseasespecific death were compared between groups, significantly elevated odds of disease-specific death were detected in the study group compared with controls (16 of 77 [20.8%] versus 13 of 192 [6.8%]; odds ratio [OR], 3.09; 95% CI, ; P ¼.007]. The odds of disease-specific death remained elevated in the study group even after adjusting for tumor stage (OR 2.99; 95% CI, ; P ¼.03). There was no significant difference in the odds of disease recurrence between the study group and controls [9 of 54 [16.7%] versus 6 of 64 [9.4%]; OR, 1.93; 95% CI, ; P ¼.24]. 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4 Characteristics Table 1. Clinicopathologic Features of Study Cases and Controls No. Study Cases Cases, No. (%) No. Controls Cases, No. (%) P Value Age, mean (SD) (15) (16),.001 Sex, Female, No. (%) (50.5) (67.4).006 Right-sided tumor 93 a 59 (63.4) 202 a 162 (80.2).002 High-grade histology 93 a 29 (31.2) 194 a 92 (47.4) MSI histologic feature present 57 a 40 (70.2) 189 a 165 (87.3).002 TILs or Crohn-like lymphocytic reaction present 57 a 31 (54.4) 174 a 138 (79.3),.001 T stage (6.0) (11.6) (12.0) (15.3) (62.7) (57.7) (19.3) (15.3) Lymph node metastases at presentation (33.3) (33.0)..99 Distant metastases at presentation (12.6) (5.2).03 Abbreviations: MSI, microsatellite instability; TIL, tumor-infiltrating lymphocyte. a Includes cases with multiple tumors. Comparison of Germline Mutation/Probable Lynch Syndrome Cases We also compared the clinicopathologic features of the germline/probable Lynch syndrome subsets of the study cases (n ¼ 40) and controls (n ¼ 35). There were no significant differences in mean patient age, percentage of female patients, tumor location, tumor grade, and histologic features of MSI (Table 2). When the analysis was restricted to the germline/probable Lynch syndrome subgroups, there were no differences between study cases and controls with respect to primary tumor (T) stage or regional lymph node metastases (N). Although the percentage of patients with distant metastases at presentation was more than twice as high in the study group as in the controls (3 of 38 [7.9%] versus 1 of 34 [2.9%]), the difference was not statistically significant (P ¼.62). When the outcomes of disease recurrence and diseasespecific death were compared between patients with germline mutation/probable Lynch syndrome in the study cases and controls, the odds of disease-specific death remained elevated but did not reach statistical significance (8 of 40 [20.0%] versus 2 of 33 [6.1%]; OR, 3.87; 95% CI, ; P ¼.10). There was no significant difference in the odds of disease recurrence between the study group and controls (2 of 19 [10.5%] versus 3 of 31 [9.7%]; OR, 1.10; 95% CI, ; P ¼.92). DISCUSSION Approximately 15% of CRCs are MMR-deficient/MSI, and these tumors exhibit distinctive clinicopathologic features, including a tendency toward a right colonic location, highgrade histology, and a relatively good prognosis. 15 However, recent studies have demonstrated that the molecular alterations underlying MMR-deficiency affect the clinicopathologic profile of these tumors. Sporadic CRCs with MSI commonly occur in older women, show a predilection for the right colon, and are often poorly differentiated. Tumors caused by germline mutations in MMR genes (ie, Lynch syndrome) occur in younger patients, affect men and women equally, occur throughout the colon, and are less likely to be poorly differentiated The risk of developing CRC also varies widely among patients with Lynch syndrome depending on the affected gene; CRC is much Table 2. Clinicopathologic Features of Germline Mutation/Probable Lynch Syndrome Cases and Controls Characteristics Study Cases With Germline Mutations/ Probable Lynch Syndrome No. Cases, No. (%) Controls With Germline Mutations/ Probable Lynch Syndrome No. Cases, No. (%) Age, mean (SD) (15) (13).06 Female sex (41.0) (37.1).73 Right-sided tumor 40 a 23 (57.5) 44 a 23 (52.3).63 High grade histology 41 a 10 (24.4) 43 a 15 (34.9).29 1 MSI histologic feature present (70.8) (82.1).30 TIL or Crohn-like lymphocytic reaction present (54.2) (69.2).23 T stage (12.1) 33 6 (18.2) (12.1) 33 5 (15.2) (63.6) (51.5) (12.1) 33 5 (15.2) Lymph node metastases at presentation 33 9 (27.3) (34.4).54 Distant metastases at presentation 38 3 (7.9) 34 1 (2.9).62 Abbreviations: MSI, microsatellite instability; TIL, tumor-infiltrating lymphocyte. a Includes cases with multiple tumors. P Value 526 Arch Pathol Lab Med Vol 142, April 2018 Isolated PMS2 Loss in Colorectal Cancer Alpert et al

5 more common among patients with mutations affecting MLH1 and MSH2, occurring in 40% to 50% of patients by age 70 years, compared with only 10% to 20% of patients with germline MSH6 and PMS2 mutations. 5,19 Such differences in distribution, appearance, and behavior suggest that the clinicopathologic features of mismatch repairdeficient tumors vary depending on the affected gene. Our study explored that possibility by examining a large series of CRCs with isolated loss of PMS2 immunohistochemical staining. Our findings suggest that the clinicopathologic features of CRCs with isolated loss of PMS2 staining are distinct from those of mismatch repair-deficient tumors with other immunohistochemical profiles. Patients with CRCs that show isolated PMS2 loss were significantly younger, more likely to be male, and less likely to have right-sided tumors compared with patients with other staining patterns (59 of 93 [63.4%] versus 162 of 202 [80.2%], P ¼.002). Tumors with isolated PMS2 loss were also less likely than other mismatch-deficient cancers to be high grade (29 of 93 [31.2%] versus 92 of 194 [47.4%], P ¼.009). Although most cases (70.2%; 40 of 57) exhibited at least one histologic feature typical of MSI, the percentage of cases with those features was significantly lower than in tumors with other types of MMR deficiency (165 of 189 [87.3%], P ¼.002). We also identified a tendency toward more aggressive behavior among CRCs with isolated PMS2 loss compared with CRCs with other types of MMR deficiency. Not only were patients with these tumors more likely to have distant metastases at presentation (11 of 87 [12.6%] versus 10 of 192 [5.2%], P ¼.03) but also the odds of disease-specific death were significantly higher (OR, 3.09; 95% CI, ; P ¼.007) and remained so after adjusting for tumor stage (OR, 2.99; 95% CI, ; P ¼.03). The differences between PMS2-deficient CRCs and CRCs with other types of MMR deficiency are likely related to a combination of factors Most CRCs with isolated loss of PMS2 immunohistochemical staining arise from germline alterations in MMR genes, particularly PMS2. 1,9,12 Consistent with that theory, 40 of 48 (83.3%) of the study cases that could be subclassified met the criteria for germline/probable Lynch syndrome, compared with only 35 of 168 (20.8%) of the tumors with other types of MMR deficiency that could be subclassified. Furthermore, when we compared the clinicopathologic features of the germline/probable Lynch syndrome subgroups of isolated PMS2-loss cases and MMR-deficient/MSI controls, the differences in age, sex, tumor location, tumor grade, and presence of MSI histologic features were no longer statistically significant. However, some of the histologic differences between PMS2-loss tumors and other MMR-deficient cancers cannot be explained by the underlying type of MMR alteration. Previous studies comparing MSI histologic features in Lynch syndrome associated and sporadic MMR-deficient CRCs have not shown major differences according to the type of MMR inactivation present. Although a study by Young et al 18 found sporadic MSI cases to be significantly more likely to exhibit a mucinous histology, Shia et al 20 detected a higher prevalence of mucinous features in Lynch syndrome associated tumors; neither group identified significant differences in the frequency of TILs or a Crohn-like lymphocytic reaction. Therefore, it seems unlikely that the significantly lower frequency of MSI histologic features in the isolated PMS2-loss group can be explained by the presence of germline versus sporadic MMR inactivation alone. Although the difference in MSI histologic features no longer reached statistical significance in the subgroup analysis, the percentage of isolated PMS2-loss cases displaying those features was nearly identical in both analyses, and the percentage of MMR-deficient/MSI cases with those features decreased only slightly, which suggests that the smaller number of cases in the subgroup analysis was the major determinant of our ability to detect a significant difference in this parameter. The lower frequency of MSI histologic features in the CRCs with isolated loss of PMS2 staining could be meaningful, both because it indicates that these tumors are less readily detected using histology-based screening methods and because it suggests a possible relationship to our other notable finding regarding these tumors: their tendency toward aggressive behavior. The percentage of cases with distant metastases at presentation was more than twice as high in the isolated PMS2-loss group, even after restriction of the analysis to the germline/probable Lynch syndrome subgroups. Although this difference no longer reached statistical significance in the subgroup analysis, there were too few cases with distant metastases in either subgroup to sufficiently analyze that parameter. Similarly, the odds of disease-specific death were much higher among patients with CRC with isolated loss of PMS2 and remained so in the subgroup analysis. We found that CRCs with isolated loss of PMS2 staining showed a lower frequency of histologic features of MSI and a tendency toward aggressive behavior, which may be related to immune activation. 21,22 Increased levels of immune activation in CRCs with MSI are manifest by increased TILs and a Crohn-like peritumoral lymphocytic infiltrate, presumably because MMR deficiency leads to accumulation of mutations and elaboration of immunestimulating neoantigens. 23 Although results have been somewhat conflicting, several studies have found that having more TILs was associated with an improved prognosis in CRC, possibly because of enhanced tumor recognition and destruction by the host immune system In our study, the frequency of immune activation-related histologic features, that is, increased TILs or Crohn-like lymphocytic infiltrates, was significantly lower in CRCs with isolated loss of PMS2 than in other MMR-deficient tumors (31 of 57 [54.4%] versus 138 of 174 [79.3%]; P,.001). A similar, although nonsignificant, trend was seen in the subgroup analysis of patients with germline/probable Lynch syndrome in the study group and controls (13 of 24 [54.2%] versus 27 of 39 [69.2%], P ¼.23). Given the proposed relationship between immune response and prognosis, the lower level of immune activation in CRCs with isolated loss of PMS2 could contribute to its biologic behavior. The lower frequency of immune activation-related histologic features in CRCs with isolated loss of PMS2 also raises the possibility that these tumors may have a lower underlying mutational burden than other MMR-deficient tumors. Although most of the isolated PMS2 loss cases tested in our study (65 of 70; 92.9%) showed a high frequency of MSI by polymerase chain reaction analysis, the mutation rate in MSI-H tumors is known to vary widely, with data from The Cancer Genome Atlas Project showing mutational rates ranging from just more than 12 per 10 6 bases to nearly 100 per 10 6 bases. 28 Therefore, it is possible that, despite the high frequency of MSI, isolated PMS2 loss CRCs develop a smaller number of neoantigen-producing mutations than do other MMR-deficient/MSI CRCs, resulting in less immune Arch Pathol Lab Med Vol 142, April 2018 Isolated PMS2 Loss in Colorectal Cancer Alpert et al 527

6 activation and possibly a worse prognosis. However, given the range of underlying molecular alterations in isolated PMS2-loss CRCs seen in our and other studies, it is likely that the mutational landscape of these tumors also varies widely. An even larger series of cases would be needed to explore this relationship and compare the clinicopathologic features and outcomes of isolated PMS2-loss cases based on their underlying molecular alterations. Several additional limitations of our study are evident; many of these limitations stem from the study s multiinstitutional nature and retrospective design. The cases did not undergo a centralized review, so there could be some inconsistency in the grading of tumor differentiation and the evaluation of the MSI histologic features. However, all cases were evaluated by gastrointestinal pathologists who are experts in the histologic assessment of CRCs, so the degree of variation in histologic evaluation among sites should not have been too great. Unfortunately, germline testing results and clinical information were lacking in many cases, which meant that only a small subset of cases were available for our subgroup analysis. In addition, follow-up data, especially regarding the presence of disease recurrence, was not available for many cases, which limited our ability to compare outcomes between groups. In conclusion, our study revealed that the clinicopathologic features of CRCs with isolated loss of PMS2 immunohistochemical staining differ from those seen in MMR-deficient/ MSI CRCs overall, but those differences are likely related to the high proportion of isolated PMS2-loss cases caused by germline mutations in PMS2 and MLH1. There was, however, a notable trend toward more aggressive behavior in the isolated PMS2-loss cases as well as a trend toward a lower frequency of MSI histologic features in these cases, including the immune activation-related histologic features. Therefore, it is possible that isolated PMS2-loss CRCs elicit less of a response from the host immune system, which could contribute to their worse prognosis. Because of this apparent potential for aggressive behavior, detecting isolated PMS2- loss cases is especially important, and such detection largely depends on immunohistochemical screening of CRCs by pathologists, because characteristic MSI histologic features are often absent and carriers of PMS2 mutations are difficult to identify clinically because of their comparatively low risk of developing CRCs and extracolonic tumors. Heather Hampel, MS, LGC, is on the Invitae Laboratory Clinical Advisory Board, is a primary investigator for a research study that receives free genetic testing from Myriad Genetic Laboratories, Inc (Salt Lake City, Utah), and has received honoraria for speaking engagements from the Beijing Genomics Institute (Yantian District, Shenzhen, China). References 1. Dudley B, Brand RE, Thull D, Bahary N, Nikiforova MN, Pai RK. Germline MLH1 mutations are frequently identified in Lynch syndrome patients with colorectal and endometrial carcinoma demonstrating isolated loss of PMS2 immunohistochemical expression. Am J Surg Pathol. 2015;39(8): Gill S, Lindor NM, Burgart LJ, et al. Isolated loss of PMS2 expression in colorectal cancers: frequency, patient age, and familial aggregation. Clin Cancer Res. 2005;11(18): Hendriks YM, Jagmohan-Changur S, van der Klift HM, et al. Heterozygous mutations in PMS2 cause hereditary nonpolyposis colorectal carcinoma (Lynch syndrome). 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