Loss of Mismatch Repair Protein Expression in Epithelial Ovarian Carcinoma: A Histomorphologic Guide to Targeted Screening

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1 Loss of Mismatch Repair Protein Expression in Epithelial Ovarian Carcinoma: A Histomorphologic Guide to Targeted Screening Eman Abdulfatah a, Sharif Sakr b, Sumi Thomas a, Sudeshna Bandyopadhyay a, Kavita Varma c, Dhananjav Chitale c, Michele Cote d, Adnan Munkarah e, Robert Morris f, Rouba Ali-Fehmi a,* Abstract Objectives: Lynch syndrome (LS) is defined by defects in mismatch repair (MMR) proteins. Histomorphologic features have been predictive of MMR protein expression loss (MMR-L) in MMR defect-associated endometrial and colon carcinomas; but none were identified for epithelial ovarian carcinomas (OCa). We evaluated the incidence and predictive histomorphologic features of MMR-L in OCa to screen for further LS testing. Methods: Patients with primary OCa (n=229) were retrospectively analyzed for peritumoral lymphoid aggregates (PTLs), tumor infiltrating lymphocytes (TILs) and nuclear pleomorphism (NP). Immunohistochemistry for 4 MMR proteins (MLH1, PMS2, MSH2 and MSH6) was performed and data was analyzed. Results: MMR-L was identified in 8/229 (3.5%) OCa; highest in undifferentiated carcinomas (1/4, 25%), clear cell (CCC) (3/14, 21%) and endometrioid carcinomas (1/8, 12%). Patients had an average age of 51.4 years, stage III-IV (57%) and were Caucasian (63%). Tumors with PTLs, ITLs and NP were more frequently associated with MMR-L(P=0.006, P=0.002 and P= 0.003, respectively). MMR-L was significantly associated with ovarian endometriosis and tumor necrosis(p=0.024 and P=0.008, respectively) and a longer five year overall survival (50% versus 29.7%, P=0.47)). Conclusions: 3.5% of patients with OCa had MMR-L; significantly higher in endometriosis associated ovarian cancers. PTLs, ITLs and NP may be used to screen patients for LS testing. Keywords ovarian cancer, MMR, Lynch syndrome, peritumoral lymphoid aggregates, tumor infiltrating lymphocytes and nuclear pleomorphism. Date of Submission: 5/1/2016 a Department of Pathology, Wayne State University, Detroit, MI, USA. b Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI, USA. c Department of Pathology, Henry Ford Hospital, Detroit, MI, USA. d Department of Epidemiology, Karmanos Cancer Institute, Detroit, MI, USA. e Department of Obstetrics and Gynecology, Henry Ford Hospital, Detroit, MI, USA. f Division of Gynecologic Oncology, Karmanos Cancer Institute, Wayne State University, Detroit, MI, USA. *Corresponding Author: Rouba Ali-Fehmi M.D. Department of Pathology, Wayne State University 540 East Canfield Avenue, Detroit, Michigan Tel: , , , Fax: rali@med.wayne.edu Conflict of Interest Disclosure: The authors have no potential conflicts of interest to disclose. INTRODUCTION Epithelial ovarian tumors account for 90% of ovarian cancers in Western countries. 1 Although different histologic subtypes have been long recognized, subtype-specific molecular abnormalities have not been fully defined. It is estimated that between 10% and 15% of ovarian cancers are associated with an underlying hereditary syndrome, with up to 90% of these attributed to BRCA1 and BRCA2 alterations; almost all of the remainder are attributable to Lynch syndrome/hereditary nonpolyposis colorectal cancer (HNPCC). 2 DNA mismatch repair (MMR) gene inactivation, either through germ-line mutations in Lynch Syndrome or through MLH-1 promoter methylation in sporadic tumors results in variation in the size of nucleotide repeats throughout the genome, a phenomenon referred to as microsatellite instability (MSI). 3 Women with MSI have a 9-12% increased lifetime risk of developing ovarian cancer, as well as 40-60% lifetime increased risk for endometrial cancer. 4 Several morphologic features have been predictive of MMR loss in both MMR defective-associated endometrial and colon carcinomas including tumor-infiltrating lymphocytes(tils), peritumoral lymphocytes (PTLs) and dedifferentiated histology; However, no correlation has been identified for ovarian tumors. 5 Among the few studies that have examined MMR protein status in ovarian carcinomas, defects have been reported in 2% to 10%, 6 with a higher incidence for endometrioid and clear cell carcinomas. 7, 8 Evaluation for the presence of hereditary cancer syndromes provides options for tailored screening and prevention strategies that may reduce morbidity associated with the development of these malignancies. Identifying epithelial ovarian tumors with MMR defects is not only important for early detection of women with Lynch syndrome, but may also have treatment implications. Several studies suggest a relation between MMR defects and decreased susceptibility to cisplatin, a common chemotherapeutic agent used to treat ovarian carcinomas. 9 Immunohistochemical (IHC) analysis with specific antibodies directed against MMR proteins has proven useful for investigating MMR defects in both familial and sporadic forms of ovarian cancer. 10 Recent studies have shown that the use of commercial antibodies against hmsh2and hmlh1 can reliably 7

2 detect MSI. 11 Guidelines for screening ovarian tumors to identify patients at risk for Lynch syndrome remain controversial. Limited data is reported in the literature regarding defective MMR proteins in ovarian carcinoma. Its significance and implications remain to be fully investigated. In this study we investigated the incidence of MMR protein defects in primary ovarian carcinomas. Specifically, we defined the immuno-expression profile of MMR proteins MLH-1, MSH-2, MSH-6, and PMS-2 in these tumors and evaluated for association with specific morphologic and clinical features. MATERIALS AND METHODS/RESULTS After obtaining Institutional Review Board (IRB) approval, patients diagnosed with primary epithelial ovarian carcinoma (n=229) between 2000 and 2013 were identified. Clinical parameters (age, race, family history of cancer, other synchronous or metachronous cancers and vital status) and pathological variables (stage and grade) were obtained from electronic medical records (EMR) and pathologic records. Borderline tumors were excluded. Tumor grade was assigned according to the MD Anderson grading criteria (MDACC) and tumor stage was assigned according to International Federation of Gynecology and Obstetrics (FIGO) criteria. 12 Mixed carcinomas were defined as carcinomas with one or more other components comprising at least 10% of the tumor. Morphologic features that have been reported in colorectal and endometrial carcinomas as potential predictors of abnormal MMR protein status were evaluated in the primary ovarian tumors and in the concurrent uterine tumors, if present. 13 These features were evaluated from review of whole-tissue sections of each tumor by two gynecological pathologists and included TILs, PTLs visible at scanning magnification and nuclear pleomorphism. TILs were assessed by enumerating the number of intercellular lymphocytes in 10 consecutive HPFs (x 400, field diameter = 0.55 mm) in the area most enriched for TILs, according to the method proposed by Soslow et al. 14 Additional pathologic features including expansile pattern invasion with pushing borders, destructive stromal invasion with desmoplastic stroma, ovarian endometriosis, psammoma bodies and necrosis were also evaluated. Information on personal and family cancer histories for each patient, with particular attention to the presence of Lynch syndrome-related tumors according to the revised Bethesda guidelines was retrieved from EMR. 15 IHC evaluation of MMR protein expression was performed on Tissue Microarrays (TMAs). TMAs were constructed from formalin-fixed, paraffin embedded blocks of primary tumors (Beecher Instruments, Silver Spring, MD) 16 using a duplicate of 1mm diameter core obtained from a representative area of each tumor. The antibodies used were MLH-1 (clone M1; Ventana, BenchMark XT/Ultra; Ready-To-Use), MSH-2 (clone G ; Ventana/Cell Marque, BenchMark GX/XT/Ultra; Ready-To-Use), MSH-6 (clone 44;Ventana, BenchMark XT ; Ready-To-Use) and PMS2 (clone EPR3947; Ventana; Ready-To-Use). Immunostaining was performed using heat induced epitope retrieval. Slides were blocked in 3% H2O2and then incubated with the primary antibody at room temperature for 48 minutes (MLH1), 120 minutes (PMS2), 48 minutes (MSH2) and 20 minutes (MSH6). Detection of antibody binding was obtained using Optiview(Ventana) after a polymer-based amplification step (Ultraview, Ventana). Slides were then counterstained with hematoxylin and cover slipped. Only cases with complete absence of nuclear staining and positive nuclear staining in internal control cells (eg. stromal fibroblasts, non-neoplastic epithelium and lymphocytes) were considered to demonstrate MMR protein loss.all abnormal results were validated and verified by staining whole-tissue sections. Statistical analyses were conducted using Fisher s Exact (2-tailed), chi-square, student t-test, log-rank tests and Kaplan- Meier survival curves. Significance was set at P < MATERIALS AND METHODS/RESULTS A total of 229 cases diagnosed with primary epithelial ovarian carcinoma were collected for TMA construction and were included in our study. The median age at diagnosis was 59 years (mean 60, range 25 to 91 years). Seventy-two percent of the ovarian carcinomas (n=164) showed serous histology. The remaining histologies included mixed (n=23,10%), clear cell (n=14, 6%), mucinous (n=14, 6%), endometrioid (n=8, 3%), undifferentiated (n=4, 2%) and malignant mixed mullerian tumor (MMMT) (n=2, 1%). Overall, the FIGO stage distribution was 16% stage I, 10% stage II, 58% stage III and 16% stage IV. The average follow-up period was 45 months (range 2 to 156 months). Loss of expression of MMR proteins was detected on TMA slides in 20/229 (9%) ovarian carcinomas with the highest percent of loss in PMS2 alone, occurring in 9/20 (45%) cases, followed by loss of MLH1 alone in 4/20 (20%), both MLH1 and PMS2 in 3/20 (15%), MSH6 alone in 3/20 (15%), and both MSH2 and MSH6 in 1/20 (5%). The whole tissue section slide staining validated 8/20 cases that had showed loss of MMR protein expression on TMA slides. The specificity of TMA as compared with whole section slides was 22.2% for MLH1, 83.3% for PMS2 and 100% for both MSH2 and MSH6. In total, 8 of 229 (3.5%) cases showed loss of MMR protein expression with the highest frequency in undifferentiated carcinoma (1/4, 25%), followed by clear cell carcinoma (3/14, 21%), endometrioid carcinoma (1/8, 12%), mixed carcinoma (1/23,4%) and serous carcinoma (2/164, 1%). No loss of MMR protein expression was identified in mucinous carcinomas (14 cases) and MMMT (2 cases) (Table 1). The frequency of MMR protein expression loss was significantly higher in endometriosis- associated (clear cell and endometrioid) 8

3 carcinomas versus other histologic subtypes (P = 0.004) (Table 2). The most common abnormality was loss of PMS2 alone, occurring in 3/8 (37.5%) cases, followed by loss of both MLH1 and PMS2 in 2/8 (25%) cases, loss of MSH6 in 2/8 (25%) cases and loss of both MSH2 and MSH6 in 1/8 (12.5%) cases (Table 3). Table 3: MMR protein expression profile and clinical features of cases demonstrating defective MMR Table 1: Incidence of MMR protein expression loss among the histologic types of ovarian carcinoma Histologic types No. of cases* Cases with MMR loss (%) Undifferentiated carcinoma 4 1 (25) Clear cell carcinoma 14 3(21) Endometrioid carcinoma 8 1 (12) Mixed 23 1 (4) Serous carcinoma (1) MMMT 2 0 (0) Mucinous carcinoma 14 0 (0) Total (3.5) * This column represents the total number of cases with the specified histologic types. Table 2: Clinicopathologic characteristics of ovarian carcinomas with and without MMR protein expression MMR Loss (n=8)* Intact MMR (n=221)* P value Age at diagnosis < 60 years 7 (87.5%) 114 (51.6%) >= 60 years 1 (12.5%) 107 (48.4%) Race Caucasian 5 (63%) 112 (62%) AA 3 (37%) 41 (22%) Others 0 (0%) 29 (16%) Stage I-II 3 (43%) 56 (26%) III-IV 4 (57%) 157 (74%) Grade Low 1 (12.5%) 13 (6%) High 7 (87.5%) 197 (94%) Histologic type Clear cell and endometrioid carcinomas 4 (50%) 18 (8%) Others 4 (50%) 203 (92%) Synchronous or metachronous cancers Present 2 (25%) 18 (10%) Absent 6 (75%) 161 (90%) Five year overall 50.0% 29.7% 0.47 survival rate *Data missing in some cases with both intact and lost MMR protein expression. For cases demonstrating loss of MMR protein expression, the average age at presentation was 51.4 years versus 60.4 years for cases with intact MMR protein. Patients less than 60 years of age showed a significantly higher incidence of MMR protein expression loss than older patients (p=0.054). Slightly more cases with defective MMR proteins (57%) presented at stages III-IV, were Caucasian (63%), and had a longer 5 year overall survival (50% vs. 29.7%, P=0.47)), without a statistical difference noted (Table 2). The median survival for patients that showed loss of MMR protein expression was 66 months versus 44 months for those with intact MMR. The majority of the cases with MMR defects (7/8 cases) were high grade. Two cases (25%) had synchronous/metachronous primary malignancies; one presented with endometrial cancer synchronously while the other had metachronous colon cancer.. Clear cell carcinomas (3/8 cases) showed only loss of MSH2and/or MSH6 proteins. There were no cases with loss of only MLH1 or only MSH2. HISTOMORPHOLOGIC FEATURES ASSOCIATED WITH DEFECTIVE MMR PROTEIN EXPRESSION Slides and blocks were available for review for 195/229 cases (thirty-four patients left for further treatment at outside institutions). Tumors with MMR defects were significantly associated with marked nuclear pleomorphism, TILs, PTLs, necrosis and ovarian endometriosis (P= 0.003, P= 0.002, P=0.006, P= and P=0.024, respectively) (Table 4). Four of 8 (50%) cases with defective MMR showed TILs and PTLs with the highest frequency associated with clear cell carcinoma (2/4 cases), followed by mixed serous and endometrioid cell carcinoma (1/4 cases) and endometrioid carcinoma (1/4 cases). No statistical association was found between the presence of desmoplastic stroma, destructive stromal invasion or psammoma bodies and MMR status (Table 4). 9

4 Table 4: Correlation of histomorphologic features of ovarian carcinoma to the MMR status MMR Intact MMR Loss (n=187)* (n=8) P value Nuclear atypia Mild 0 (0%) 72 (38%) Moderate 2 (25%) 72 (38%) Severe 6 (75%) 43 (24%) TILs < 40 4 (50%) 175 (94%) (50%) 12 (6%) PTLs Present 4 (50%) 18 (10%) Absent 4 (50%) 169 (90%) Necrosis Present 8 (100%) 96 (51%) Absent 0 (100%) 91 (49%) Psammoma bodies Present 1 (13 %) 17 (9%) Absent 7 (87 %) 169 (91%) Desmoplastic stroma Present 5 (63%) 89 (48%) Absent 3 (37%) 98 (52%) Invasion borders Pushing 6 (75%) 87 (47%) borders Infiltrative borders 2 (25%) 100 (53%) Endometriosis Present 3 (38%) 17 (8 %) Absent 5 (62 %) 203 (92 %) * Slides for 34 cases were unavailable for review Figure 2: A. Loss of MSH2 (200X). B. Loss of MSH6 (200X). C. Intact MLH 1(200X). D. Intact PMS2 (200X) Figure 3: Kaplan Meier survival curve comparing survival in patients with MMR loss vs. MMR intact ovarian carcinomas Figure 1: A. PTLs visible at scanning magnification (40X). B. CCC showing significant nuclear pleomorphism (200X). C and D. Two examples of EOC with TILs (100X and 200X respectively). DISCUSSION Hereditary gynecologic cancers are relatively uncommon, accounting for approximately 5% of endometrial cancers and 5% to 15% of ovarian cancers. Germline mutations in BRCA1 or BRCA2 genes accounts for most inherited ovarian cancers, 17 however, % are attributable to mutations in MMR genes and Lynch syndrome. 2 The lifetime risk for ovarian cancer in women with Lynch syndrome is 9% -12%, compared with 40% -60% for endometrial cancer and 60% -80% for colorectal cancer. 4 On basis of these risks, cancer prevention strategies (screening and/or intervention) have been proposed for patients 10

5 suspected or known to have Lynch syndrome. 18 Formal genetic evaluation, including pedigree analysis and germline mutation testing, is offered for patients who meet clinical screening criteria, and/or whose tumors demonstrate loss of MMR protein immuno-expression or MSI. 18 Tumor morphologic markers including TILs, PTLs, dedifferentiated histology, primary origin in lower uterine segment and association with ovarian clear cell carcinoma can be used in conjunction with clinical factors to guide which endometrial cancers should undergo testing for MMR protein status and/or MSI. 19 Whether these morphological markers can be applied to screening patients with ovarian cancer has not been fully studied previously. The aim of our study is to identify a relationship between histology and loss of MMR protein expression, which might serve as a platform for targeted screening. Among studies of unselected types of ovarian cancer, the incidence of MMR protein expression loss ranges from 2 to 10%. 6 Endometrioid and clear cell carcinomas were the most frequent ovarian cancer subtypes with defective MMR and/or MSI-high status in 3 studies, none of which reported any abnormalities in serous carcinomas. 6 Studies restricted to endometrioid-type ovarian cancer reported 14% to 20% MSI-high and/or abnormal MMR immuno-expression, 8 whereas those restricted to clear cell carcinomas reported a range of 6% to 37.5%. 7, 8 Furthermore, Lu et al demonstrated loss of MMR protein expression in 10.1% of carcinoma subtypes that had shown a strong association with endometriosis in the literature, 20 namely clear cell and endometrioid carcinomas. 1 The present cohort demonstrated loss of MMR protein expression in 3.5% of unselected ovarian carcinomas with the highest frequency in undifferentiated carcinomas (25%), followed by clear cell (21%) and endometrioid carcinomas (12%).The frequency of defective MMR was significantly higher in endometriosis associated ovarian carcinomas, as shown in multiple reports previously. Endometrial undifferentiated carcinomas and dedifferentiated carcinomas previously described by Silva et al, 20 are associated with MMR protein abnormalities and Lynch syndrome. 21 Tafe et al 21 showed MMR protein defects in 7 of 12 (58%) endometrial undifferentiated carcinomas and accordingly they recommend staining all cases of undifferentiated carcinomas for MMR proteins, irrespective of the patient s age. Similarly, our study detected the highest incidence of MMR protein defects among ovarian undifferentiated carcinomas. This was not addressed previously in ovarian undifferentiated carcinomas and future studies are recommended to further investigate and validate this finding. Previously, it has been suggested that ovarian carcinoma presents at younger age in Lynch syndrome patients than typically observed in the general population, with the mean age across studies ranging from 41 to 55 years. 22 In the present cohort, the average age at presentation was 51.4 years versus 60.4 years for cases with intact MMR protein. Patients less than 60 years of age showed a significantly higher incidence of MMR protein expression loss than older patients. Although there is a conflicting data, several studies suggests that Lynch syndrome associated endometrial carcinoma presents at a higher stage and higher histologic grade and thus may be more virulent than sporadic endometrial cancer. 23 Data are limited concerning grade and stage at presentation for Lynch syndrome associated ovarian cancer. 22 The International Collaborative Group on HNPCC found a predilection for low grade and low stage HNPCC associated ovarian tumors compared with the general population. 22 King et al 24 also found a higher frequency of MSI in low stage (defined as stage I) ovarian carcinoma (75% of stage I tumors as compared with 11% of tumors greater than stage I). In contrast, Cai and colleagues, 7 in evaluating only ovarian clear cell carcinomas, found no correlation between MSI and tumor stage. In our study, 4/7 (57%) cases with MMR protein expression loss had high stage disease (defined as stage III-IV). Although slightly more cases showed a higher stage, no statistical difference was noted. However, stage of disease may be a surrogate for a subtype-specific bias in MMR protein defects and Lynch syndrome population, with these tumors being predominantly clear cell and endometrioid subtypes. Larger studies are needed to clarify this further. Overall, the literature suggests that ovarian cancer subtypes (endometrioid or clear cell type) may be a useful screening variable in conjunction with young patient age and family pedigree. However, none of these studies to date examined whether any morphologic markers beside tumor histologic type may correlate with MMR/MSI status. Several studies of endometrial carcinoma have demonstrated an association between tumor morphology and status of MMR/MSI. 19 Published data on colorectal carcinomas have demonstrated a poorer sensitivity and specificity when using guidelines that are solely based on clinical criteria (age and family history) as compared with pathological criteria known to be associated with Lynch syndrome. 25 In line with these findings, combining morphologic features to clinical data in endometrial cancer have showed a sensitivity for detecting MSI ranging up to 85%. 19 The present cohort was designed to evaluate whether morphologic markers carry the same association with MMR/MSI status in ovarian cancer. We identified a significant correlation with a diffuse inflammatory infiltrate (TILs and PTLs) and marked nuclear pleomorphism. The majority of ovarian carcinomas with MMR protein expression loss that showed TILs and PTLs were of the clear cell type (2/4 cases). A significant relationship was also identified between tumor necrosis, endometriosis and MMR defects. In contrast, Aysal et al 26 showed no association between morphologic predictors of MMR status with MMR protein expression loss in ovarian endometrioid carcinomas. The fact that they only included the endometrioid subtype may account for the differences observed. In 2004, the revised Bethesda guidelines recommended that patients with synchronous or metachronous colorectal or other HNPCC-associated tumors, regardless of the age, should undergo MSI testing or immunohistochemical analysis for MMR gene expression. 15 Several studies to determine whether there is utility in testing all women with synchronous 11

6 endometrial and ovarian cancers as per the revised Bethesda Criteria were published. 27 Soliman et al 28, found only 7% of women with synchronous endometrial and ovarian cancer to have clinical or molecular criteria suggestive of Lynch syndrome, an incidence lower than that seen in women with synchronous or metachronous colon and endometrial cancer. In our study, 25% (2/8) cases with loss of MMR protein expression had synchronous/metachronous HNPCC-associated tumors with one case having ovarian and endometrial carcinomas synchronously and the other ovarian and metachronous colorectal carcinoma. Another study by Aysal et al 26 found five of seven patients with MMR deficient endometrioid ovarian cancer that presented with synchronous primary endometrial carcinomas. These findings would argue against conservative surgery and fertility preservation in young patients with ovarian cancers demonstrating loss of MMR protein expression and could be used to trigger further diagnostic evaluation for potential endometrial involvement. Molecular analysis used to identify MSI in sporadic and familial tumors involves time-consuming and expensive techniques, hampering its use as a screening tool. Moreover, it does not identify the genes involved, a question that would require further molecular level tests. IHC analysis, on the other hand, is readily available, relatively inexpensive, fast and simple. Furthermore, published data describes a high correlation between MSI and IHC analysis of MMR proteins ranging from 80% to 100%. 29 Nevertheless, increasing evidence suggests the use of MMR IHC as a screen for Lynch Syndrome. 30 The present cohort did not specifically confirm germline mutations because of the study design, however future studies are needed to clarify possible correlations between MMR IHC and germline mutation status. Clinical outcome did not correlate with MMR status in the present cohort. Although not statistically significant, the five year overall survival was longer in cases with defective MMR versus cases with intact MMR (50% vs 29.7%). Similarly, Aysal et al 26 demonstrated no correlation between MMR status and outcome. Most of the patients who died during their follow-up period had intact MMR. Given that the highest frequency of MMR protein defects in our case was detected in undifferentiated carcinoma which typically has an unfavorable prognosis and an aggressive behavior, perhaps association with Lynch syndrome portends a more favorable outcome. We recognize that the major limitations of this study include the use of IHC only without other confirmatory test. However, it illustrates the feasibility of use of IHC along with histologic parameters in screening patients for the need of further testing. Additionally, because of the inherent rare nature of the MMR protein expression loss in ovarian cancer patients, only a retrospective review could be carried out on the data obtained from a single institution (as with the rest of the literature addressing this subject). Ideally, prospective analysis of data from multiple institutions shall provide a reliable confirmation of the current understanding of MMR protein loss in ovarian cancer. A major strength of this study is the easy applicability of such histologic and IHC parameters for reflex MMR testing, once the results were validated on a larger scale. In conclusion, as demonstrated in the literature and the present study, the frequency of MMR protein expression loss among unselected ovarian cancer patients was significantly associated with specific tumor subtypes, namely clear cell and endometrioid carcinoma (known to be endometriosis associated). Previous studies have been unable to identify a relationship between histology and MMR loss in ovarian cancer; however, the present cohort identified a significant correlation with a diffuse inflammatory infiltrate, marked nuclear pleomorphism, tumor necrosis and ovarian endometriosis. Potentially, ovarian carcinomas with these histologic characteristics can be selected for MMR analysis which is cost effective and convenient and may serves as tool for discovering other tumors and screening family members. The college of American pathologists has stated that pathologists should recognize the histologic and clinical features that should prompt a recommendation for MMR testing. Reflex testing of colorectal cancer with MMR IHC is becoming standard of care and similar recommendations are being made for endometrial cancer. This stems from the appreciably high life time risk for colorectal and endometrial cancer in patients with Lynch syndrome. On the other hand, because of the lower lifetime risk of ovarian cancer in such patients, the use of identified histologic and IHC features in conjunction with clinical features would provide the most convenient and cost-effective tool in screening for the need for reflex MMR testing. 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