Published OnlineFirst November 15, 2011; DOI: 10.1158/1940-6207.CAPR-11-0288 Cancer Prevention Research Article Research Identification of Cancer Patients with Lynch Syndrome: Clinically Significant Discordances and Problems in Tissue-Based Mismatch Repair Testing Angela N. Bartley1, Rajyalakshmi Luthra1, Devki S. Saraiya3, Diana L. Urbauer2, and Russell R. Broaddus1 Abstract Tissue-based microsatellite instability (MSI) analysis and immunohistochemistry for DNA mismatch repair proteins are accepted screening tools to evaluate patients with cancer for Lynch syndrome. These laboratory analyses are thus important tools in cancer prevention. Quality assurance review was conducted to identify test discordances and problems. These results were then analyzed in conjunction with genetic testing outcomes. Six hundred and forty-six consecutive tumors from 2002 to 2010 were examined. MSI-low tumors were excluded so that 591 tumors comprised the final analyses. Discordance was defined as a discrepancy between immunohistochemical and MSI analysis. Problem was defined as indeterminate or questionable immunohistochemical or MSI results. All results and clinical and family histories were centrally reviewed by two pathologists and one genetics counselor. Discordances and problems were identified in 23 of 591 (3.9%) of the tumors. Twelve of 102 MSI-high carcinomas (11.8%) and one of 489 microsatellite stable tumors had discordant immunohistochemistry. Of these 13 tumors, 11 were from patients who had personal and/or family cancer histories concerning for a germline mismatch repair gene mutation. In addition to discordances, 10 tumors with problematic immunohistochemical profiles were identified. Accurate evaluation of MSI was possible in all tumors. In summary, concordance between immunohistochemistry and MSI was high, particularly for tumors that are microsatellite stable. Greater frequency of test discordance was identified in the tumors that were MSI-high. Thus, a major consequence of the use of immunohistochemistry by itself as a screen is the failure to identify colorectal and endometrial cancer patients who likely have Lynch syndrome. Cancer Prev Res; 5(2); 1–8. Ó2011 AACR. Introduction become important for the identification of patients with Lynch syndrome and has treatment and prognostic signifi- Defects in the DNA mismatch repair (MMR) system cause cance for patients with sporadic colorectal cancer. Screening replication errors or instability in DNA microsatellites. for Lynch syndrome can not only effectively prevent sub- Germline mutations in MMR genes MLH1, MSH2, MSH6, sequent cancers in the screened individual, but it can also and PMS2 cause the autosomal dominant hereditary disease prevent cancer development in relatives who also harbor a Lynch syndrome. More than 90% of tumors from patients germline mutation of an MMR gene. with this syndrome show high levels of microsatellite insta- Traditionally, clinical screening methods to help identify bility (MSI-high; ref. 1). MSI also occurs in 10% to 15% of families at risk for Lynch syndrome included the use of the sporadic tumors, especially colorectal and endometrial Amsterdam and Amsterdam II criteria. Because these criteria carcinomas, due to hypermethylation of the MLH1 pro- have been thought to lack sensitivity, particularly when an moter (2). Screening for defects in the MMR genes has extensive family history is unavailable, the revised Bethesda criteria were developed. The Bethesda criteria are more sensitive, focus on the patient medical history, and intro- Authors' Affiliations: Divisions of 1Pathology and Laboratory Medicine duced the category of MSI and tumor morphology into and 2Quantitative Sciences and 3Department of Surgical Oncology, Uni- clinical guidelines. However, these criteria do not address versity of Texas MD Anderson Cancer Center, Houston Texas patients with endometrial cancer and may fail to identify a Note: This original research was presented in part at the 100th United States large number of mutation carriers that do not meet the and Canadian Academy of Pathology Annual Meeting, February 27 to March 4, 2011, San Antonio, TX. clinical criteria (3). To further help identify individuals at risk for Lynch syndrome, several clinical prediction models Corresponding Author: Russell R. Broaddus, Department of Pathology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe have been developed to quantify the risk for germline MMR Blvd. Houston, TX 77030. Phone: 713-795-2794; Fax: 713-792-5532; mutations (4–6). These have comparable sensitivities to the E-mail: [email protected] Bethesda criteria and can provide quantitative risk assess- doi: 10.1158/1940-6207.CAPR-11-0288 ment (7). However, such prediction models require subse- Ó2011 American Association for Cancer Research. quent genetic testing and are not helpful in identifying www.aacrjournals.org OF1 Downloaded from cancerpreventionresearch.aacrjournals.org on October 2, 2021. © 2011 American Association for Cancer Research. Published OnlineFirst November 15, 2011; DOI: 10.1158/1940-6207.CAPR-11-0288 Bartley et al. patients with sporadic MSI-high colorectal cancers requir- Molecular analyses ing alternative treatment regimens. All immunohistochemical and PCR-based molecular Two tissue-based screening methods used to assess analyses were conducted in Clinical Laboratory Improve- tumors for MMR defects include PCR-based MSI testing ment Amendments (CLIA)- and College of American and immunohistochemistry. In general, a number of large Pathology–approved laboratories. Immunohistochemistry studies have concluded that MSI and immunohistochem- was carried out for DNA MMR gene products MLH1 (G168- istry have comparable sensitivity and specificity in detecting 15, 1:25; BD Biosciences Pharmingen), MSH2 (FE11, 1:100; MMR defects (8–12). Because it is generally associated with Calbiochem), MSH6 (44, 1:300; BD Biosciences Pharmin- lower cost, faster turnaround time, and wider availability in gen), and PMS2 (Alb-4, 1:125; BD Biosciences Pharmingen) smaller pathology laboratories, some advocate that immu- as previously described (18). Tumors showing loss of nohistochemistry could be used as the sole method of nuclear MLH1, MSH2, PMS2, or MSH6 were classified as screening in certain cancers (9, 13–16). negative for the protein expression of each respective mark- At our institution, we conduct both PCR-based MSI and er. Stromal and normal tissues within the tumor served as immunohistochemistry concurrently. Immunohistochem- internal positive controls. ical–MSI analysis discordances have been acknowledged in MSI analysis was conducted using 6 National Cancer the research setting, but the frequency and nature of these Institute (Bethesda, MD) recommended microsatellites problems have not been formally examined in a quality (19) with the addition of analysis for the mutational inac- assurance fashion in the clinical laboratory. In surgical tivation of TGF-b receptor type II (TGFBR2). Therefore, 7 pathology practice, correlation reviews (cytology/histology microsatellites comprise the MSI panel. A tumor was des- and frozen section final pathology correlations) are com- ignated as MSI-high if it showed allelic shift at 3 or more monly used tools for quality assurance (17). This type of markers and as MSI-low with allelic shift in one or 2 quality assurance review has not been previously applied to markers. Those without allelic shifts were designated micro- the tissue-based approach to MMR testing. Therefore, we satellite (MS)-stable. A methylation-specific PCR MLH1 examined sources of discordances and problems in tissue- promoter methylation assay and BRAF V600E mutational based testing to help optimize this approach in our patient testing by pyrosequencing were conducted on a subset of population. cases (20, 21). Materials and Methods Statistical analysis It is controversial whether MSI analysis or immuno- Patient population and study design histochemistry represents the "gold standard" when con- After obtaining Institutional Board approval, we sidering the tissue testing approach. Therefore, tradition- obtained the results and clinical records for all cancer al measures of testing efficacy, such as sensitivity and patients who had MSI and/or immunohistochemical test- specificity, are likely not appropriate here. Instead, the ing between August 2002, and August 2010, at the Uni- measures calculated were concordance (percentage of versity of Texas MD Anderson Cancer Center (Houston, pairs in agreement) and discordance (percentage of pairs TX; n ¼ 736 patients). For 107 of these patients, only in disagreement). Specifically,wecalculatedconcordance immunohistochemical or MSI analysis had been previ- and discordance and their exact 95% confidence intervals ously conducted, so these patients are excluded from (CI) for MSI-high tumors, MS-stable tumors, tumors further consideration. Only those patients with both MSI with loss of an MMR protein by immunohistochemistry, testing and immunohistochemical results for the same and tumors with retained expression of all 4 MMR tumor were included (n ¼ 629). Demographic, clinical, proteins. and pathologic data were collected. Initial immunohis- tochemical slides were examined by a group of 14 dif- Results ferent gastrointestinal and gynecologic pathologists, whereas initial MSI analyses were evaluated by 7 gastro- Patient tumor population intestinal pathologists and 5 molecular pathologists. Dis- From August 2002 to August 2010, 629 patients had cordance was defined as a discrepancy between