Detection of DNA mismatch repair (MMR) deficiencies by immunohistochemistry can effectively diagnose the microsatellite instability (MSI) phenotype in endometrial carcinomas
Introduction
Microsatellite instability (MSI) describes a molecular phenotype that arises secondary to defects in the post-replicative DNA mismatch repair (MMR) system [1], [2]. Microsatellite regions are repetitive sequences throughout the genome consisting of mono-, di- or higher order nucleotide repeats. These repeats are more frequently transcribed incorrectly as DNA polymerases cannot bind efficiently. The MMR system is responsible for the identification and correction of these errors. Defects in the system can be secondary to genetic or epigenetic mechanisms, through germline or somatic mutations in one of the MMR genes (four highest frequency = MLH1, MSH2, MSH6, PMS2), or methylation of a promoter region, most commonly MLH1, respectively. MSI has been well characterized in colorectal cancer (CRC) but is also commonly recognized within specific histologic subtypes of ovarian cancer and endometrial cancers [3], [4], [5], [6], [7].
Identification of the MSI-high phenotype may trigger hereditary testing for Lynch Syndrome (LS) for the index case, and if confirmed, will initiate testing for first degree relatives. Detection of LS enables an individual to consider changes in lifestyle, screening, and/or risk reducing surgery [8], [9], [10], [11], [12], [13]. The MSI-high or hypermutated phenotype was one of the four categories of EC distinguished by genomic characterization in The Cancer Genome Atlas (TCGA)[14], and categorization of MSI status proposed as an early step in molecular classification of tumors. Identification of MSI has both prognostic and predictive implications in colorectal carcinomas and similar associations in gynecologic malignancies are being elucidated [7], [14], [15], [16], [17], [18], [19], [20]. Categorization of tumors can be achieved by either immunohistochemistry for MMR-associated proteins or MSI assay.
In colorectal cancers and more recently in endometrial cancers and endometriosis-associated ovarian cancer MSI phenotype is commonly tested by mismatch repair protein immunohistochemistry (MMR IHC) in the post-operative pathology specimen. Standardization of methods and published guidelines in interpretation has greatly improved consistency [21], [22], [23], [24], [25], [26]. MMR IHC cost is ~$20–130/MMR protein tested (usually two-six proteins tested).
In contrast, in the research setting MSI is assessed utilizing PCR amplification of a set of nucleotide repeat markers. These microsatellite markers are then compared between tumor and normal DNA to detect somatic changes. Historically, choice of microsatellite markers varied widely; thus in 1998 a National Cancer Institute consensus panel recommended the assessment of two mononucleotide markers (BAT25 and BAT26) and three dinucleotide markers (D5S346, D17S250 and D2S123). MSI-H (MSI-high) is assigned if size alterations or shifts are observed in two or more markers, and MSI-L (MSI-low) or low microsatellite instability phenotype if just one marker shows instability. If none of the markers show instability the phenotype is considered MSS (MicroSatellite Stable). There are no FDA-approved MSI tests, and private, academic or commercial labs must validate their own assays independently. This has led to variation in techniques and challenges in interpreting data from across different centers. The PCR MSI assay requires normal DNA for comparison, which limits the number of cases easily tested. Minimal cost estimates from an in-house lab include DNA extraction and MSI assay sum to ~$40/case but commercial cost is over $400 with additional costs accrued for interpretation (~$25–50) and microdissection (~$200–250).
Although a high level of concordance of MSI assay testing and MMR IHC has been demonstrated in CRCs, data in other Lynch-associated cancers is absent or sparse. We wished to determine the level of agreement between these two methodologies in endometrial cancers, with the ultimate goal of supporting MMR IHC in research as the primary means of determining MSI phenotype.
Section snippets
Materials and methods
157 endometrial tumors from the OvCaRe Tissue Biobank Repository were obtained for study based on availability of frozen tissue and where possible matched buffy coat for comparison of normals. Mutational profiling data on this cohort has been previously published [27]. Research ethics approval for the Tissue/Biospecimen Bank and this project was granted from the University of British Columbia Institutional Review Board and all patients underwent informed written consent for use of their
Results
Although 157 endometrial tumors were obtained for study from the OvCaRe Tissue Biobank Repository there were 68 cases with insufficient normal material to report MSI status using the MSI assay. There were only 2 cases with MMR status missing due to insufficient evaluable material. For the comparison, we must consider complete observations for both assays. Therefore, the final data consists of 89 tumors (Table 1, Table 2).
The overall accuracy is 93.3% with a 95% confidence interval of
Discussion
Data directly comparing methods of MSI assessment in Lynch-associated cancers other than CRC are sparse. In endometrial cancer, several studies have assessed the clinical validity of MSI assay testing by direct comparison to germline results in endometrial cancers, with sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) ranging from 77 to 100%, 38 to 81%, 9 to 76% and 97 to 100% respectively. The validity of IHC testing for determining germline
Conflict of interest statement
The authors have not conflict of interest to declare.
Acknowledgments
We are grateful for the work of Christine Chow, Margaret Luk, Winnie Yang, Janine Senz and Ying Ng. Partial funding for this work comes from the Sarabjit Gill Fund in Endometrial Cancer through the BC Cancer Foundation. Dr. McConechy receives funding through the CIHR Frederick Banting and Charles Best Canada Graduate Scholarships Doctoral Award and Dr. McAlpine through the CIHR New Investigator Award.
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