YGYNO-975783; No. of pages: 5; 4C: Gynecologic Oncology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Gynecologic Oncology journal homepage: www.elsevier.com/locate/ygyno
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M.K. McConechy a,c, A. Talhouk a, H.H. Li-Chang a, S. Leung b, D.G. Huntsman a, C.B. Gilks a, J.N. McAlpine a,⁎
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Keywords: Mismatch repair Microsatellite instability Lynch syndrome Endometrial cancer
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Department of Pathology and Laboratory Medicine, University of British Columbia and BC Cancer Agency, 509-2660 Oak Street, Vancouver V6H 3Z6, BC, Canada Genetic Pathology Evaluation Centre, Department of Pathology and Laboratory Medicine, University of British Columbia, 509-2660 Oak Street, Vancouver V6H 3Z6, BC, Canada Department of Gynecology and Obstetrics, Division of Gynecologic Oncology, University of British Columbia, 2775 Laurel St. 6th Floor, Vancouver V5Z 1M9, BC, Canada
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Background. A proportion of endometrial carcinomas (ECs) are associated with deficient DNA mismatch repair (MMR). These tumors are characterized by high levels of microsatellite instability (MSI). Identification of MSI is important in identifying women who should be tested for Lynch syndrome and identifying a phenotype that may have specific prognostic and predictive implications. Genomic characterization of ECs has shown that MSI tumors form a distinct subgroup. The two most common methodologies for MSI assessment have not been compared in EC. Methods. Pentaplex mononucleotide PCR for MSI testing was compared to MMR IHC (presence/absence of MLH1, MSH2, MSH6, PMS2) in a cohort of patients with EC. Concordance, Kappa statistic, sensitivity, specificity, positive and negative predictive values were obtained on the cross-tabulation of results. Results. Comparison of both MSI and MMR status was complete for 89 cases. Overall agreement between methods (concordance) was 93.3% (95% CI[85.9%–97.5%]). A one-sided test to determine whether the accuracy is better than the “no information rate,” which is taken to be the largest class percentage in the data, is significant (p b 0.00001). Unweighted Kappa was 0.84, along with the sensitivity (88.5%), specificity (95.2%), PPV (88.5%), and NPV (95.2%). The balanced accuracy (i.e. the average between sensitivity and specificity) was 92%. Discussion. We show the equivalence of MSI testing and MMR IHC. We advocate the implementation of MMR IHC in future EC classification schemes, enabling stratification of cases for future clinical trials as well as assisting identification of Lynch syndrome, so that screening and risk reducing interventions can be undertaken. © 2015 Published by Elsevier Inc.
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Article history: Received 29 November 2014 Accepted 21 January 2015 Available online xxxx
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Detection of DNA mismatch repair (MMR) deficiencies by immunohistochemistry can effectively diagnose the microsatellite instability (MSI) phenotype in endometrial carcinomas
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Introduction
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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
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⁎ Corresponding author at: Department of Gynecology and Obstetrics, Division of Gynecologic Oncology, 2775 Laurel St, 6th Fl, Vancouver V5Z-1M9, BC, Canada. Fax: +1 604 875 4869. E-mail address: [email protected] (J.N. McAlpine).
recognized within specific histologic subtypes of ovarian cancer and endometrial cancers [3–7]. 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 [8–13]. 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 [7,14–20]. 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
http://dx.doi.org/10.1016/j.ygyno.2015.01.541 0090-8258/© 2015 Published by Elsevier Inc.
Please cite this article as: McConechy MK, et al, Detection of DNA mismatch repair (MMR) deficiencies by immunohistochemistry can effectively diagnose the microsatellite instability..., Gynecol Oncol (2015), http://dx.doi.org/10.1016/j.ygyno.2015.01.541
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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 biospecimens for research purposes.
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DNA isolation
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Genomic DNA was extracted from flash frozen tumors from hysterectomy specimens using the Ambion DNA extraction kit as per manufacturer's protocol (Ambion, Life Technologies) and as previously described [27].
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Microsatellite instability (MSI) assay
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The analysis of tumor MSI was performed following the recommendations of the National Cancer Institute (NCI). The Bethesda Marker panel of five microsatellite loci; Bat 25, Bat 26, DI7S250, D5S346, and D2S123 were used to assess the MSI status. MSI-high is reported if 2 or more markers have MSI, 1 or less markers with MSI were considered MSI-low, and MSS (Microsatellite Stable) indicates that no markers were positive. Cases reported as N/A did not have sufficient normal available to assess the MSI status of these tumors. Details of these methods have been previously reported [27]. Results of MSI assay were then binarized as MSI-high or MSS with MSI-low considered MSS.
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Tissue microarray (TMA) and immunohistochemistry
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To construct the endometrial TMA, tumors were annotated by a pathologist (BG) and two 0.6 mm cores per case were arrayed. TMAs were cut at 4 μm thickness onto Superfrost + glass slides, and were processed
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MLH1 promoter methylation testing
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Cases where discordance was noted between MSI assay results and MMR IHC were sent for testing for MLH1 promoter methylation at Mayo Medical Laboratories, Rochester, MN who perform a PCR-based assay to test tumor DNA for the presence of hypermethylation of the MLH1 promoter. This is a modification of the method described by Grady et al. [28].
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Statistics
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The data under consideration consists of two assignment of MSIStatus on 157 cases of EC; one of the assignments was done using the MSI assay and one using MMR. We were interested in measuring the degree of agreement between the two methods, while accounting for the agreement that occurs by chance. We began by calculating concordance. We then calculated reliability statistics such as Cohen's kappa, the accuracy of MMR relative to the MSI assay, the sensitivity, specificity, and the positive and negative predictive value. We also used a test statistic to compare the observed accuracy using MMR relative to the no information rate, which is the accuracy obtained by a method that would assign all samples to the largest class. P values smaller than 0.05 were considered statistically significant. All statistical analyses were performed using R (ver. 3.1.0) [29] and packages caret (ver. 6.0-30) for reliability analysis, Gmisc for plot and table output (ver. 0.6.4), and knitr (ver 1.6) [30–32] for reproducible research. For comparison of clinicopathologic factors in the MSI-high vs. MSS cohorts (as assessed by both methods) P-values were computed using a one way analysis of variance in the case of continuous variables (Age) and Fisher's exact test with Monte Carlo simulated P-values for categorical variables.
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Results
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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 (Tables 1 and 2). The overall accuracy is 93.3% with a 95% confidence interval of (85.9%–97.5%). A one-sided test was conducted to see if the accuracy is better than the “no information rate” (in this case 70.8% which is
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using the Ventana Discovery XT, and the Ventana Benchmark XT automated system (Ventana Medical Systems, Tucson, AZ, USA) as per manufacturer's protocol with proprietary reagents. After slides were baked at 60 °C for 1 h, they were deparaffinized on the automated system with EZ Prep solution (Ventana). Heat induced antigen retrieval method was used in Cell Conditioning solution (CC1-Tris based EDTA buffer, pH 8.0, Ventana). Anti-hMLH1 (1:150, ES05, Novacastra, Vector Labs Burlington ON), hMSH2 (1:100, 25D12, Novacastra, Vector Labs), hMSH6 (1:600, PU29, Novacastra, Vector Labs), nadhPMS@ (1:150, MOR4G, Novacastra, Vector Labs), with appropriate positive and negative controls according to the manufacturers' standardized staining protocols. Tumor was considered aberrant if tumor cells showed complete absence of nuclear staining with positive non-neoplastic internal control, and intact if tumor cells show nuclear positivity. Aberrant, equivocal, and uninterpretable cases (e.g., no tumor tissue) on TMA had whole sections requisitioned for repeat staining of the MMR protein or proteins needed. Results were ultimately binarized e.g., MMR absent = 0 (one or more than one of four MMR proteins missing and confirmed on whole section), or intact = 1 (all four proteins present). Comparison of four protein (MLH1, MSH2, MSH6, PMS2) and two protein (MSH6, PMS2) panel IHC results was also undertaken.
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and published guidelines in interpretation has greatly improved consistency [21–26]. 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.
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Please cite this article as: McConechy MK, et al, Detection of DNA mismatch repair (MMR) deficiencies by immunohistochemistry can effectively diagnose the microsatellite instability..., Gynecol Oncol (2015), http://dx.doi.org/10.1016/j.ygyno.2015.01.541
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Missing
Total
23 (88.5%) 2 (16.7%) 1 (2.0%) 20 (29.4%) 47 (29.9%)
3 (11.5%) 10 (83.3%) 50 (98.0%) 46 (67.6%) 108 (68.8%)
0 (0.0%) 0 (0.0%) 0 (0.0%) 2 (2.9%) 2 (1.3%)
26 (16.6%) 12 (7.6%) 51 (32.5%) 68 (43.3%) 157(100%)
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MSI MSI-high MSI-low MSS Missing Total
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Discussion
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Data directly comparing methods of MSI assessment in Lynchassociated 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 mutations has also been evaluated yielding a sensitivity of 86–100%, specificity of 48–81%, PPV of 10–80% and NPV of 79–100%. PPV improved for both methodologies when testing was restricted to younger cohorts [33–39]. These were population-based or more
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Table 2 Confusion matrix of the complete data (n = 89) where both MMR IHC and MSI assay results were available for comparison of methods.
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taken to be the largest class percentage in the data). The difference between the observed accuracy and that obtained using assignment to the largest class percentage, i.e. the no information rate, is significant (P value b 1.52e − 07). Un-weighted Kappa was 0.84, with the sensitivity (88.5%), specificity (95.2%), PPV (88.5%), and NPV (95.2%). The balanced accuracy, the average between sensitivity and specificity, was 91.8%. Four protein and two protein IHC results were identical (100% concordance) in the cohort. Discordance between MSI assay and MMR IHC was observed for 6 cases in the following scenarios (Table 3): i) Isolated MSH6 loss (MSI-high phenotype on MMR IHC) with MSI assay result MSS (one case), ii) MLH1 and PMS2 loss (MSI-high phenotype on MMR IHC) with MSI assay result MSI-low (two cases), and iii) MMR IHC revealing presence of all proteins (MSS phenotype) with MSI-high per MSI assay (three cases). For discordant cases MLH1 promoter methylation testing was requested. In 3 cases results suggest somatic/epigenetic changes that could account for abnormal MMR IHC. Specifically, in the two cases where MSI assay read was low (MSI-low was grouped with MSS in comparing results of the two methods) and MMR IHC revealed loss of MLH1 and PMS2 (interpreted as MSI-high on IHC) promoter hypermethylation of MLH1 was found explaining the discordant results between the two methods. In one case no promoter methylation was detected, in one the assay failed and one was not sent out in time for publication. The relative frequency of histologic subtype, tumor grade, stage, and age parameters of the total cohort, and within the MSI-high (as determined by MMR IHC) and MSS are shown in Table 4, and are similar to the TCGA endometrial data cohort.
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23 (88.5%) 3 (4.8%) 27(30.4%)
3 (11.5%) 60 (95.2%) 62(69.6%)
26 (29.2%) 63 (70.8%) 89 (100%)
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MSI-high MSI-high MSI-high MSI-high MSI-high MSI-high MSS MSS MSS
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often institution/clinic-based cohorts. There is little data on the number of mutation carriers and best methods of testing in unselected endometrial cancer patients not restricted by age criteria. Collaborative dual or multi-institutional studies that assessed MSI [14,27] used only one method of assessment (MSI assay) and neither MMR IHC nor germline status of the cohort was known for comparison. Direct comparison of the MSI assay to MMR IHC in endometrial cancers has not previously been examined. Our cohort, consists of unselected endometrial cancer cases (“all comers”) with banked frozen tissue (n = 157) and normal material (buffy coat) for comparison (n = 89). We demonstrated a high level of concordance (N93%) between testing methodologies. These results are as good as or better than observed in the CRC series. Concordance rate between the two tests in CRC is estimated at ~92%. Possible weaknesses of both methods that may contribute to imperfect agreement include presence of abnormal MMR genes that are not covered in antibody panels, present but non-functioning MMR genes, antigen degradation, tumor heterogeneity, or the inability for MSI testing to detect MSH6 mutations [21,2]. In a comprehensive review comparing the predictive value of IHC versus MSI testing to determine germline MMR mutation status in CRC it was concluded that methods were equivalent[2]. Advantages of IHC cited included easy availability, efficient testing at the time of colectomy/pathology, ability to identify which gene is mutated (compared with overall tumor status MSI-high
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Table 3 Details and distribution of microsatellite instability (MSI) assay and mismatch repair immunohistochemistry (MMR IHC) results across the cohort. Note: Cases with discordant MSI Assay and MMR IHC results are shown within the box (total of 5). Criteria for MSI-high, MSI-low, and MSS for both MSI assay results and MMR IHC results are outlined in the methods. Presence (denoted as “1”) or loss (denoted as “0”) of each protein tested by IHC is shown. Summary of MSI assay status and MMR IHC status assigned are shaded in gray.
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Table 1 Confusion matrix of the raw data (n = 157) of endometrial cancer cases to undergo MMR IHC and MSI assay.
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Table 4 t4:1 Clinical and pathological characteristics by mismatch repair immunohistochemistry t4:2 (MMR IHC) status (n = 89). t4:3 Total
Age (in years) Mean (SD) Missing Histology Endometrioid Serous Other Missing Tumor grade Grade 1 Grade 2 Grade 3 Missing Stage I II III IV Missing
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62.6 (±12.7) 2 (2.2%)
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71 (79.8%) 12 (13.5%) 4 (4.5%) 2 (2.2%)
22 (81.5%) 2 (7.4%) 1 (3.7%) 2 (7.4%)
49 (79.0%) 10 (16.1%) 3 (4.8%) 0
30 (33.7%) 27 (30.3%) 30 (33.7%) 2 (2.2%)
6 (22.2%) 11 (40.7%) 8 (29.6%) 2 (7.4%)
24 (38.7%) 16 (25.8%) 22 (35.5%) 0
61 (68.5%) 6 (6.7%) 17 (19.1%) 3 (3.4%) 2 (2.2%)
16 (59.3%) 3 (11.1%) 6 (22.2%) 0 (0.0%) 2 (7.4%)
45 (72.6%) 3 (4.8%) 11 (17.7%) 3 (4.8%) 0
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0.2319
0.3843
Please cite this article as: McConechy MK, et al, Detection of DNA mismatch repair (MMR) deficiencies by immunohistochemistry can effectively diagnose the microsatellite instability..., Gynecol Oncol (2015), http://dx.doi.org/10.1016/j.ygyno.2015.01.541
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The authors have not conflict of interest to declare.
Acknowledgments
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Conflict of interest statement
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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Specific methodology costs are comparable but the efficiency, feasibility and level of sophistication required for interpretation favor MMR IHC. Ultimately, if we are to move to practical, affordable, and reproducible categorization of endometrial cancers, MMR IHC would be an integral and very achievable first step [54]. The identification of Lynch syndrome cases would enable risk reducing interventions that may improve outcomes for the index case or her relatives. Future clinical trial design should consider molecular subtypes and MSI-high tumors would ideally be assessed separately both in terms of response to treatment (predictive implications) and outcomes (prognosis). We suggest that MMR IHC is adopted in research, as it has been in the clinical setting as a step towards achieving molecular characterization of endometrial cancer.
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from assay results), and the ability to detect MMR-deficient cases that can potentially be missed by MSI testing, specifically MSH6 mutations that tend to result in weaker or no MSI in the tumors and which are in fact more common in EM cancer than CRC [4]. We had six discordant cases in our series, the details of which are outlined in the results, and the majority of which can be explained with the addition of MLH1 promoter methylation testing or can be attributed to known deficiencies in MSI assay and IHC as outlined for CRCs in the preceding paragraph [21, 34, 36, 38, 40–44]. It is worth highlighting that there are a higher proportion of MSH6 mutations among EC patients (vs. CRC) with Lynch syndrome. As MSH6 carriers have been demonstrated to exhibit a lower frequency of MSI, with assay results MSI-low or even MSS and only demonstrated in the mononucleotide markers. MMR IHC should be the preferred method of testing in order to identify this specific MSI-high phenotype that might otherwise be missed [34, 36, 37, 44, 46, 47] The improved feasibility of MMR IHC for interpretation of MSI-phenotype is evident even in this small series where out of 157 cases MSI assay results were achievable in only 89 cases as no normal DNA was available (buffy coat) for comparison whereas MMR IHC was interpretable in almost 100% of the cohort. Although in this series of cases where archival material is available we could have returned to the hysterectomy specimen for retrieval of “normal” DNA this would require extra cost and effort and highlights the additional deficiency of MSI assay as matched normal may only be available for hysterectomies, and will not be possible for endometrial biopsies. MMR IHC can readily be performed on FFPE endometrial biopsy material. Cost comparison of methodologies should also be considered. Estimates of in-house and commercial costs were outlined above but vary across different health care systems and countries. Published reviews estimate that IHC is approximately threefold less expensive than MSI testing when all components of analysis and interpretation are considered [7]. MMR IHC testing has been adopted by pathologists in our center as the method of choice for the assessment of MSI phenotype in CRC and for endometrial cancers and endometriosis-associated ovarian cancers since January 2012. There are practical implications and potentially empowering opportunities for women with MSI tumors. The literature on the prognostic impact of MSI endometrial cancers is discordant, with both favorable and adverse outcomes reported [14, 19, 8, 49]. The predictive implications of MMR deficient endometrial cancers have not been well characterized but in CRC and ovarian cancers treatment stratification and alternate regimens are recommended [7,15,17,18]. Multiple hereditary cancer referral strategies for MSI tumors have been debated [50] but it is agreed that identification of lynch syndrome is very impactful, both for the index case, and her family members, enabling increased screening, and/or risk reducing surgery. Providing patients and their clinicians with MSI status promptly, as exemplified by MMR IHC results which are dictated concurrently with pathology reports, (usually within 5–10 days of surgery) provides an advantage over off-site requests for MSI assay. Recent investigations in genomic characterization of endometrial cancers support MSI phenotype as a distinct subgroup, with a distinct molecular profile and outcomes [14]. This is consistent with our understanding that inactivation of MMR genes leads to the accumulation of mutations throughout the genome, and can lead to the development of cancer [51]. Given that histomorphologic classification of high grade endometrial cancers is plagued by insoluble problems of irreproducibility [52, 53] molecular classification becomes even more important, and identifying MSI phenotype is a critical part of this classification. In summary, MMR IHC is highly concordant with MSI assay and may be more accurate at identifying MSI phenotype than MSI assay when discordant cases are scrutinized. It can be performed at any pathology laboratory, on FFPE material, in endometrial biopsy specimen (does not require matched normal material), and with fast turnaround.
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References
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[1] Thibodeau SN, Bren G, Schaid D. Microsatellite instability in cancer of the proximal colon. Science 1993;260:816–9. [2] Jiricny J. The multifaceted mismatch-repair system. Nat Rev Mol Cell Biol 2006;7: 335–46. [3] Chui MH, Gilks CB, Cooper K, Clarke BA. Identifying Lynch syndrome in patients with ovarian carcinoma: the significance of tumor subtype. Adv Anat Pathol 2013;20: 378–86. [4] Chui MH, Ryan P, Radigan J, Ferguson SE, Pollett A, Aronson M, et al. The histomorphology of Lynch syndrome-associated ovarian carcinomas: toward a subtypespecific screening strategy. Am J Surg Pathol 2014. [5] Nelson GS, Pink A, Lee S, Han G, Morris D, Ogilvie T, et al. MMR deficiency is common in high-grade endometrioid carcinomas and is associated with an unfavorable outcome. Gynecol Oncol 2013;131:309–14. [6] Rabban JT, Calkins SM, Karnezis AN, Grenert JP, Blanco A, Crawford B, et al. Association of tumor morphology with mismatch-repair protein status in older endometrial cancer patients: implications for universal versus selective screening strategies for Lynch syndrome. Am J Surg Pathol 2014;38:793–800. [7] Xiao X, Melton DW, Gourley C. Mismatch repair deficiency in ovarian cancer—molecular characteristics and clinical implications. Gynecol Oncol 2014;132:506–12. [8] Hadley DW, Jenkins JF, Steinberg SM, Liewehr D, Moller S, Martin JC, et al. Perceptions of cancer risks and predictors of colon and endometrial cancer screening in women undergoing genetic testing for Lynch syndrome. J Clin Oncol 2008;26: 948–54. [9] Lynch HT, Lynch J. Lynch syndrome: genetics, natural history, genetic counseling, and prevention. J Clin Oncol 2000;18:19S–31S. [10] Meyer LA, Broaddus RR, Lu KH. Endometrial cancer and Lynch syndrome: clinical and pathologic considerations. Cancer Control 2009;16:14–22. [11] Schmeler KM, Lu KH. Gynecologic cancers associated with Lynch syndrome/HNPCC. Clin Transl Oncol 2008;10:313–7. [12] Schmeler KM, Lynch HT, Chen LM, Munsell MF, Soliman PT, Clark MB, et al. Prophylactic surgery to reduce the risk of gynecologic cancers in the Lynch syndrome. N Engl J Med 2006;354:261–9. [13] Bonadona V, Bonaiti B, Olschwang S, Grandjouan S, Huiart L, Longy M, et al. Cancer risks associated with germline mutations in MLH1, MSH2, and MSH6 genes in Lynch syndrome. JAMA 2011;305:2304–10. [14] Cancer Genome Atlas Research N, Kandoth C, Schultz N, Cherniack AD, Akbani R, Liu Y, et al. Integrated genomic characterization of endometrial carcinoma. Nature 2013; 497:67–73. [15] Sinicrope FA, Mahoney MR, Smyrk TC, Thibodeau SN, Warren RS, Bertagnolli MM, et al. Prognostic impact of deficient DNA mismatch repair in patients with stage III colon cancer from a randomized trial of FOLFOX-based adjuvant chemotherapy. J Clin Oncol 2013;31:3664–72.
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Please cite this article as: McConechy MK, et al, Detection of DNA mismatch repair (MMR) deficiencies by immunohistochemistry can effectively diagnose the microsatellite instability..., Gynecol Oncol (2015), http://dx.doi.org/10.1016/j.ygyno.2015.01.541
M.K. McConechy et al. / Gynecologic Oncology xxx (2015) xxx–xxx
[28] Grady WM, Rajput A, Lutterbaugh JD, Markowitz S. Detection of aberrantly methylated hMLH1 promoter DNA in the serum of patients with microsatellite unstable colon cancer. Cancer Res 2001;61:900. [29] Computing. RCTRFfS. R: A Language and Environment for Statistical Computing; 2014. [30] Xie Y. Knitr: A General-Purpose Package for Dynamic Report Generation in R. R Package Version 17; 2014. [31] Gordon M. Gmisc: Collection of Functions for Plotting Relations, Generating Tables, and More. R Package Version 0.6.7; 2014. [32] Kuhn M, Contributions from, Wing Jed, Weston Stew, Williams Andre, Keefer Chris, Engelhardt Allan, Cooper Tony, Mayer Zachary, R Core Team. caret: Classification and Regression Training. R package version 6.0-35; 2014. [33] Mercado RC, Hampel H, Kastrinos F, Steyerberg E, Balmana J, Stoffel E, et al. Performance of PREMM(1,2,6), MMRpredict, and MMRpro in detecting Lynch syndrome among endometrial cancer cases. Genet Med 2012;14:670–80. [34] Lu KH, Schorge JO, Rodabaugh KJ, Daniels MS, Sun CC, Soliman PT, et al. Prospective determination of prevalence of lynch syndrome in young women with endometrial cancer. J Clin Oncol 2007;25:5158–64. [35] Berends MJ, Wu Y, Sijmons RH, van der Sluis T, Ek WB, Ligtenberg MJ, et al. Toward new strategies to select young endometrial cancer patients for mismatch repair gene mutation analysis. J Clin Oncol 2003;21:4364–70. [36] Ryan P, Mulligan AM, Aronson M, Ferguson SE, Bapat B, Semotiuk K, et al. Comparison of clinical schemas and morphologic features in predicting Lynch syndrome in mutation-positive patients with endometrial cancer encountered in the context of familial gastrointestinal cancer registries. Cancer 2012;118:681–8. [37] de Leeuw WJ, Dierssen J, Vasen HF, Wijnen JT, Kenter GG, Meijers-Heijboer H, et al. Prediction of a mismatch repair gene defect by microsatellite instability and immunohistochemical analysis in endometrial tumours from HNPCC patients. J Pathol 2000;192:328–35. [38] Kuismanen SA, Moisio AL, Schweizer P, Truninger K, Salovaara R, Arola J, et al. Endometrial and colorectal tumors from patients with hereditary nonpolyposis colon cancer display different patterns of microsatellite instability. Am J Pathol 2002;160:1953–8. [39] Goodfellow PJ, Buttin BM, Herzog TJ, Rader JS, Gibb RK, Swisher E, et al. Prevalence of defective DNA mismatch repair and MSH6 mutation in an unselected series of endometrial cancers. Proc Natl Acad Sci U S A 2003;100:5908–13. [40] Laghi L, Bianchi P, Malesci A. Differences and evolution of the methods for the assessment of microsatellite instability. Oncogene 2008;27:6313–21.
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[16] Sinicrope FA, Foster NR, Thibodeau SN, Marsoni S, Monges G, Labianca R, et al. DNA mismatch repair status and colon cancer recurrence and survival in clinical trials of 5-fluorouracil-based adjuvant therapy. J Natl Cancer Inst 2011;103:863–75. [17] Sargent DJ, Marsoni S, Monges G, Thibodeau SN, Labianca R, Hamilton SR, et al. Defective mismatch repair as a predictive marker for lack of efficacy of fluorouracil-based adjuvant therapy in colon cancer. J Clin Oncol 2010;28:3219–26. [18] Zaanan A, Flejou JF, Emile JF, Des GG, Cuilliere-Dartigues P, Malka D, et al. Defective mismatch repair status as a prognostic biomarker of disease-free survival in stage III colon cancer patients treated with adjuvant FOLFOX chemotherapy. Clin Cancer Res 2011;17:7470–8. [19] Diaz-Padilla I, Romero N, Amir E, Matias-Guiu X, Vilar E, Muggia F, et al. Mismatch repair status and clinical outcome in endometrial cancer: a systematic review and meta-analysis. Crit Rev Oncol Hematol 2013;88:154–67. [20] Shih KK, Garg K, Levine DA, Kauff ND, Abu-Rustum NR, Soslow RA, et al. Clinicopathologic significance of DNA mismatch repair protein defects and endometrial cancer in women 40 years of age and younger. Gynecol Oncol 2011;123:88–94. [21] Shia J, Ellis NA, Klimstra DS. The utility of immunohistochemical detection of DNA mismatch repair gene proteins. Virchows Arch 2004;445:431–41. [22] Shia J, Klimstra DS, Nafa K, Offit K, Guillem JG, Markowitz AJ, et al. Value of immunohistochemical detection of DNA mismatch repair proteins in predicting germline mutation in hereditary colorectal neoplasms. Am J Surg Pathol 2005;29:96–104. [23] Muller A, Giuffre G, Edmonston TB, Mathiak M, Roggendorf B, Heinmoller E, et al. Challenges and pitfalls in HNPCC screening by microsatellite analysis and immunohistochemistry. J Mol Diagn 2004;6:308–15. [24] Arends M IM, Happerfield S, Frayling I, Miller K. Interpretation of immunohistochemical analysis of mismatch repair (MMR) protein expression in tissue sections for investigation of suspected Lynch/Hereditary Non-Polyposis Colorectal Cancer (HNPCC) Syndrome. UKNEQAS ICC & ISH Recommendations. [25] Overbeek LI, Ligtenberg MJ, Willems RW, Hermens RP, Blokx WA, Dubois SV, et al. Interpretation of immunohistochemistry for mismatch repair proteins is only reliable in a specialized setting. Am J Surg Pathol 2008;32:1246–51. [26] Klarskov L, Ladelund S, Holck S, Roenlund K, Lindebjerg J, Elebro J, et al. Interobserver variability in the evaluation of mismatch repair protein immunostaining. Hum Pathol 2010;41:1387–96. [27] McConechy MK, Ding J, Cheang MC, Wiegand KC, Senz J, Tone AA, et al. Use of mutation profiles to refine the classification of endometrial carcinomas. J Pathol 2012; 228:20–30.
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Please cite this article as: McConechy MK, et al, Detection of DNA mismatch repair (MMR) deficiencies by immunohistochemistry can effectively diagnose the microsatellite instability..., Gynecol Oncol (2015), http://dx.doi.org/10.1016/j.ygyno.2015.01.541
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M.K. McConechy a,c, A. Talhouk a, H.H. Li-Chang a, S. Leung b, D.G. Huntsman a, C.B. Gilks a, J.N. McAlpine a,⁎
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Keywords: Mismatch repair Microsatellite instability Lynch syndrome Endometrial cancer
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Department of Pathology and Laboratory Medicine, University of British Columbia and BC Cancer Agency, 509-2660 Oak Street, Vancouver V6H 3Z6, BC, Canada Genetic Pathology Evaluation Centre, Department of Pathology and Laboratory Medicine, University of British Columbia, 509-2660 Oak Street, Vancouver V6H 3Z6, BC, Canada Department of Gynecology and Obstetrics, Division of Gynecologic Oncology, University of British Columbia, 2775 Laurel St. 6th Floor, Vancouver V5Z 1M9, BC, Canada
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Background. A proportion of endometrial carcinomas (ECs) are associated with deficient DNA mismatch repair (MMR). These tumors are characterized by high levels of microsatellite instability (MSI). Identification of MSI is important in identifying women who should be tested for Lynch syndrome and identifying a phenotype that may have specific prognostic and predictive implications. Genomic characterization of ECs has shown that MSI tumors form a distinct subgroup. The two most common methodologies for MSI assessment have not been compared in EC. Methods. Pentaplex mononucleotide PCR for MSI testing was compared to MMR IHC (presence/absence of MLH1, MSH2, MSH6, PMS2) in a cohort of patients with EC. Concordance, Kappa statistic, sensitivity, specificity, positive and negative predictive values were obtained on the cross-tabulation of results. Results. Comparison of both MSI and MMR status was complete for 89 cases. Overall agreement between methods (concordance) was 93.3% (95% CI[85.9%–97.5%]). A one-sided test to determine whether the accuracy is better than the “no information rate,” which is taken to be the largest class percentage in the data, is significant (p b 0.00001). Unweighted Kappa was 0.84, along with the sensitivity (88.5%), specificity (95.2%), PPV (88.5%), and NPV (95.2%). The balanced accuracy (i.e. the average between sensitivity and specificity) was 92%. Discussion. We show the equivalence of MSI testing and MMR IHC. We advocate the implementation of MMR IHC in future EC classification schemes, enabling stratification of cases for future clinical trials as well as assisting identification of Lynch syndrome, so that screening and risk reducing interventions can be undertaken. © 2015 Published by Elsevier Inc.
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Article history: Received 29 November 2014 Accepted 21 January 2015 Available online xxxx
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Detection of DNA mismatch repair (MMR) deficiencies by immunohistochemistry can effectively diagnose the microsatellite instability (MSI) phenotype in endometrial carcinomas
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Introduction
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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
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⁎ Corresponding author at: Department of Gynecology and Obstetrics, Division of Gynecologic Oncology, 2775 Laurel St, 6th Fl, Vancouver V5Z-1M9, BC, Canada. Fax: +1 604 875 4869. E-mail address: [email protected] (J.N. McAlpine).
recognized within specific histologic subtypes of ovarian cancer and endometrial cancers [3–7]. 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 [8–13]. 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 [7,14–20]. 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
http://dx.doi.org/10.1016/j.ygyno.2015.01.541 0090-8258/© 2015 Published by Elsevier Inc.
Please cite this article as: McConechy MK, et al, Detection of DNA mismatch repair (MMR) deficiencies by immunohistochemistry can effectively diagnose the microsatellite instability..., Gynecol Oncol (2015), http://dx.doi.org/10.1016/j.ygyno.2015.01.541
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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 biospecimens for research purposes.
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DNA isolation
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Genomic DNA was extracted from flash frozen tumors from hysterectomy specimens using the Ambion DNA extraction kit as per manufacturer's protocol (Ambion, Life Technologies) and as previously described [27].
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Microsatellite instability (MSI) assay
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The analysis of tumor MSI was performed following the recommendations of the National Cancer Institute (NCI). The Bethesda Marker panel of five microsatellite loci; Bat 25, Bat 26, DI7S250, D5S346, and D2S123 were used to assess the MSI status. MSI-high is reported if 2 or more markers have MSI, 1 or less markers with MSI were considered MSI-low, and MSS (Microsatellite Stable) indicates that no markers were positive. Cases reported as N/A did not have sufficient normal available to assess the MSI status of these tumors. Details of these methods have been previously reported [27]. Results of MSI assay were then binarized as MSI-high or MSS with MSI-low considered MSS.
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Tissue microarray (TMA) and immunohistochemistry
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To construct the endometrial TMA, tumors were annotated by a pathologist (BG) and two 0.6 mm cores per case were arrayed. TMAs were cut at 4 μm thickness onto Superfrost + glass slides, and were processed
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MLH1 promoter methylation testing
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Cases where discordance was noted between MSI assay results and MMR IHC were sent for testing for MLH1 promoter methylation at Mayo Medical Laboratories, Rochester, MN who perform a PCR-based assay to test tumor DNA for the presence of hypermethylation of the MLH1 promoter. This is a modification of the method described by Grady et al. [28].
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The data under consideration consists of two assignment of MSIStatus on 157 cases of EC; one of the assignments was done using the MSI assay and one using MMR. We were interested in measuring the degree of agreement between the two methods, while accounting for the agreement that occurs by chance. We began by calculating concordance. We then calculated reliability statistics such as Cohen's kappa, the accuracy of MMR relative to the MSI assay, the sensitivity, specificity, and the positive and negative predictive value. We also used a test statistic to compare the observed accuracy using MMR relative to the no information rate, which is the accuracy obtained by a method that would assign all samples to the largest class. P values smaller than 0.05 were considered statistically significant. All statistical analyses were performed using R (ver. 3.1.0) [29] and packages caret (ver. 6.0-30) for reliability analysis, Gmisc for plot and table output (ver. 0.6.4), and knitr (ver 1.6) [30–32] for reproducible research. For comparison of clinicopathologic factors in the MSI-high vs. MSS cohorts (as assessed by both methods) P-values were computed using a one way analysis of variance in the case of continuous variables (Age) and Fisher's exact test with Monte Carlo simulated P-values for categorical variables.
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Results
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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 (Tables 1 and 2). The overall accuracy is 93.3% with a 95% confidence interval of (85.9%–97.5%). A one-sided test was conducted to see if the accuracy is better than the “no information rate” (in this case 70.8% which is
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using the Ventana Discovery XT, and the Ventana Benchmark XT automated system (Ventana Medical Systems, Tucson, AZ, USA) as per manufacturer's protocol with proprietary reagents. After slides were baked at 60 °C for 1 h, they were deparaffinized on the automated system with EZ Prep solution (Ventana). Heat induced antigen retrieval method was used in Cell Conditioning solution (CC1-Tris based EDTA buffer, pH 8.0, Ventana). Anti-hMLH1 (1:150, ES05, Novacastra, Vector Labs Burlington ON), hMSH2 (1:100, 25D12, Novacastra, Vector Labs), hMSH6 (1:600, PU29, Novacastra, Vector Labs), nadhPMS@ (1:150, MOR4G, Novacastra, Vector Labs), with appropriate positive and negative controls according to the manufacturers' standardized staining protocols. Tumor was considered aberrant if tumor cells showed complete absence of nuclear staining with positive non-neoplastic internal control, and intact if tumor cells show nuclear positivity. Aberrant, equivocal, and uninterpretable cases (e.g., no tumor tissue) on TMA had whole sections requisitioned for repeat staining of the MMR protein or proteins needed. Results were ultimately binarized e.g., MMR absent = 0 (one or more than one of four MMR proteins missing and confirmed on whole section), or intact = 1 (all four proteins present). Comparison of four protein (MLH1, MSH2, MSH6, PMS2) and two protein (MSH6, PMS2) panel IHC results was also undertaken.
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and published guidelines in interpretation has greatly improved consistency [21–26]. 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.
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Please cite this article as: McConechy MK, et al, Detection of DNA mismatch repair (MMR) deficiencies by immunohistochemistry can effectively diagnose the microsatellite instability..., Gynecol Oncol (2015), http://dx.doi.org/10.1016/j.ygyno.2015.01.541
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Missing
Total
23 (88.5%) 2 (16.7%) 1 (2.0%) 20 (29.4%) 47 (29.9%)
3 (11.5%) 10 (83.3%) 50 (98.0%) 46 (67.6%) 108 (68.8%)
0 (0.0%) 0 (0.0%) 0 (0.0%) 2 (2.9%) 2 (1.3%)
26 (16.6%) 12 (7.6%) 51 (32.5%) 68 (43.3%) 157(100%)
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Discussion
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Data directly comparing methods of MSI assessment in Lynchassociated 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 mutations has also been evaluated yielding a sensitivity of 86–100%, specificity of 48–81%, PPV of 10–80% and NPV of 79–100%. PPV improved for both methodologies when testing was restricted to younger cohorts [33–39]. These were population-based or more
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Table 2 Confusion matrix of the complete data (n = 89) where both MMR IHC and MSI assay results were available for comparison of methods.
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taken to be the largest class percentage in the data). The difference between the observed accuracy and that obtained using assignment to the largest class percentage, i.e. the no information rate, is significant (P value b 1.52e − 07). Un-weighted Kappa was 0.84, with the sensitivity (88.5%), specificity (95.2%), PPV (88.5%), and NPV (95.2%). The balanced accuracy, the average between sensitivity and specificity, was 91.8%. Four protein and two protein IHC results were identical (100% concordance) in the cohort. Discordance between MSI assay and MMR IHC was observed for 6 cases in the following scenarios (Table 3): i) Isolated MSH6 loss (MSI-high phenotype on MMR IHC) with MSI assay result MSS (one case), ii) MLH1 and PMS2 loss (MSI-high phenotype on MMR IHC) with MSI assay result MSI-low (two cases), and iii) MMR IHC revealing presence of all proteins (MSS phenotype) with MSI-high per MSI assay (three cases). For discordant cases MLH1 promoter methylation testing was requested. In 3 cases results suggest somatic/epigenetic changes that could account for abnormal MMR IHC. Specifically, in the two cases where MSI assay read was low (MSI-low was grouped with MSS in comparing results of the two methods) and MMR IHC revealed loss of MLH1 and PMS2 (interpreted as MSI-high on IHC) promoter hypermethylation of MLH1 was found explaining the discordant results between the two methods. In one case no promoter methylation was detected, in one the assay failed and one was not sent out in time for publication. The relative frequency of histologic subtype, tumor grade, stage, and age parameters of the total cohort, and within the MSI-high (as determined by MMR IHC) and MSS are shown in Table 4, and are similar to the TCGA endometrial data cohort.
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often institution/clinic-based cohorts. There is little data on the number of mutation carriers and best methods of testing in unselected endometrial cancer patients not restricted by age criteria. Collaborative dual or multi-institutional studies that assessed MSI [14,27] used only one method of assessment (MSI assay) and neither MMR IHC nor germline status of the cohort was known for comparison. Direct comparison of the MSI assay to MMR IHC in endometrial cancers has not previously been examined. Our cohort, consists of unselected endometrial cancer cases (“all comers”) with banked frozen tissue (n = 157) and normal material (buffy coat) for comparison (n = 89). We demonstrated a high level of concordance (N93%) between testing methodologies. These results are as good as or better than observed in the CRC series. Concordance rate between the two tests in CRC is estimated at ~92%. Possible weaknesses of both methods that may contribute to imperfect agreement include presence of abnormal MMR genes that are not covered in antibody panels, present but non-functioning MMR genes, antigen degradation, tumor heterogeneity, or the inability for MSI testing to detect MSH6 mutations [21,2]. In a comprehensive review comparing the predictive value of IHC versus MSI testing to determine germline MMR mutation status in CRC it was concluded that methods were equivalent[2]. Advantages of IHC cited included easy availability, efficient testing at the time of colectomy/pathology, ability to identify which gene is mutated (compared with overall tumor status MSI-high
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Table 3 Details and distribution of microsatellite instability (MSI) assay and mismatch repair immunohistochemistry (MMR IHC) results across the cohort. Note: Cases with discordant MSI Assay and MMR IHC results are shown within the box (total of 5). Criteria for MSI-high, MSI-low, and MSS for both MSI assay results and MMR IHC results are outlined in the methods. Presence (denoted as “1”) or loss (denoted as “0”) of each protein tested by IHC is shown. Summary of MSI assay status and MMR IHC status assigned are shaded in gray.
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Table 1 Confusion matrix of the raw data (n = 157) of endometrial cancer cases to undergo MMR IHC and MSI assay.
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Table 4 t4:1 Clinical and pathological characteristics by mismatch repair immunohistochemistry t4:2 (MMR IHC) status (n = 89). t4:3 Total
Age (in years) Mean (SD) Missing Histology Endometrioid Serous Other Missing Tumor grade Grade 1 Grade 2 Grade 3 Missing Stage I II III IV Missing
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71 (79.8%) 12 (13.5%) 4 (4.5%) 2 (2.2%)
22 (81.5%) 2 (7.4%) 1 (3.7%) 2 (7.4%)
49 (79.0%) 10 (16.1%) 3 (4.8%) 0
30 (33.7%) 27 (30.3%) 30 (33.7%) 2 (2.2%)
6 (22.2%) 11 (40.7%) 8 (29.6%) 2 (7.4%)
24 (38.7%) 16 (25.8%) 22 (35.5%) 0
61 (68.5%) 6 (6.7%) 17 (19.1%) 3 (3.4%) 2 (2.2%)
16 (59.3%) 3 (11.1%) 6 (22.2%) 0 (0.0%) 2 (7.4%)
45 (72.6%) 3 (4.8%) 11 (17.7%) 3 (4.8%) 0
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Please cite this article as: McConechy MK, et al, Detection of DNA mismatch repair (MMR) deficiencies by immunohistochemistry can effectively diagnose the microsatellite instability..., Gynecol Oncol (2015), http://dx.doi.org/10.1016/j.ygyno.2015.01.541
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The authors have not conflict of interest to declare.
Acknowledgments
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Conflict of interest statement
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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Specific methodology costs are comparable but the efficiency, feasibility and level of sophistication required for interpretation favor MMR IHC. Ultimately, if we are to move to practical, affordable, and reproducible categorization of endometrial cancers, MMR IHC would be an integral and very achievable first step [54]. The identification of Lynch syndrome cases would enable risk reducing interventions that may improve outcomes for the index case or her relatives. Future clinical trial design should consider molecular subtypes and MSI-high tumors would ideally be assessed separately both in terms of response to treatment (predictive implications) and outcomes (prognosis). We suggest that MMR IHC is adopted in research, as it has been in the clinical setting as a step towards achieving molecular characterization of endometrial cancer.
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from assay results), and the ability to detect MMR-deficient cases that can potentially be missed by MSI testing, specifically MSH6 mutations that tend to result in weaker or no MSI in the tumors and which are in fact more common in EM cancer than CRC [4]. We had six discordant cases in our series, the details of which are outlined in the results, and the majority of which can be explained with the addition of MLH1 promoter methylation testing or can be attributed to known deficiencies in MSI assay and IHC as outlined for CRCs in the preceding paragraph [21, 34, 36, 38, 40–44]. It is worth highlighting that there are a higher proportion of MSH6 mutations among EC patients (vs. CRC) with Lynch syndrome. As MSH6 carriers have been demonstrated to exhibit a lower frequency of MSI, with assay results MSI-low or even MSS and only demonstrated in the mononucleotide markers. MMR IHC should be the preferred method of testing in order to identify this specific MSI-high phenotype that might otherwise be missed [34, 36, 37, 44, 46, 47] The improved feasibility of MMR IHC for interpretation of MSI-phenotype is evident even in this small series where out of 157 cases MSI assay results were achievable in only 89 cases as no normal DNA was available (buffy coat) for comparison whereas MMR IHC was interpretable in almost 100% of the cohort. Although in this series of cases where archival material is available we could have returned to the hysterectomy specimen for retrieval of “normal” DNA this would require extra cost and effort and highlights the additional deficiency of MSI assay as matched normal may only be available for hysterectomies, and will not be possible for endometrial biopsies. MMR IHC can readily be performed on FFPE endometrial biopsy material. Cost comparison of methodologies should also be considered. Estimates of in-house and commercial costs were outlined above but vary across different health care systems and countries. Published reviews estimate that IHC is approximately threefold less expensive than MSI testing when all components of analysis and interpretation are considered [7]. MMR IHC testing has been adopted by pathologists in our center as the method of choice for the assessment of MSI phenotype in CRC and for endometrial cancers and endometriosis-associated ovarian cancers since January 2012. There are practical implications and potentially empowering opportunities for women with MSI tumors. The literature on the prognostic impact of MSI endometrial cancers is discordant, with both favorable and adverse outcomes reported [14, 19, 8, 49]. The predictive implications of MMR deficient endometrial cancers have not been well characterized but in CRC and ovarian cancers treatment stratification and alternate regimens are recommended [7,15,17,18]. Multiple hereditary cancer referral strategies for MSI tumors have been debated [50] but it is agreed that identification of lynch syndrome is very impactful, both for the index case, and her family members, enabling increased screening, and/or risk reducing surgery. Providing patients and their clinicians with MSI status promptly, as exemplified by MMR IHC results which are dictated concurrently with pathology reports, (usually within 5–10 days of surgery) provides an advantage over off-site requests for MSI assay. Recent investigations in genomic characterization of endometrial cancers support MSI phenotype as a distinct subgroup, with a distinct molecular profile and outcomes [14]. This is consistent with our understanding that inactivation of MMR genes leads to the accumulation of mutations throughout the genome, and can lead to the development of cancer [51]. Given that histomorphologic classification of high grade endometrial cancers is plagued by insoluble problems of irreproducibility [52, 53] molecular classification becomes even more important, and identifying MSI phenotype is a critical part of this classification. In summary, MMR IHC is highly concordant with MSI assay and may be more accurate at identifying MSI phenotype than MSI assay when discordant cases are scrutinized. It can be performed at any pathology laboratory, on FFPE material, in endometrial biopsy specimen (does not require matched normal material), and with fast turnaround.
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[1] Thibodeau SN, Bren G, Schaid D. Microsatellite instability in cancer of the proximal colon. Science 1993;260:816–9. [2] Jiricny J. The multifaceted mismatch-repair system. Nat Rev Mol Cell Biol 2006;7: 335–46. [3] Chui MH, Gilks CB, Cooper K, Clarke BA. Identifying Lynch syndrome in patients with ovarian carcinoma: the significance of tumor subtype. Adv Anat Pathol 2013;20: 378–86. [4] Chui MH, Ryan P, Radigan J, Ferguson SE, Pollett A, Aronson M, et al. The histomorphology of Lynch syndrome-associated ovarian carcinomas: toward a subtypespecific screening strategy. Am J Surg Pathol 2014. [5] Nelson GS, Pink A, Lee S, Han G, Morris D, Ogilvie T, et al. MMR deficiency is common in high-grade endometrioid carcinomas and is associated with an unfavorable outcome. Gynecol Oncol 2013;131:309–14. [6] Rabban JT, Calkins SM, Karnezis AN, Grenert JP, Blanco A, Crawford B, et al. Association of tumor morphology with mismatch-repair protein status in older endometrial cancer patients: implications for universal versus selective screening strategies for Lynch syndrome. Am J Surg Pathol 2014;38:793–800. [7] Xiao X, Melton DW, Gourley C. Mismatch repair deficiency in ovarian cancer—molecular characteristics and clinical implications. Gynecol Oncol 2014;132:506–12. [8] Hadley DW, Jenkins JF, Steinberg SM, Liewehr D, Moller S, Martin JC, et al. Perceptions of cancer risks and predictors of colon and endometrial cancer screening in women undergoing genetic testing for Lynch syndrome. J Clin Oncol 2008;26: 948–54. [9] Lynch HT, Lynch J. Lynch syndrome: genetics, natural history, genetic counseling, and prevention. J Clin Oncol 2000;18:19S–31S. [10] Meyer LA, Broaddus RR, Lu KH. Endometrial cancer and Lynch syndrome: clinical and pathologic considerations. Cancer Control 2009;16:14–22. [11] Schmeler KM, Lu KH. Gynecologic cancers associated with Lynch syndrome/HNPCC. Clin Transl Oncol 2008;10:313–7. [12] Schmeler KM, Lynch HT, Chen LM, Munsell MF, Soliman PT, Clark MB, et al. Prophylactic surgery to reduce the risk of gynecologic cancers in the Lynch syndrome. N Engl J Med 2006;354:261–9. [13] Bonadona V, Bonaiti B, Olschwang S, Grandjouan S, Huiart L, Longy M, et al. Cancer risks associated with germline mutations in MLH1, MSH2, and MSH6 genes in Lynch syndrome. JAMA 2011;305:2304–10. [14] Cancer Genome Atlas Research N, Kandoth C, Schultz N, Cherniack AD, Akbani R, Liu Y, et al. Integrated genomic characterization of endometrial carcinoma. Nature 2013; 497:67–73. [15] Sinicrope FA, Mahoney MR, Smyrk TC, Thibodeau SN, Warren RS, Bertagnolli MM, et al. Prognostic impact of deficient DNA mismatch repair in patients with stage III colon cancer from a randomized trial of FOLFOX-based adjuvant chemotherapy. J Clin Oncol 2013;31:3664–72.
349 350 351 352 353 354 355 356 357 358 Q11 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390
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[28] Grady WM, Rajput A, Lutterbaugh JD, Markowitz S. Detection of aberrantly methylated hMLH1 promoter DNA in the serum of patients with microsatellite unstable colon cancer. Cancer Res 2001;61:900. [29] Computing. RCTRFfS. R: A Language and Environment for Statistical Computing; 2014. [30] Xie Y. Knitr: A General-Purpose Package for Dynamic Report Generation in R. R Package Version 17; 2014. [31] Gordon M. Gmisc: Collection of Functions for Plotting Relations, Generating Tables, and More. R Package Version 0.6.7; 2014. [32] Kuhn M, Contributions from, Wing Jed, Weston Stew, Williams Andre, Keefer Chris, Engelhardt Allan, Cooper Tony, Mayer Zachary, R Core Team. caret: Classification and Regression Training. R package version 6.0-35; 2014. [33] Mercado RC, Hampel H, Kastrinos F, Steyerberg E, Balmana J, Stoffel E, et al. Performance of PREMM(1,2,6), MMRpredict, and MMRpro in detecting Lynch syndrome among endometrial cancer cases. Genet Med 2012;14:670–80. [34] Lu KH, Schorge JO, Rodabaugh KJ, Daniels MS, Sun CC, Soliman PT, et al. Prospective determination of prevalence of lynch syndrome in young women with endometrial cancer. J Clin Oncol 2007;25:5158–64. [35] Berends MJ, Wu Y, Sijmons RH, van der Sluis T, Ek WB, Ligtenberg MJ, et al. Toward new strategies to select young endometrial cancer patients for mismatch repair gene mutation analysis. J Clin Oncol 2003;21:4364–70. [36] Ryan P, Mulligan AM, Aronson M, Ferguson SE, Bapat B, Semotiuk K, et al. Comparison of clinical schemas and morphologic features in predicting Lynch syndrome in mutation-positive patients with endometrial cancer encountered in the context of familial gastrointestinal cancer registries. Cancer 2012;118:681–8. [37] de Leeuw WJ, Dierssen J, Vasen HF, Wijnen JT, Kenter GG, Meijers-Heijboer H, et al. Prediction of a mismatch repair gene defect by microsatellite instability and immunohistochemical analysis in endometrial tumours from HNPCC patients. J Pathol 2000;192:328–35. [38] Kuismanen SA, Moisio AL, Schweizer P, Truninger K, Salovaara R, Arola J, et al. Endometrial and colorectal tumors from patients with hereditary nonpolyposis colon cancer display different patterns of microsatellite instability. Am J Pathol 2002;160:1953–8. [39] Goodfellow PJ, Buttin BM, Herzog TJ, Rader JS, Gibb RK, Swisher E, et al. Prevalence of defective DNA mismatch repair and MSH6 mutation in an unselected series of endometrial cancers. Proc Natl Acad Sci U S A 2003;100:5908–13. [40] Laghi L, Bianchi P, Malesci A. Differences and evolution of the methods for the assessment of microsatellite instability. Oncogene 2008;27:6313–21.
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F
[16] Sinicrope FA, Foster NR, Thibodeau SN, Marsoni S, Monges G, Labianca R, et al. DNA mismatch repair status and colon cancer recurrence and survival in clinical trials of 5-fluorouracil-based adjuvant therapy. J Natl Cancer Inst 2011;103:863–75. [17] Sargent DJ, Marsoni S, Monges G, Thibodeau SN, Labianca R, Hamilton SR, et al. Defective mismatch repair as a predictive marker for lack of efficacy of fluorouracil-based adjuvant therapy in colon cancer. J Clin Oncol 2010;28:3219–26. [18] Zaanan A, Flejou JF, Emile JF, Des GG, Cuilliere-Dartigues P, Malka D, et al. Defective mismatch repair status as a prognostic biomarker of disease-free survival in stage III colon cancer patients treated with adjuvant FOLFOX chemotherapy. Clin Cancer Res 2011;17:7470–8. [19] Diaz-Padilla I, Romero N, Amir E, Matias-Guiu X, Vilar E, Muggia F, et al. Mismatch repair status and clinical outcome in endometrial cancer: a systematic review and meta-analysis. Crit Rev Oncol Hematol 2013;88:154–67. [20] Shih KK, Garg K, Levine DA, Kauff ND, Abu-Rustum NR, Soslow RA, et al. Clinicopathologic significance of DNA mismatch repair protein defects and endometrial cancer in women 40 years of age and younger. Gynecol Oncol 2011;123:88–94. [21] Shia J, Ellis NA, Klimstra DS. The utility of immunohistochemical detection of DNA mismatch repair gene proteins. Virchows Arch 2004;445:431–41. [22] Shia J, Klimstra DS, Nafa K, Offit K, Guillem JG, Markowitz AJ, et al. Value of immunohistochemical detection of DNA mismatch repair proteins in predicting germline mutation in hereditary colorectal neoplasms. Am J Surg Pathol 2005;29:96–104. [23] Muller A, Giuffre G, Edmonston TB, Mathiak M, Roggendorf B, Heinmoller E, et al. Challenges and pitfalls in HNPCC screening by microsatellite analysis and immunohistochemistry. J Mol Diagn 2004;6:308–15. [24] Arends M IM, Happerfield S, Frayling I, Miller K. Interpretation of immunohistochemical analysis of mismatch repair (MMR) protein expression in tissue sections for investigation of suspected Lynch/Hereditary Non-Polyposis Colorectal Cancer (HNPCC) Syndrome. UKNEQAS ICC & ISH Recommendations. [25] Overbeek LI, Ligtenberg MJ, Willems RW, Hermens RP, Blokx WA, Dubois SV, et al. Interpretation of immunohistochemistry for mismatch repair proteins is only reliable in a specialized setting. Am J Surg Pathol 2008;32:1246–51. [26] Klarskov L, Ladelund S, Holck S, Roenlund K, Lindebjerg J, Elebro J, et al. Interobserver variability in the evaluation of mismatch repair protein immunostaining. Hum Pathol 2010;41:1387–96. [27] McConechy MK, Ding J, Cheang MC, Wiegand KC, Senz J, Tone AA, et al. Use of mutation profiles to refine the classification of endometrial carcinomas. J Pathol 2012; 228:20–30.
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