Mismatch Repair Immunohistochemistry Research Articles

No Racial Disparities Observed Using Point-of-Care Genetic Counseling and Testing for Endometrial and Ovarian Cancer in a Diverse Patient Population: A Retrospective Cohort Study.

We investigated genetic counseling and testing rates for patients with gynecologic malignancy at a tertiary care center with a large minority population. Our retrospective cohort included newly diagnosed epithelial ovarian, fallopian tube, peritoneal, or endometrial cancer patients between January 2014 and June 2022. For endometrial cancer, 373 patients were identified. A total of 207 (55%) patients were screened using mismatch repair immunohistochemistry (MMR IHC). A total of 82 (40%) had MMR deficiencies on IHC. Of these, 63 (77%) received genetic counseling. A total of 62 (98%) underwent genetic testing, and ultimately, 7 (11%) were diagnosed with Lynch syndrome (LS). The overall rate of LS was 1.9%. MMR IHC testing increased steadily, reaching 100% in 2022. For ovarian cancer, 144 patients were identified. A total of 104 (72%) patients received genetic counseling, and 99 (95%) underwent genetic testing. Rates were not influenced by race, ethnicity, insurance type, or family history of cancer. They were significantly different by cancer stage (p < 0.01). The proportion of patients who received genetic counseling increased from 47% in 2015 to 100% in 2022 (p < 0.01). Most counseling was performed by a gynecologic oncologist (93%) as opposed to a genetic counselor (6.7%). Overall, 12 (8.3%) patients were BRCA+. High rates of counseling and testing were observed with few disparities.

Challenging Standard Protocols: A New Perspective on MMR Deficiency from a Longitudinal Regional Study

The role of DNA mismatch repair (MMR) tumor testing in colon and endometrial carcinomas is well-established. However, the universal application of this testing faces challenges. These challenges are due to limited data on its efficacy in diverse populations. This retrospective study was conducted at a community hospital in Northern Indiana, USA. It examined MMR immunohistochemistry (IHC) results across 549 cases. Our analysis found that 469 cases had intact MMR expression. Meanwhile, 80 cases demonstrated MMR deficiency. This translates to MMR deficiency rates of 10% in colon cancers and 22% in uterine carcinomas. Notably, only 16 patients (2.9%) were categorized as having high microsatellite instability (MSI-H). This rate is significantly lower than the 3-5-fold higher incidences reported in other Midwestern populations, including Ohio. These findings suggest that the benefits of universal cascade methylation testing may vary significantly. This variation is particularly notable in MLH1/PMS2 IHC-negative tumors, and it depends heavily on regional demographics. The notably lower MSI-H incidence in this study suggests a need to re-evaluate reflex testing protocols in low-resource settings. A more targeted, cost-effective approach could be developed without compromising diagnostic accuracy. This study challenges the one-size-fits-all approach to MMR testing. It highlights the need for tailored strategies that consider regional variations in cancer genetics

Microsatellite instability and mismatch repair protein deficiency: equal predictive markers?

Current clinical guidelines recommend mismatch repair (MMR) protein immunohistochemistry (IHC) or molecular microsatellite instability (MSI) tests as predictive markers of immunotherapies. Most of the pathological guidelines consider MMR protein IHC as the gold standard test to identify cancers with MMR deficiency and recommend molecular MSI tests only in special circumstances or to screen for Lynch syndrome. However, there are data in the literature which suggest that the two test types may not be equal. For example, molecular epidemiology studies reported different rates of deficient MMR (dMMR) and MSI in various cancer types. Additionally, direct comparisons of the two tests revealed relatively frequent discrepancies between MMR IHC and MSI tests, especially in non-colorectal and non-endometrial cancers and in cases with unusual dMMR phenotypes. There are also scattered clinical data showing that the efficacy of immune checkpoint inhibitors is different if the patient selection was based on dMMR versus MSI status of the cancers. All these observations question the current dogma that dMMR phenotype and genetic MSI status are equal predictive markers of the immunotherapies.

Prognosis and immunotherapy efficacy in dMMR&MSS colorectal cancer patients and an MSI status predicting model.

The inconsistency between mismatch repair (MMR) protein immunohistochemistry (IHC) and microsatellite instability PCR (MSI-PCR) methods has been widely reported. We aim to investigate the prognosis and the effect of immunotherapy in dMMR by IHC but MSS by MSI-PCR (dMMR&MSS) colorectal cancer (CRC) patients. A microsatellite instability (MSI) predicting model was established to help find dMMR&MSS patients. MMR and MSI states were detected by the IHC and MSI-PCR in 1622 CRC patients (ZS6Y-1 cohort). Logistic regression analysis was used to screen clinical features to construct an MSI-predicting nomogram. We propose a new nomogram-based assay to find patients with dMMR&MSS, in which the MSI-PCR assay only detects dMMR patients with MSS predictive results. We applied the new strategy to a random cohort of 248 CRC patients (ZS6Y-2 cohort). The consistency of MMR IHC and MSI-PCR in the ZS6Y-1 cohort was 95.7% (1553/1622). Both pMMR&MSS and dMMR&MSS groups experienced significantly shorter overall survival (OS) than those in dMMR by IHC and MSI-H by MSI-PCR (dMMR&MSI-H) group (hazard ratio [HR] = 2.429, 95% confidence interval [CI]: 1.89-3.116, p < .01; HR = 21.96, 95% CI: 7.24-66.61, p < .01). The dMMR&MSS group experienced shorter OS than the pMMR&MSS group, but the difference did not reach significance (log rank test, p = .0686). In the immunotherapy group, the progression-free survival of dMMR&MSS patients was significantly shorter than that of dMMR&MSI-H patients (HR = 13.83, 95% CI: 1.508-126.8, p < .05). The ZS6Y-MSI-Pre nomogram (C-index = 0.816, 95% CI: 0.792-0.841, already online) found 66% (2/3) dMMR&MSS patients in the ZS6Y-2 cohort. There are significant differences in OS and immunotherapy effect between dMMR&MSI-H and dMMR&MSS patients. Our prediction model provides an economical way to screen dMMR&MSS patients.

Implementation of endometrial cancer molecular subtyping into a hybrid community-academic practice.

We sought to confirm utility of our institution's modified Proactive Molecular Risk Classifier for Endometrial Cancer protocol in our daily practice, which includes mismatch repair (MMR), p53, and L1 cell adhesion molecule (L1CAM) immunohistochemistry with in-house next-generation sequencing for POLE, TP53, and CTNNB1. We conducted a retrospective review of all patients in our institution who underwent primary endometrial carcinoma resection from the year prior to protocol implementation (PRE; October 1, 2020, to September 30, 2021) through first year of implementation (POST; October 1, 2021, to September 30, 2022) to compare the distribution of molecular and traditional staging factors using GOG-249 criteria to assign clinical risk. In total, 136 of 260 PRE patients were classified as clinically low risk (LR), of whom 31 were MMR deficient. Of the 157 LR POST patients with endometrioid-type carcinoma, 45 were MMR deficient, 5 were POLE mutant, 5 were TP53 mutant, 56 were of no specific molecular profile (NSMP), and 46 did not receive full protocol testing. Of all 79 POST NSMP endometrioid-type cases, 18 were CTNNB1 mutated and 8 showed L1CAM expression. Our protocol identified 22 (14%) of 157 LR tumors that harbored incipient intermediate- to high-risk molecular aberrations in TP53, CTNNB1, or L1CAM. Moving forward, results of ongoing trials assessing adjuvant therapy decisions based on molecular classification are necessary to confirm protocol utility and identify appropriate modifications.

Abstract B014: Patient-derived organoid models for drug screening in molecular subtypes of endometrial cancer

Abstract Introduction: The lack of reliable pre-clinical models has significantly impeded the progress of drug discovery for specific molecular subtypes of endometrial cancer. This study aimed to develop patient-derived organoid models for use in preclinical screening of novel and repurposed therapies in endometrial cancer molecular subtypes. Method: Endometrial cancer tissue (n=27) and ascites fluid (n=1) were collected for this study (ethics approval SESLHD HREC 2021_ETH11975 and 19/001) from patients of various subtypes (17 endometrioid, 3 high grade serous, 2 carcinosarcoma, 2 clear cell, 3 mixed) undergoing treatment at the Royal Hospital for Women and Prince of Wales Private Hospital in Sydney, Australia. Tumor cells were isolated, embedded in 70% Matrigel and cultured in defined culture medium to form organoids. Molecular subtyping of organoids and matched parental tissue was undertaken by mismatch repair (MMR) and p53 immunohistochemistry (IHC), and sequencing of POLE exonuclease domain mutations (EDMs). A unique matched two-dimensional (2D) cell line and three-dimensional (3D) organoid culture derived from ascites isolated from a high grade serous endometrial cancer patient was established. These molecularly stratified pre-clinical models were then used to test drug activity. As a proof of principle, response to paclitaxel and carboplatin was undertaken. Result: 20 organoids representing all major molecular subtypes were established (71% success rate), including one derived from ascites fluid. Organoids are presented with either a cystic or solid structure that highly resembles the morphology of the original tissue. Organoid culture can faithfully retain key features including MMR, p53 status as well as POLE EDMs of the parental tissue. The drug screening results identified contrasting responses of 2D and 3D models obtained from the same patient tissue, highlighting the importance of selecting appropriate pre-clinical models for drug development and testing. Conclusion: This study demonstrated that patient-derived organoids can successfully recapitulate the molecular characterization of endometrial cancer. Molecularly defined organoids represent a promising preclinical resource for drug screening in endometrial cancer. In addition, the model structure should be taken into account while conducting drug screening. Citation Format: Dongli Liu, Elyse Powell, King Man Wan, Ramanand Athavale, Christine Loo, Caroline E. Ford. Patient-derived organoid models for drug screening in molecular subtypes of endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B014.

A164 OPTIMIZING A PROVINCIAL SCREENING PROGRAM FOR DETECTION OF LYNCH SYNDROME

Abstract Background Up to 30% of those with colorectal cancer (CRC) have an affected family member. Lynch syndrome (LS) is the most common inherited condition predisposing patients to CRC. LS is an autosomal dominant condition associated with microsatellite instability (MSI) and mutations in DNA mismatch repair (MMR) genes, and screening CRC cases ≤ age 70 can identify LS in a cost-effective manner. Manitoba launched Canada's first provincial reflex screening program for all CRC cases diagnosed ≤ age 70 using MMR immunohistochemistry (MMR-IHC) in 2017. Our prior 2022 study evaluating the program showed compliance rates of 89.4% for MMR-IHC reflex testing and a 75.8% overall referral rate of screen-positive cases to genetics. However, only 50% were referred directly by pathologists. Several modifications have since been implemented to enhance the program. These included providing feedback to pathologists, creating an “additional testing comment section” on synoptic pathology reports, and regular review of the pathology reports by a pathology assistant. Information materials were developed to encourage genetic testing among relatives. Aims Assess whether modifications to Manitoba’s universal screening protocol improved LS patient outcomes and determine any remaining factors to optimize. We specifically aimed to identify current compliance rates for MMR-IHC testing, the overall referral rate of screen-positive cases to genetics, uptake of genetic referrals and genetic testing as well as rate of cascade testing among relatives. Methods Data has been obtained by searching the provincial pathology database for “adenocarcinoma” in the colorectal specimen pathology reports. All specimens from 2022 and Q1/Q2 of 2023 were reviewed, meeting the criteria of: a) specimen from colon biopsy/excision containing the term “adenocarcinoma”, b) patient age ≤70 years, and c) missing MMR centralized by the IT/Pathology department. Additionally, a random sample of cases categorized as “MMR performed” will be audited. We are in the process of reviewing genetic referrals, genetic uptake and the frequency of reminders triggered to each pathologist. Results Currently, a total of 1,308 colonic adenocarcinoma specimens (811 from 2022, 497 for 2023) have been reviewed. Appropriate MMR testing was missed in only 1.5% of cases (13/811 =1.6% missed in 2022, 7/497= 1.4% missed in 2023). Conclusions Current MMR testing rates for colonic adenocarcinoma specimens within the Manitoba LS screening program have reached a current compliance rate of 98.5%, a substantial improvement from the prior audit (89.4% previously). Further data collection and analysis is ongoing and will also be presented. The reported rates of compliance are the highest that have been reported from any jurisdiction. This study highlights the sustained efforts required to improve detection of LS. Funding Agencies None

Benchmarking mismatch repair testing for patients with cancer receiving immunotherapy.

179 Background: Mismatch repair deficiency (MMR-D) serves as a theranostic marker for directing immune checkpoint inhibitor (ICI) treatment across various cancer stages and tumor types. The College of American Pathologists endorses immunohistochemistry (IHC) as the primary approach for assessing mismatch repair (MMR) testing in colorectal (CRC) and endometrial cancer (EC) patients. An alternative method involves genetic evaluation of microsatellite instability (MSI). Previous studies have shown that resistance to immune checkpoint inhibitors (ICIs) may occur due to inaccurate MSI or MMR IHC status determination. Accurate assessment of MMR status is critical for treatment decision-making. Methods: We hypothesized that tumor-only next generation sequencing (NGS) for MMR mutation signature assessment for identification of MMR-D tumors is more sensitive than MMR IHC testing. Using a standardized, clinically validated, tumor-only NGS mutation signature pipeline in place in the Oncopanel assay at DFCI/BWH, we compared the concordance in MMR determination between NGS and IHC. We then evaluated a subset of ICI-treated patients to assess the clinical implications of these findings. Results: 1655 colorectal cancer (CRC) and endometrial carcinoma (EC) patients who had both NGS MSI (NGSd = deficient or NGSp = proficient) and MMR IHC (IHC- = absent or IHC+ = intact) assays performed were assessed. Discordant NGSd/IHC+ results were identified in 1% of CRC and 5% of EC cases that were IHC+. Importantly, patients with discordant NGSd/IHC+ (median OS: not reached [NR], 95% CI, 9.9 months-NR; median TTF: 44.4 months, 95% CI, 4.1-NR) and concordant NGSd/IHC- results, showed a similar overall survival and time to treatment failure with ICI treatment (p=non-significant) which contrasted with results for patients with concordant NGSp/IHC+ results (median OS: 14.9 months, 95% CI, 7.3-24.6, p-value 0.0025 and &lt; 0.0001; median TTF: 5.0 months, 95% CI, 3.2-7.3; p-value 0.0014 and &lt; 0.0001). Patients with EC or CRC and discordant NGSd/IHC+ results had better survival when treated with ICI-based regimens, compared to those who received non-ICI treatments (p= 0.054 and 0.017). Conclusions: In 2022, 151,030 new CRC and 65,950 new EC cases were diagnosed in the USA. Extrapolating our findings to this larger population, use of NGS mutational signature analysis instead of IHC would amount to about 1510 additional MMR-D CRC cases and 3297 additional MMR-D endometrial cases identified each year for whom first-line ICI therapy is preferred. Our findings support revisiting guideline recommendations for diagnostic testing of MMR to incorporate both IHC and targeted NGS tumor panel testing using sensitive and specific mutation signature calling algorithms. Larger prospective studies are warranted to confirm these findings in patients with mCRC and mEC treated with ICIs. Evaluation of this approach in other cancer types is also warranted.

Evaluation of mismatch-repair and microsatellite-instability status in a Chinese colorectal cancer Cohort

BackgroundImmunohistochemistry (IHC) and traditional polymerase chain reaction (PCR) are the methods of choice in clinical practice to identify the mismatch repair (MMR) and microsatellite instability (MSI) status in colorectal cancer (CRC). In some previous researches, the concordance rate between two methods was different and discordance existed in about 1 %–9.7 %. MethodsWe retrospectively reviewed 406 patients received surgical CRC resections and tests of both MMR IHC and MSI PCR from January 2019 to April 2022 in Shanghai Changzheng Hospital. The incidence of deficient mismatch repair (dMMR) or microsatellite instability-high (MSI-H) CRCs, the concordance rate between two methods, and the reasons for discordant results were evaluated with clinicopathological data, immunochemical staining, whole-exome sequencing, and MLH1 methylation analysis. ResultsAmong 406 patients, the incidence of MSI-H CRCs was 7.88 %. Nearly a quarter of the cases under reexamination of IHC was initial misinterpreted. Besides, the concordance rate between MMR IHC and MSI PCR was 99.26 % (401 of 404) and the Kappa value was 0.945 (p < 0.001). Finally, some somatic variants of MMR and POLE genes which may explain the discordance were identified. ConclusionThe incidence rate of MSI-H in Chinese patients with CRC might be relatively low owing to tumor location. Although MSI and IHC analyses are highly concordant, both MMR IHC and MSI PCR tests should be simultaneously performed and MMR IHC should be interpreted by experienced pathologists. In the future, further studies on discordant results should be carried out to improve the personalized management of CRC.

B-210 Spectrum of Germline Mutation with Characteristic MLH1 Hotspots and High Concordance with MMR Deficiency Phenotype in Lynch Syndrome Patients in Taiwan

Abstract Background Patients carried defects on cancer susceptibility genes or had cancer family history have at least twice the risk of developing cancer than the general population, with both early onset and multiple cancers features. For elucidating the carrier rate of cancer susceptibility genes in patients with inherited colorectal cancer (CRC) and endometrial cancer (EnCa) in Taiwan, we established a NCCN guided 30-gene panel which included four DNA Mismatch Repair (MMR) genes to detect the germline mutation of patients with Lynch Syndrome. Correlation of genotype and phenotype of MMR enzymes are under investigation as well. Methods Sixty-eight blood samples were collected from unrelated cancer patients, 42 of them were from patients with inherited colon cancer, 13 from patients with diagnosis of both CRC and endometrial cancer and 13 from patients with early onset EnCa. 30-gene panel was performed by next generation sequencing (NGS) platform and variants were annotated following ACMG guideline. MMR protein expression in tumor tissue sections were identified by conventional immunohistochemistry (IHC) stain. Loss of nuclear staining were classified as MMR deficiency. Results Average age of first tumor onset of 68 patients were 49.2 years old. 54% (37/68) of the subjects had at least one germline mutations with pathogenic effect or deleterious variant predicted by in silico protein function software. Five of them carried two heterozygous mutations. Total 42 mutations appeared majority on three MMR genes, MLH1 (17/42, 40%), MSH2 (10/42, 24%) and MSH6 (6/42, 11%) with notified three hotspots on MLH1 p.R265C (7/17, 41%); p.T117M (2/17, 12%) and p.K618del(2/17, 12%). The rest mutations spread sporadically on nine genes (PMS2, TP53, CHEK2, POLE, POLD1, ATM, EPCAM, MLH3 and RNF43). For exploring the effect of germline mutation on MMR protein expression status on tissue, we compared the MMR IHC results and the germline mutation pattern. As high as 57% concordance between phenotype and genotype were found. We also collected six blood samples from relatives of our CRC cohort to demonstrate the inheritance and pathogenesis of the germline mutation. Exactly the same germline mutations can be found in diseased relatives among different generation or siblings. Conclusion In spite of having founder mutations on MLH1 gene in Taiwanese Lynch syndrome, most of the germline mutations randomly occurred in MMR-related genes. Comprehensive cancer susceptible gene screening panel by NGS is a cost-effective way to identify the inherited status of individuals with family cancer history and serves as a surrogate tool to predict MMR protein expression. Genetic counseling and prophylactic therapeutic information can be provided for positive cases for better cancer prevention.

Both MLH1 deficiency and BRAFV600E mutation are a unique characteristic of colorectal medullary carcinoma: An observational study.

Although immunohistochemistry (IHC) for mismatch repair (MMR) proteins (MMR IHC) is used to identify DNA MMR status, universal screening of all patients with colorectal cancer (CRC) using a combination of both MMR IHC and genetic testing for the BRAFV600E mutation is limited in Japan. This study aimed to better understand the histopathological characteristics of CRCs, which exhibit both deficient mismatch repair (dMMR) and BRAFV600E mutation. MMR IHC of formalin-fixed paraffin-embedded tissues from tumor areas obtained from 651 patients with CRC who underwent surgical resection at Hamamatsu University Hospital (Hamamatsu, Japan) between August 2016 and March 2022 were used to evaluate MMR status, which was determined by staining for the expression of 4 MMR proteins (MLH1, MSH2, PMS2, and MSH6). All dMMR tumors were additionally evaluated for BRAFV600 mutation status via Sanger sequencing. Patient clinical characteristics (age, sex, tumor location, size, and tumor pathology) were then classified using their dMMR and BRAFV600 mutation statuses. Among the 651 patients with CRC, 58 carried tumors with dMMR, of which 52 were deficiency in MLH1 (dMLH1). Interestingly, all 16 medullary carcinomas that were analyzed showed characteristics corresponding to the presence of both dMLH1 and BRAFV600E mutation (P = .01). These results suggest that colorectal medullary carcinomas can be diagnosed based on their unique characteristics of harboring the BRAFV600E mutation and exhibiting dMLH1 expression.

Adoption of Universal Testing in Endometrial Cancers for Microsatellite Instability Using Next-Generation Sequencing.

To assess implementation of a next-generation sequencing (NGS) assay to detect microsatellite instability (MSI) as a screen for Lynch syndrome (LS) in endometrial cancer (EC), while determining and comparing characteristics of the four molecular subtypes. A retrospective review was performed of 408 total patients with newly diagnosed EC: 140 patients who underwent universal screening with NGS and 268 patients who underwent screening via mismatch repair immunohistochemistry (MMR IHC) as part of a historical screening paradigm. In the NGS cohort, incidental POLE and TP53 mutations along with MSI were identified and used to characterize EC into molecular subtypes: POLE-ultramutated, MSI high (MSI-H), TP53-mutated, and no specific molecular profile (NSMP). In historical cohorts, age- and/or family history-directed screening was performed with MMR IHC. Statistical analysis was performed using a t-test for continuous variables and chi-square or Fisher's exact test for categorical variables. In the NGS cohort, 38 subjects (27%) had MSI-H EC, 100 (71%) had microsatellite stable EC, and two (1%) had an indeterminate result. LS was diagnosed in two subjects (1%), and all but five patients completed genetic screening (96%). Molecular subtypes were ascertained: eight had POLE-ultramutated EC, 28 had TP53-mutated EC (20%), and 66 (47%) had NSMP. MSI-H and TP53-mutated EC had worse prognostic features compared with NSMP EC. Comparison with historical cohorts demonstrated a significant increase in follow-up testing after an initial positive genetic screen in the MSI NGS cohort (56% v 89%; P = .001). MSI by NGS allowed for simultaneous screening for LS and categorization of EC into molecular subtypes with prognostic and therapeutic implications.

Mismatch Repair Protein Expression in Endometrial Cancer: Assessing Concordance and Unveiling Pitfalls in Two Different Immunohistochemistry Assays.

This study aimed to compare the concordance and interchangeability of the Dako/Agilent and Ventana/Roche mismatch repair (MMR) immunohistochemistry (IHC) assays commonly used in pathology. It also aimed to provide diagnostic insights by examining the frequency and characteristics of the dot-like artifact observed in MLH1 M1 clone staining in endometrial cancer. Fifty endometrial cancer cases with MMR deficiency, excised between 2011 and 2018, were included in the study. IHC was performed using primary antibody clones from Ventana/Roche (MLH1, clone M1; MSH2, G219-1129; MSH6, SP93; PMS2, A16-4) and Dako/Agilent (MLH1, ES05; MSH2, FE11; MSH6, EP49; PMS2, EP51). Both assays were conducted using respective autostainers. The Dako/Agilent assay showed a loss of MLH1 in 26 cases, MSH2 in 12 cases, MSH6 in 23 cases, and PMS2 in 28 cases. The two assays had a complete agreement in MMR protein expression or loss. The dot-like artifact in MLH1 M1 clone staining was observed in 77% (20/26) of cases, predominantly in the surface area of the tumor, ranging from 5% to 40% (median: 10%). These findings highlight the high concordance between the MMR-IHC assays and emphasize the importance of considering the dot-like artifact in MLH1 M1 clone staining when diagnosing endometrial cancer with MMR deficiency.

Harmonized molecular classification; assessment of a single-test ProMisE NGS tool

ObjectivesDespite recommendations for integrating molecular classification of endometrial cancers (EC) into pathology reporting and clinical management, uptake is inconsistent. To assign ProMisE subtype, all molecular components must be available (POLE mutation status, mismatch repair (MMR) and p53 immunohistochemistry (IHC)) and often these are assessed at different stages of care and/or at different centres resulting in delays in treatment. We assessed a single-test DNA-based targeted next generation sequencing (NGS) molecular classifier (ProMisE NGS), comparing concordance and prognostic value to the original ProMisE classifier. MethodsDNA was extracted from formalin-fixed paraffin embedded (FFPE) ECs that had previously undergone ProMisE molecular classification (POLE sequencing, IHC for p53 and MMR). DNA was sequenced using the clinically validated Imagia Canexia Health Find It™ amplicon-based NGS gene panel assay to assess for pathogenic POLE mutations (unchanged from original ProMisE), TP53 mutations (in lieu of p53 IHC), and microsatellite instability (MSI) (in lieu of MMR IHC),with the same order of segregation as original ProMisE used for subtype assignment. Molecular subtype assignment of both classifiers was compared by concordance metrics and Kaplan-Meier survival statistics. ResultsThe new DNA-based NGS molecular classifier (ProMisE NGS) was used to determine the molecular subtype in 164 ECs previously classified with ProMisE. 159/164 cases were concordant with a kappa statistic of 0.96 and an overall accuracy of 0.97. Prognostic differences in progression-free, disease-specific and overall survival between the four molecular subtypes were observed for the new NGS classifier, recapitulating the survival curves of the original ProMisE classifier. ProMisE NGS was 100% concordant between matched biopsy and hysterectomy samples. ConclusionProMisE NGS is feasible on standard FFPE material, demonstrates high concordance with the original ProMisE classifier and maintains prognostic value in EC. This test has the potential to facilitate implementation of molecular classification of EC at the time of first diagnosis.

Improving genetic testing following abnormal mismatch repair immunohistochemistry results in endometrial cancer

ObjectivesAlthough universal mismatch repair (MMR) immunohistochemistry (IHC) in endometrial cancer began at our institution in July 2015, not all eligible patients were referred for genetic testing (GT). In April 2017, genetic counselors obtained IHC data and contacted physicians to approve genetic counseling referrals (GCRs) for Lynch Syndrome (LS) in eligible patients. We assessed if this protocol increased frequency of GCRs and GT in patients with abnormal MMR IHC. MethodsWe retrospectively (7/2015–5/2022) identified patients with abnormal MMR IHC at a large urban hospital. GCRs and GT were compared between cases from 7/2015–4/2017 (pre-protocol) and 5/2017–5/2022 (post-protocol) with chi-square and Fisher's exact tests. ResultsOf 794 patients with IHC testing, 177 (22.3%) had abnormal MMR results with 46 (26.0%) meeting criteria for LS screening with GT. Of 46 patients, 16 (34.8%) were identified prior to and 30 (65.2%) after the protocol initiation. GCRs significantly increased from 11/16 (68.8%) to 29/30 (96.7%) in the pre-protocol versus post-protocol groups, p = 0.02. There was no statistically significant difference in GT between groups (10/16, 62.5% vs 26/30, 86.7%, p = 0.07). Of 36 patients who underwent GT, 16 (44.4%) had LS: MSH6, 9; MSH2, 4; PMS2, 2; MLH1, 1. ConclusionsIncreased frequency of GCRs was observed following the change in protocol, which is important as LS screening has clinical implications for patients and their families. Despite this additional effort, approximately 15% who met criteria did not undergo GT; further efforts such as universal germline testing in patients with endometrial cancer should be considered.

Lynch screening and genetic testing in endometrial cancer in a diverse population.

e17619 Background: Lynch syndrome (LS) accounts for most of the inherited endometrial cancers. Universal screening of endometrial tumors for defects in DNA mismatch repair (MMR) is recommended using immunohistochemistry (IHC) to identify which patients qualify for genetic counseling and testing. We report Lynch screening and genetic testing rates at a New York City tertiary care center with a large minority population, while investigating any potential disparities. Methods: This is a retrospective cohort study of patients with newly diagnosed endometrial cancer between January 2014 and June 2022. Data were obtained from pathology and chemotherapy databases at the NewYork-Presbyterian Brooklyn Methodist Hospital. Variables including age, race, ethnicity, BMI, insurance, language, family history of cancer, date of diagnosis, stage, MMR IHC, and genetic counseling/testing status were manually extracted. Descriptive statistics were used. Chi-square/Fisher’s exact tests were used to examine the association between genetic counseling/testing and categorical variables. Wilcoxon rank-sum/t-tests were used for continuous factors. The Mann-Kendall trend test was used to determine the significance of time trends. Results: 373 patients were identified. 45% identified as white, 42% black, 1.3% Asian, 12% other/unknown; 8.3% were of Hispanic ethnicity and 18% were non-English speaking. Mean age at diagnosis was 66 years (SD:10). 207 (55%) patients were screened using MMR IHC. 82 (40%) of these patients had MMR deficiencies on IHC. Of these, 63 (77%) received genetic counseling. 62 (98%) of those counseled subsequently underwent genetic testing, and ultimately 7 (11%) were diagnosed with LS. The overall rate of LS detected was 1.9% in the study population. The rate of MMR IHC testing has been increasing, reaching 95% in 2021 and 100% in 2022. It was associated with significant differences in mean age at diagnosis (p &lt; 0.01) and insurance type (p = 0.04). 45% of patients that received MMR IHC had private insurance, compared to 34% of patients that did not. The rate was not influenced by race, language, BMI, family history of cancer, or stage. The proportion of patients that received genetic counseling and testing for endometrial cancer also showed a significant upwards trend over time (p &lt; 0.01). Genetic counseling and testing rates were significantly different by ethnicity (p = 0.03), with only 3.0% of patients receiving genetic counseling/testing identifying as Hispanic. 98% of genetic counseling was performed by a gynecologic oncologist, as opposed to a genetic counselor. Conclusions: There were no disparities in access to initial IHC screening in this diverse patient population, however more work must be done to reach all ethnicities for genetic counseling and testing. The rate of LS detected was less than the known prevalence in endometrial cancer, possibly indicating demographic differences or gaps in screening.

Integration of clinical sequencing and immunohistochemistry for the molecular classification of endometrial carcinoma

PurposeUsing next generation sequencing (NGS), The Cancer Genome Atlas (TCGA) found that endometrial carcinomas (ECs) fall under one of four molecular subtypes, and a POLE mutation status, mismatch repair (MMR) and p53 immunohistochemistry (IHC)-based surrogate has been developed. We sought to retrospectively classify and characterize a large series of unselected ECs that were prospectively subjected to clinical sequencing by utilizing clinical molecular and IHC data. Experimental designAll patients with EC with clinical tumor-normal MSK-IMPACT NGS from 2014 to 2020 (n = 2115) were classified by integrating molecular data (i.e., POLE mutation, TP53 mutation, MSIsensor score) and MMR and p53 IHC results. Survival analysis was performed for primary EC patients with upfront surgery at our institution. ResultsUtilizing our integrated approach, significantly more ECs were molecularly classified (1834/2115, 87%) as compared to the surrogate (1387/2115, 66%, p < 0.001), with an almost perfect agreement for classifiable cases (Kappa 0.962, 95% CI 0.949–0.975). Discrepancies were primarily due to TP53 mutations in p53-IHC-normal ECs. Of the 1834 ECs, most were of copy number (CN)-high molecular subtype (40%), followed by CN-low (32%), MSI-high (23%) and POLE (5%). Histologic and genomic variability was present amongst all molecular subtypes. Molecular classification was prognostic in early- and advanced-stage disease, including early-stage endometrioid EC. ConclusionsThe integration of clinical NGS and IHC data allows for an algorithmic approach to molecularly classifying newly diagnosed EC, while overcoming issues of IHC-based genetic alteration detection. Such integrated approach will be important moving forward given the prognostic and potentially predictive information afforded by this classification.

Mutation-specific Mismatch Repair-deficient Benign Endometrial Glands in Endometrial Biopsies and Curettings Are a Biomarker of Lynch Syndrome and Associate With Endometrial Carcinoma Development.

Endometrial carcinoma is the most common extraintestinal cancer in Lynch syndrome (LS). Recent studies have demonstrated mismatch repair (MMR) deficiency can be detected in benign endometrial glands in LS. We performed MMR immunohistochemistry in benign endometrium from endometrial biopsies and curettings (EMCs) from a study group of 34 confirmed LS patients and a control group of 38 patients without LS who subsequently developed sporadic MLH1-deficient or MMR-proficient endometrial carcinoma. MMR-deficient benign glands were only identified in patients with LS (19/34, 56%) and were not identified in any control group patient (0/38, 0%) ( P < 0.001). MMR-deficient benign glands were identified as large, contiguous groups in 18 of 19 cases (95%). MMR-deficient benign glands were identified in patients with germline pathogenic variants in MLH1 (6/8, 75%), MSH6 (7/10, 70%), and MSH2 (6/11, 55%) but not in patients with variants in PMS2 (0/4). MMR-deficient benign glands were seen in all EMC samples (100%) but in only 46% of endometrial biopsy samples ( P =0.02). Patients with MMR-deficient benign glands were significantly more likely to have endometrial carcinoma (53%) compared with LS patients with only MMR-proficient glands (13%) ( P =0.03). In conclusion, we demonstrated that MMR-deficient benign endometrial glands are frequently identified in EMB/EMC in women with LS and are a specific marker for LS. Women with LS with MMR-deficient benign glands were more likely to have endometrial carcinoma suggesting that MMR-deficient benign glands may be a biomarker of increased risk of endometrial carcinoma development in LS.

Clinical predominance of whole-exome sequencing to evaluate microsatellite instability status.

The microsatellite instability (MSI)/mismatch repair (MMR) status is one of the critical genomic biomarkers for predicting patient response to immune checkpoint inhibitors (ICIs). In this study, we aimed to investigate the concordance among the MSIsensor score obtained from whole-exome sequencing (WES), which could be a futuristic clinical cancer sequencing method, using only tumor tissues, MSI-PCR results, and immunohistochemistry (IHC) results to analyze various solid cancer types. We first endeavored to set the cut-off value of MSIsensor to determine functional deficient mismatch repair (f-dMMR) status. The MSI status of 1054 patients analyzed using WES was evaluated using MSIsensor. In addition, 87 of these patients were further analyzed using MSI-PCR and MMR IHC to calculate the sensitivity and specificity of the MSIsensor cut-off score. Our results showed that score 12.5 was an adequate cut-off score equivalent to PCR-confirmed MSS/MSI-low and MSI-high statuses, with sensitivity, specificity, and area under the curve values of 95.2%, 100%, and 0.998, respectively. Moreover, we identified false-positive cases of tumors with high mutational burden with an MSIsensor score <12.5, and optional IHC examination could rescue these cases. In conclusion, the MSIsensor score obtained using WES with tumor tissue showed a high clinical validity, with a cut-off value of 12.5 for f-dMMR detection, in combination with optional IHC analysis for MMR. Our novel algorithm will provide insights into the development of ICIs for cancer treatment, particularly when WES becomes a more common cancer genomic test in the near future.

A tumor focused approach to resolving the etiology of DNA mismatch repair deficient tumors classified as suspected Lynch syndrome

Routine screening of tumors for DNA mismatch repair (MMR) deficiency (dMMR) in colorectal (CRC), endometrial (EC) and sebaceous skin (SST) tumors leads to a significant proportion of unresolved cases classified as suspected Lynch syndrome (SLS). SLS cases (n = 135) were recruited from Family Cancer Clinics across Australia and New Zealand. Targeted panel sequencing was performed on tumor (n = 137; 80×CRCs, 33×ECs and 24xSSTs) and matched blood-derived DNA to assess for microsatellite instability status, tumor mutation burden, COSMIC tumor mutational signatures and to identify germline and somatic MMR gene variants. MMR immunohistochemistry (IHC) and MLH1 promoter methylation were repeated. In total, 86.9% of the 137 SLS tumors could be resolved into established subtypes. For 22.6% of these resolved SLS cases, primary MLH1 epimutations (2.2%) as well as previously undetected germline MMR pathogenic variants (1.5%), tumor MLH1 methylation (13.1%) or false positive dMMR IHC (5.8%) results were identified. Double somatic MMR gene mutations were the major cause of dMMR identified across each tumor type (73.9% of resolved cases, 64.2% overall, 70% of CRC, 45.5% of ECs and 70.8% of SSTs). The unresolved SLS tumors (13.1%) comprised tumors with only a single somatic (7.3%) or no somatic (5.8%) MMR gene mutations. A tumor-focused testing approach reclassified 86.9% of SLS into Lynch syndrome, sporadic dMMR or MMR-proficient cases. These findings support the incorporation of tumor sequencing and alternate MLH1 methylation assays into clinical diagnostics to reduce the number of SLS patients and provide more appropriate surveillance and screening recommendations.