MLH1 Hypermethylation Research Articles

High-risk histology endometrial cancer and mismatch repair status: The impact of histologic subtype and MLH1 hypermethylation on outcomes

High-risk histology endometrial cancer and mismatch repair status: The impact of histologic subtype and MLH1 hypermethylation on outcomes

Immune checkpoint inhibition for mismatch repair deficient endometrial cancer: MLH1 hypermethylation does not impact outcome in a multi-institutional retrospective database

Immune checkpoint inhibition for mismatch repair deficient endometrial cancer: MLH1 hypermethylation does not impact outcome in a multi-institutional retrospective database

Retained PAX2 expression associated with DNA mismatch repair deficiency in endometrial endometrioid adenocarcinoma.

Loss of expression of tumour suppressor PAX2 and MMR deficiency (dMMR) has been frequently seen in endometrial endometrioid adenocarcinoma (EEC). However, the relationship between PAX2 expression and MMR status is unknown. We studied the PAX2 expression and examined its association with MMR status at the protein and genetic levels in 180 cases of EEC. Overall, total loss of PAX2 expression was found in about 70%, while retained PAX2 expression was seen in 30% of EEC. Among 125 cases with loss of PAX2, 68.8% were found in EECs with pMMR, while 31.2% were seen in those with dMMR. Among 55 cases of EECs with retained PAX2 expression, 92.7% were EECs with dMMR and 7.3% were those with pMMR (P < 0.001). While dMMR cases with MLH1 hypermethylation show almost equal retained or loss of PAX2 expression (52% versus 48%), dMMR with genetic alterations had significantly more retained PAX2 expression than loss of PAX2 (92.3% versus 7.7%), regardless of somatic or germline mutations. Loss of PAX2 was observed in 97.3% of dMMR with MLH1 hypermethylation compared to 2.7% of dMMR with genetic alterations (P < 0.001). Aggressive features such as higher tumour grades (FIGO 2-3) and advanced clinical stage (T2-T4) were significantly more frequently seen in dMMR with retained PAX2 expression, compared those to pMMR with loss of PAX2 expression. Our study demonstrates a close correlation between retained PAX2 expression and dMMR in EEC. The molecular mechanism and clinical significance linking these two pathways in EEC remains to be unravelled.

Predictors for dMMR colorectal cancer in patients with serrated lesions and polyps – A register-based cohort study

BackgroundSerrated lesions and polyps (SP) are precursors of up to 30 % of colorectal cancers (CRC) through the serrated pathway. This often entails early BRAF mutations and MLH1 hypermethylation leading to mismatch repair deficient (dMMR) CRC. We investigated predictors of dMMR CRC among patients with co-occurrence of CRC and SP to increase our knowledge on the serrated pathway. MethodsWe used data from The Danish Pathology Registry and Danish Colorectal Cancer Groups Database from the period 2010–2021 to investigate risk factors for development of dMMR CRC. We used logistic regression models to identify difference in risk factors of developing dMMR CRC in comparison to CRC with proficient MMR (pMMR). ResultsWe included 3273 patients with a median age of 70.7 years [64.3,76.4] of which 1850 (56.5 %) were male. dMMR CRC was present in 592 patients (18.1 %), with loss of MLH1/PMS2 being most common. The risk of dMMR CRC was significantly higher in females OR 3.47 [2.87;4.20]. When adjusting for age, SP subtype, conventional adenomas (CA), anatomical location and lifestyle factors, female sex remained the strongest predictor OR 2.84 [2.27;3.56]. The presence of sessile serrated lesions with or without dysplasia was related to higher risk OR 1.60 [1.11;2.31] and OR 1.42 [1.11;1.82] respectively, while conventional adenomas constituted a lower risk OR 0.68 [0.55;0.84]. ConclusionIn conclusion we found several predictors of whom female sex had the strongest correlation with dMMR CRC in patients with SP.

A registry-based study on universal screening for defective mismatch repair in colorectal cancer in Denmark highlights disparities in screening uptake and counselling referrals

Universal screening for defective mismatch repair (dMMR) in colorectal cancer utilizes immunohistochemical staining for MLH1, MSH2, MSH6 and PSM2. Additionally, BRAF V600E mutations status and MLH1 hypermethylation should be performed to distinguish germline and somatic dMMR alterations. A decade of Danish population-based registries has been analysed regarding screening uptake, detection rate and referral to genetic counselling. MMR testing was performed in 71·8% (N = 34,664) of newly diagnosed colorectal cancers with an increasing trend to 88·8% coverage in the study's final year. The likelihood of undergoing MMR testing was reduced in males with 2% (95% CI 0·4–2·7, p = 0·008), with 4·1% in patients above age 70 years (95% CI 1·5–6·6, p = 0·003) compared in patients below age 51 years, with 16·3% in rectal cancers (95% CI 15·1–17·6, p < 0·001) and 1·4% left-sided colon cancers (95% CI 0·1–1·7, p = 0·03) compared to right-sided colon cancers. Tumour stage II and III increased the likelihood of being tested, with 3·7% for stage II (95% CI 2·2–5·6, p < 0·001) and 3·3% for stage III tumours (95% CI 1·8–4·8, p < 0·001) compared to stage I tumours, whereas the likelihood for stage IV tumours is reduced by 35·7% (95% CI 34·2–37·2, p < 0·001). Test rates significantly differed between the Danish health care regions. dMMR was identified in 15·1% (95% CI 14·8–15·6, p < 0·001) cases with somatic MMR inactivation in 6·7% of the cases. 8·3% tumours showed hereditary dMMR expression patterns, and 20·0% of those were referred to genetic counselling. Despite the high uptake rates, we found disparities between patient groups and missed opportunities for genetic diagnostics.

Abstract B026: Substratification of mismatch repair deficient (MMRd) endometrial cancers can provide prognostic and predictive refinement

Abstract Background: Mismatch repair deficient (MMRd) endometrial cancer (EC) represents one third of all ECs. Identifying patients with MMRd EC enables testing for Lynch Syndrome (LS) and access to FDA-approved immune checkpoint blockade (ICB) therapy. Recent data have highlighted the diversity within MMRd tumors, suggesting worse outcomes and lower response to ICB in patients with MLH1 loss. Our aim was to characterize a cohort of MMRd ECs to elucidate if clinically meaningful substratification of this molecular subtype (MLH1 loss vs. other MMRd) could be achieved. Methods: MMRd ECs were identified from retrospective institutional and population-based cohorts (1994-2016) with clinicopathologic data, immunohistochemistry (IHC) assessment of estrogen receptor (ER) and L1CAM and CTNNB1 mutation status recorded. Multiplex IHC for immune markers (CD3, CD8, CD79a, CD138, PD-1, PD-L1, FoxP3, IDO-1) was assessed and compared in patients with MLH1 loss or PMS2/MLH1 loss (when 2 antibody MMRd testing performed) vs. isolated MSH2, MSH6, PMS2 or MSH2/MSH6 loss (remainder of MMRd). Results: 655 MMRd ECs were identified, 52% of cases assessed with 4 antibody IHC (MLH1, PMS2, MSH2, MSH6) and 48% with 2 (PMS2 and MSH6). This included 32 (5%) patients with MLH1 loss and 488 (75%) with PMS2/MLH1 loss (together 80% of MMRd cohort), 1% (n= 9) MSH2, 11% (n=75) MSH6, 4% (n=24) PMS2, and 4% (n=24) with loss of MSH2/MSH6). 76 cases were confirmed to have MLH1 hypermethylation but 67% of cases with loss of MLH1 or PMS2/MLH1 did not have methylation testing performed. The majority MMRd ECs were FIGO stage I (75%) and endometrioid histotype (90%). Patients with loss of MLH1 or PMS2/MLH1 were older (p&amp;lt;0.001), had higher BMI (p&amp;lt;0.001) and had tumors with more LVI (p=0.031), deep myoinvasion (p&amp;lt;0.001) and grade 3 (p=0.046) compared to remainder of MMRd EC. 9% of the MMRd cohort were ER negative, 8% with loss of MLH1 or PMS2/MLH1 and 16% in remainder of MMRd. Inferior outcomes were observed for progression-free survival and overall survival in patients with MLH1 or PMS2/MLH1 loss compared with the remainder of MMRd. ER, L1CAM and CTNNB1 status were not associated with clinical outcomes across the total MMRd cohort, nor within MLH1 or PMS2/MLH1 loss. There was diversity in the immune landscape within MMRd EC with significantly lower CD8 levels in MLH1 or PMS2/MLH1 loss compared to the remainder of MMRd EC. Furthermore, 25% of the MLH1 or PMS2/MLH1 loss group were tumor infiltrating lymphocyte (TIL) ‘low’/immune cold by cluster analysis compared to 11% in the remainder of MMRd EC. Only 16 patients (2.4%) were identified as having LS but 82% of these MMRd patients had not been tested (31% non-LS testing attributable to identification of hypermethylation of MLH1). Conclusion: Substratification within MMRd ECs can provide prognostic and predictive information, with loss of MLH1 and/or its dimer partner PMS2 identifying a subset of MMRd EC with inferior outcomes that may be attributed to lower TIL. ER, L1CAM, and CTNNB1 mutation status do not add prognostic refinement within MMRd EC. Citation Format: Amy Jamieson, Jennifer Pors, Samuel Leung, Derek Chiu, Stefan Kommoss, Aline Talhouk, David G. Huntsman, Naveena Singh, Blake Gilks, Jessica N. McAlpine. Substratification of mismatch repair deficient (MMRd) endometrial cancers can provide prognostic and predictive refinement [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 B026.

Aberrant PRDM2 methylation as an early event in serrated lesions destined to evolve into microsatellite-instable colorectal cancers.

Up to 30% of colorectal cancers (CRCs) develop from sessile serrated lesions (SSLs). Within the serrated neoplasia pathway, at least two principally distinct oncogenetic routes exist generating microsatellite-stable and microsatellite-instable CRCs, respectively. Aberrant DNA methylation (DNAm) is found early in the serrated pathway and might play a role in both oncogenetic routes. We studied a cohort of 23 SSLs with a small focus (<10 mm) of dysplasia or cancer, 10 of which were MLH1 deficient and 13 MLH1 proficient. By comparing, for each SSL, the methylation status of (1) the region of dysplasia or cancer (SSL-D), (2) the nondysplastic SSL (SSL), and (3) adjacent normal mucosa, differentially methylated probes (DMPs) and regions (DMRs) were assessed both genome-wide as well as in a tumor-suppressor gene-focused approach. By comparing DNAm of MLH1-deficient SSL-Ds with their corresponding SSLs, we identified five DMRs, including those annotating for PRDM2 and, not unexpectedly, MLH1. PRDM2 gene promotor methylation was associated with MLH1 expression status, as it was largely hypermethylated in MLH1-deficient SSL-Ds and hypomethylated in MLH1-proficient SSL-Ds. Significantly increased DNAm levels of PRDM2 and MLH1, in particular at 'critical' MLH1 probe sites, were to some extent already visible in SSLs as compared to normal mucosa (p = 0.02, p = 0.01, p < 0.0001, respectively). No DMRs, nor DMPs, were identified for SSLs destined to evolve into MLH1-proficient SSL-Ds. Our data indicate that, within both arms of the serrated CRC pathway, the majority of the epigenetic alterations are introduced early during SSL formation. Promoter hypermethylation of PRDM2 and MLH1 on the other hand specifically initiates in SSLs destined to transform into MLH1-deficient CRCs suggesting that the fate of SSLs may not necessarily result from a stochastic process but possibly is already imprinted and predisposed.

Retrospective review of compliance with guideline-directed germline testing in microsatellite instability-high colorectal cancer treated at the Veterans Health Administration.

86 Background: Lynch syndrome (LS) is an autosomal dominant inherited cancer syndrome that confers an increased risk of colorectal cancer (CRC). As clinical and history features under-recognize LS cases (3-5% of CRC), universal screening of CRCs for LS is recommended. Patients whose CRCs are mismatch repair protein deficient (dMMR) and/or microsatellite instability high (MSI-H) are recommended to have subsequent genetics evaluation to screen for germline mutations. Confirmed LS changes cancer surveillance recommendations for affected patients and their relatives. Within Veterans Health Administration (VHA), GT is ordered by genetic counselors at genomic medicine telehealth centers (GMTC), after initial consultation. We aim to quantify rates at which CRC patients treated at the VHA complete germline testing (GT) when indicated. Methods: The electronic health records (EHRs) of VHA patients with known dMMR/MSI-H CRC were reviewed. Cases were extracted from both Veterans Affairs (VA) Puget Sound and referral VA MSI testing laboratories. Surgical pathology reports and clinical documentation were reviewed in Joint Longitudinal Viewer (JLV) to determine if GT was indicated and reason for non-testing, if not performed. Patients with known LS prior to development of CRC were excluded, and patients with dMMR in MLH1 or MSI-H with concurrent BRAFV600 mutation and/or MLH1 hypermethylation were considered to have sporadic CRC. Results: Of 110 patients with abnormal tumor LS screening; 39 (36%) patients’ tumor tissue underwent IHC for dMMR, 16 (14%) underwent PCR for MSI, and 55 (50%) had both. 45 (41%) patients were classified as somatic MSI-H. 65 (59%) patients met criteria for referral for GMTC and GT. Of these 65, 49 ultimately did not undergo GT and 16 did, which confirmed 4 cases of LS and revealed 1 variant of uncertain significance in the MLH1 gene. Reasons for non-testing were: 7 (14%) declined all cancer testing and treatment after CRC diagnosis or were lost to follow-up/declined establishment of cancer care, 3 (6%) declined referral to GMTC, 14 (29%) accepted referral to GMTC but never had consultation, 7 (14%) accepted referral to GMTC but declined GT when offered, and 18 (37%) were never offered GMTC referral or GT. Conclusions: A majority of dMMR/MSI-H cases with an indication for GT were referred for genetic counseling, but only a small subset completed testing. This analysis identified multiple areas for improvement, including a lack of recognition by providers for the need for GT, and lack of completion of testing by patients at multiple levels. Further evaluation of VHA-specific barriers is needed to improve the quality of delivered care.

BRAF variants and oncogenic fusions to define sporadic MMR-deficient colorectal cancer subtypes.

211 Background: Mismatch-repair deficient (MMRd) colorectal cancers (CRC) are rare tumors exquisitely sensitive to immunotherapy. The molecular events that initiate and sustain MMRd tumors remain controversial. The majority of sporadic MMRd CRCs exhibit BRAF-V600E variants with MLH1 hypermethylation, however BRAF-wild-type(wt) tumors exhibit high rates of rare gene fusions. Methods: We performed an integrated molecular and clinical analysis for 253 patients with MMRd sporadic CRC who received MSK-IMPACT genomic profiling at Memorial Sloan Kettering Cancer Center. Tumors were classified based on BRAF-V600E variants and gene fusion events. We evaluated the tumor burden of molecular alterations and disproportionate co-occurrence of specific driver variants of significance between each subtype. We used allele-specific FACETS analysis and MLH1 promoter hypermethylation assessments to infer the MMR-gene inactivation mechanism per tumor. We also examined differences in tumor phenotype and patient clinical outcomes. Patient progression-free-survival after treatment and overall survival was assessed with the Kaplan-Meier curves, and multivariable analyses were performed with Cox proportional hazards regression modeling. Results: Patients exhibited BRAF-V600E-mutated (mut: n=178), BRAF-V600E-wt/gene-fusion positive (n=42), and BRAF-V600E-wt/fusion-negative (n=33) disease. Gene fusion events were mutually exclusive with BRAF-V600E variants and occurred in NTRK1 (n=13), NTRK3 (n=8; one with co-occurring NTRK1 fusion), RET (n=8), BRAF (n=8), ALK (n=5), and FGFR2 (n=1). BRAF-V600E-mut and fusion-positive tumors display similar predominant MLH1 silencing, WNT-signaling activators, and co-alterations. In contrast, BRAF-V600E-wt/fusion-negative tumors exhibit MLH1 inactivation associated with increased copy number alterations, and exhibit distinct co-occurring APC and TCFF7L2 variants. Both fusion-positive and BRAF-V600E-wt/fusion-negative tumors disproportionately arise in the transverse and left colon. In a multivariable analysis of 73 patients with stage IV MMRd CRC, fusion-positive status was an independently-significant, positive prognostic marker. For patients treated with immune checkpoint inhibitors (ICIs), tumor regression rates were high in BRAF-V600E-mut (63%; n=22/35) and fusion-positive (62.5%, n=5/8) subtypes, with slightly poorer outcomes in BRAF-V600E-wt/fusion-negative (40%, n=2/5) tumors. Patients with fusion-positive tumors exhibited clinical benefit to NTRK inhibition. Conclusions: In sporadic MMRd CRC, gene fusion-positive tumors exhibit an improved prognosis with similar molecular and treatment outcomes to BRAF-V600E-mut tumors. BRAF-V600E-wt/fusion-negative tumors are a unique sporadic MMRd subtype driven by alternative WNT-activation and potentially lower immunotherapy responses in small numbers of patients.

MLH1 Promoter Methylation Could Be the Second Hit in Lynch Syndrome Carcinogenesis.

(1) Background: MLH1 hypermethylation is an epigenetic alteration in the tumorigenesis of colorectal cancer (CRC) and endometrial cancer (EC), causing gene silencing, and, as a consequence, microsatellite instability. Commonly, MLH1 hypermethylation is considered a somatic and sporadic event in cancer, and its detection is recognized as a useful tool to distinguish sporadic from inherited conditions (such as, Lynch syndrome (LS)). However, MLH1 hypermethylation has been described in rare cases of CRC and EC in LS patients. (2) Methods: A total of 61 cancers (31 CRCs, 27 ECs, 2 ovarian cancers, and 1 stomach cancer) from 56 patients referred to cancer genetic counselling were selected for loss of MLH1 protein expression and microsatellite instability. All cases were investigated for MLH1 promoter methylation and MLH1/PMS2 germline variants. (3) Results: Somatic MLH1 promoter hypermethylation was identified in 16.7% of CRC and in 40% of EC carriers of MLH1 germline pathogenic variants. In two families, primary and secondary MLH1 epimutations were demonstrated. (4) Conclusions: MLH1 hypermethylation should not be exclusively considered as a sporadic cancer mechanism, as a non-negligible number of LS-related cancers are MLH1 hypermethylated. Current flow charts for universal LS screening, which include MLH1 methylation, should be applied, paying attention to a patient's family and personal history.

Differential outcomes and immune checkpoint inhibitor response among endometrial cancer patients with MLH1 hypermethylation versus MLH1 “Lynch-like” mismatch repair gene mutation

ObjectivesTo identify differential survival outcomes and immune checkpoint inhibitor (ICI) response in MLH1 hypermethylated versus MLH1 mutated (“Lynch-like”) endometrial tumors and determine whether their molecular profiles can elucidate the differential outcomes. Methods1673 mismatch repair deficient endometrial tumors were analyzed by next-generation sequencing and whole transcriptome sequencing (Caris Life Sciences, Phoenix, AZ). PD-L1, ER, and PR were tested by immunohistochemistry and immune cell infiltrates were calculated using MCP-counter. Significance was determined using Chi-square and Mann-Whitney U tests and adjusted for multiple comparisons. Overall survival (OS) was depicted using Kaplan-Meier survival curves. ResultsThe endometrial cancer cohort comprised 89.2% patients with MLH1 hypermethylated tumors and 10.8% with MLH1 mutated tumors, with median ages of 67 and 60 years, respectively (p < 0.01). Patients with MLH1 hypermethylated tumors had significantly worse OS and trended toward worse OS following ICI treatment than patients with MLH1 mutated tumors. The immune microenvironment of MLH1 hypermethylated relative to MLH1 mutated was characterized by decreased PD-L1 positivity, immune checkpoint gene expression, immune cell infiltration, T cell inflamed scores, and interferon gamma (IFNγ) scores. MLH1 hypermethylation was also associated with decreased mutation rates in TP53 and DNA damage repair genes, but increased rates of JAK1, FGFR2, CCND1, and PTEN mutations, as well as increased ER and PR positivity. ConclusionsEndometrial cancer patients with MLH1 hypermethylation display significantly decreased survival and discrepant immunotherapy responses compared to patients with MLH1 mutated tumors, which was associated with differential mutational profiles, a more immune cold phenotype, and increased ER/PR expression in MLH1 hypermethylated tumors. Providers may consider early transition from single agent ICI to a multi-agent regimen or hormonal therapy for patients with MLH1 hypermethylated tumors.

Differential response to immune checkpoint inhibitors in patients with endometrial cancer according to MLH1 hypermethylation vs MLH1 “Lynch-like” mismatch repair gene mutations (1264)

Differential response to immune checkpoint inhibitors in patients with endometrial cancer according to MLH1 hypermethylation vs MLH1 “Lynch-like” mismatch repair gene mutations (1264)

Association Between Microsatellite Instability (MSI) based on MLH1 and PMS2 Marker with Clinicopathological Features of Colorectal Cancer Patients

Background: Microsatellite instability (MSI) is one of the important pathways involved in development of colorectal cancer (CRC). MSI occurs due to mutations or hypermethylation of negative MMR proteins MLH1 and PMS2. The accumulation of mutations at microsatellite locus accelerated the development of CRC. Characteristics of CRC patients with MSI are not sensitive to 5FU chemotherapy and have a good clinical outcomes that can be used as a prognostic factor and a predictor of therapy. Therefore MSI detection is needed. Method: A retrospective cross-sectional study of 80 CRC slides obtained from DR. Sardjito Hospital Yogyakarta and clinical laboratory in 2010-2016. Immunohistochemical staining with anti-MLH1 and anti PMS2 antibodies to see MSI status. Negative MLH1 expression is called MSI MLH1 positive and PMS2 negative expression is called positive MSI PMS2. The association between MSI MLH1 and PMS2 with clinicopathology parameters was analyzed using Chi square. Results: Colorectal cancer patients MSI MLH1 as much as 44 (61.1%), MSI PMS2 as much as 21 (29.2%) and, MSI MLH1 and PMS2 simultaneously as much as 18 (25.0%). MSI MLH1 and PMS2 positive are found in ≥50 years old. The number of male patients is more than women. Most of the patients had a T3-T4 tumor size with advanced stage and well differentiated. MSI MLH1 associated with tumor differentiation (p = 0.011). Conclusion: MSI MLH1 is associated with tumor differentiation (p = 0.011), but, no association with the other clinicopathology parameters. MSI PMS2 is not associated to the overall clinicopathology parameters. MSI MLH1 and PMS2 are not simultaneously associated with all clinicopathology parameters (p&gt; 0.05)

Somatic Sequencing and Microsatellite Instability Results From Mismatch Repair-deficient Endometrial Carcinoma Patients Without Lynch Syndrome ("Lynch-like" tumors): Implications for Heritable Cancer Screening, Molecular Prognostication, and Immunotherapeutic Vulnerability.

Immunostaining of endometrial carcinomas for mismatch repair (MMR) protein loss is standard-of-care for Lynch syndrome screening, but also identifies MMR-deficient tumors without germline pathogenic variants. While the majority show MLH1 hypermethylation (MLH1hm), somatic MMR pathogenic variants are increasingly recognized drivers of immunohistochemistry-germline discordance. Because MMR abnormalities with both germline and somatic origins have prognostic significance and impart susceptibility to immune checkpoint inhibitors, it is important to understand how frequently tumors with MMR immunohistochemical loss and normal germline testing ("Lynch-like" tumors) have underlying somatic MMR pathogenic variants. Somatic tumor sequencing±microsatellite instability (MSI) testing was performed on 18 endometrial cancers with MMR immunohistochemical loss but negative MMR germline results and negative MLH1hm where relevant. Tumor sequencing and MSI testing were successful in 94%. Where successful, 80% were MSI-high and 94% had a molecular correlate for the initial immunohistochemical interpretation. The single case without an identified somatic pathogenic variant was MSI-low and initially showed loss of MSH6 by immunohistochemistry but with extremely limited internal control staining. On review, MSH6 immunohistochemistry was reclassified as equivocal, and repeat staining revealed improved control expression with intact MSH6. Following reclassification of this case, 100% tumors with MMR deficiency by immunohistochemistry had at least 1 confirmed somatic MMR pathogenic variant, and 86% were MSI-high. These results demonstrate that when correctly interpreted immunohistochemistry is a strong surrogate for somatic MMR pathogenic variants and support its use as the frontline MMR biomarker in endometrial cancer for heritable screening, molecular prognostic classification, and immunotherapeutic biomarker testing purposes.

Epigenetic MMR defect identifies a risk group not accounted for through traditional risk stratification algorithms in endometrial cancer.

We sought to evaluate the contribution of mismatch repair (MMR) status to traditional risk stratification algorithms used to predict nodal involvement and recurrence in a large single-institution cohort. Endometrioid endometrial cancer (EC) cases from 2014-2020 were evaluated. MMR immunohistochemistry (IHC) was performed universally. Uterine factors assessed in the Mayo criteria were used to retrospectively classify patients as low or high risk for lymphatic spread. Patients were classified according to risk for recurrence using GOG 99 and PORTEC criteria. Associations were evaluated using chi-square and t-tests and contributing factors assessed using logistic regression models. 1,514 endometrioid EC were evaluated; 392 (25.9%) were MMR (MMR) deficient of which 80.4% of MMR defects were associated with epigenetic silencing of MLH1. Epigenetic MMR defects were significantly more likely to be high risk for lymph node (LN) metastasis based on Mayo criteria (74.9% vs 60.6%, p=<0.001) and with the presence of LN metastasis (20.3 vs 10.5%, p=0.003) compared to MMR proficient tumors. Tumors with epigenetic MMR defects were significantly more likely to be classified as high or high intermediate risk using GOG99 and PORTEC criteria. Furthermore, cases with epigenetic MMR defects classified as low or low intermediate risk were significantly more likely to recur (GOG99 p=0.013; PORTEC p=0.008) and independently associated with worse disease-free survival (DFS). MMR status was found to be independently associated with worse DFS (HR 1.90; 95% CI 1.34-2.70; p=0.003) but not overall survival. While MMR deficient EC has been associated with poor prognostic features in prior reports; we demonstrate that only epigenetic MMR defects have poorer outcomes. Epigenetic MMR defect were independently associated with lymph node metastasis after controlling for risk criteria. Epigenetic MMR deficiency was found to be an independent predictor of recurrence beyond the factors considered in traditional risk stratification algorithms. Traditional uterine-based risk stratification algorithms may not fully reflect the risk for recurrence in MMR deficient tumors. Consideration should be given to implementing MMR status and MLH1 hypermethylation alongside traditional risk stratification algorithms. Performing MMR IHC on preoperative pathologic specimens may aid in risk stratification and patient counseling.

High frequency of MLH1 promoter methylation mediated by gender and age in colorectal tumors from Mexican patients.

Several genes determine the development of colorectal cancer (CRC), such as MLH1, which encodes a protein that participates in DNA repair. MLH1 hypermethylation has been associated with gene silencing. To analyze the methylation of five regions of MLH1 CpG island in colorectal tumors from Mexican patients. One hundred and one tumor tissue samples were obtained from Mexican patients with CRC who provided informed consent. DNA was subjected to bisulfite conversion. Methylation of all five regions of the CpG island was evaluated using methylation-specific PCR. The frequency of methylation in Mexican patients with CRC was 25%. Regions A and B methylation was the main observed pattern (60%). Female patients showed a higher frequency of methylation (71%; OR 3.085; CI: 1.85-8.03; p = 0.02), and out of total methylated samples, 80% corresponded to individuals older than 45 years (p < 0.05). We calculated a methylation frequency for the MLH1 gene of 25% in Mexican patients with CRC, with this being the first report for this population. Female patients and patients older than 45 years showed a higher frequency of methylation.

Epigenomics may begin to explain in vitro differential response to hypomethylating agents in MMR-D hypermethylated endometrial cancer.

This work examines differences in chromatin accessibility, methylation, and response to DNA hypomethylating agents between mismatch repair-deficient and non-mismatch repair-deficient endometrial cancer. Next-generation sequencing of a stage 1B, grade 2 endometrioid endometrial cancer tumor revealed microsatellite instability and a variant of unknown significance in POLE along with global and MLH1 hypermethylation. Inhibition of viability by decitabine in the study and comparison tumors was minimal, as shown by an inhibitory effect of 0 and 17.9, respectively. Conversely, the inhibitory effect of azacitidine on the study tumor was more pronounced, at 72.8versus 41.2. In vitro, mismatch repair-deficient endometrial cancer with MLH1 hypermethylation respond better to DNAmethyltransferase inhibition by azacytidine (DNA/RNA inhibition), than to decitabine (DNA-only inhibition). Additional large studies are needed to substantiate our findings.

Rates and outcomes of testing for lynch syndrome in a national colorectal cancer screening programme

BackgroundLynch Syndrome (LS), the most common cause of hereditary colorectal cancer (CRC), is characterised by pathogenic variants in mismatch repair (MMR) genes. Universal testing of all CRCs for LS can increase detection. Rates and outcomes of testing in Ireland’s national CRC screening programme have not been examined previously. MethodsCRCs diagnosed at two screening sites between 2015 and 2020 were identified. Patient records were used to determine if CRCs had been tested for MMR deficiency and if detected, what downstream testing to rule out LS or genetic testing to confirm LS was undertaken. ResultsOver five years, 206 CRCs were diagnosed. Testing for LS was carried out for 100% of CRCs at site A and 69% of CRCs at site B. Of CRCs tested for LS, 14 (8%) were MMR deficient. After downstream testing for BRAF mutation or hypermethylation of MLH1, three CRCs were identified as potentially LS-related. Of these two individuals declined genetic testing and one was lost to follow-up. ConclusionsBy 2020 both sites had implemented universal testing of all CRCs for LS. A small number of individuals were identified as being eligible for genetic testing for LS, however those offered declined testing and one individual was lost to follow up. This highlights the importance of universal testing and the need for referral pathways to ensure all appropriate individuals are referred onwards to genetic services.

DNA Mismatch Repair Proteins and BRAF V600E Detection by Immunohistochemistry in Colorectal Cancer Demonstrates Concordance with Next Generation Sequencing

Background and Aims: Multiple laboratory methods are used to screen patients with colorectal cancer (CRC) for mismatch repair (MMR) protein deficiency to identify possible Lynch syndrome patients. The goal of this study was to compare the agreement between ready-to-use immunohistochemistry (IHC) assays for MLH-1, PMS-2, MSH-2, MSH-6, and mutated BRAF at V600E and molecular methods in CRC cases. The inclusion of the BRAF V600E mutation testing is important for the identification of patients with sporadic CRC, as the BRAF V600E mutation is very rarely observed in patients with Lynch syndrome tumors. Methods: CRC cases were analyzed by ColoSeqTM tumor sequencing assay and VENTANA MMR IHC Panel that included anti-MLH1, anti-PMS2, anti-MSH2, anti-MSH6, and anti-BRAF V600E antibodies. Additionally, CRC cases with MLH1 IHC loss were evaluated for MLH1 promoter hypermethylation. Results: One hundred and eighteen cases were analyzed. The overall percent agreement (OPA) for each evaluated marker status compared to next-generation sequencing (NGS) exceeded 96%. Twenty-three cases were positive for the BRAF V600E mutation by IHC and NGS, and twenty cases showed loss of MLH1 protein and were positive for MLH1 hypermethylation. Samples with loss of MMR protein expression by IHC demonstrated genetic and/or epigenetic alterations that were consistent with the observed protein expression patterns. Conclusions: The results of this study indicate that ready-to-use IHC assays can correctly identify the loss of MMR proteins and the presence of mutated BRAF V600E protein, supporting the utility of the VENTANA MMR IHC Panel as an aid to stratify patients with sporadic CRC vs. potential Lynch syndrome.

MLH1 hypermethylation predicts poor outcomes with pembrolizumab in recurrent endometrial cancer (087)

Objectives: To determine response rate, recurrence-free survival, and overall survival to pembrolizumab in recurrent endometrial adenocarcinomas based upon the classification of mismatch repair (MMR) deficiency. Methods: A retrospective study was conducted among all women with recurrent endometrial adenocarcinomas treated with single-agent pembrolizumab at our institution from 2017 to 2021. Cases were included if complete mismatch repair data were available and were categorized based upon MMR status – germline Lynch Syndrome mutation (gLS), somatic Lynch Syndrome mutation (sLS), or MLH1 promoter hypermethylation (hMLH1). Clinical and pathologic data were abstracted by chart review and included patient demographics, tumor characteristics, recurrence date, and treatment. Continuous predictors were compared between groups using two-sample t-tests. Ordinal predictors such as stage and grade were compared using Wilcoxon rank-sum tests. Categorical predictors were compared using Fisher’s exact tests due to small counts. Unadjusted differences in survival were compared with log-rank tests and visualized with Kaplan-Meier curves. Cox proportional hazards models were used to compare differences in survival between groups after adjusting for BMI. All statistical tests were conducted in R v3.5.1 with an alpha level of 0.05. Results: Eighteen patients were categorized into two groups based upon results of tumor MMR testing and germline genetic testing: gLS/sLS (n = 6, 33.3%) or hMLH1 (n = 12, 66.7%). The hMLH1 patients had a higher mean BMI than gLS/sLS patients (40.9 vs 28.5, p = 0.003). Demographic and tumor characteristics were otherwise well-matched between groups. Median follow-up was 9.45 months with a response rate of 55.6%. When stratified by MLH1 hypermethylation status, response rate was 83.3% in gLS/sLS patients and 41.7% in hMLH1 patients, though this did not reach statistical significance (p = 0.152). Recurrence-free survival (RFS) was significantly decreased among hMLH1 patients (p = 0.031). Similarly, overall survival (OS) was significantly decreased for these patients (p = 0.037), as shown in Figure 1. Objectives: To determine response rate, recurrence-free survival, and overall survival to pembrolizumab in recurrent endometrial adenocarcinomas based upon the classification of mismatch repair (MMR) deficiency. Methods: A retrospective study was conducted among all women with recurrent endometrial adenocarcinomas treated with single-agent pembrolizumab at our institution from 2017 to 2021. Cases were included if complete mismatch repair data were available and were categorized based upon MMR status – germline Lynch Syndrome mutation (gLS), somatic Lynch Syndrome mutation (sLS), or MLH1 promoter hypermethylation (hMLH1). Clinical and pathologic data were abstracted by chart review and included patient demographics, tumor characteristics, recurrence date, and treatment. Continuous predictors were compared between groups using two-sample t-tests. Ordinal predictors such as stage and grade were compared using Wilcoxon rank-sum tests. Categorical predictors were compared using Fisher’s exact tests due to small counts. Unadjusted differences in survival were compared with log-rank tests and visualized with Kaplan-Meier curves. Cox proportional hazards models were used to compare differences in survival between groups after adjusting for BMI. All statistical tests were conducted in R v3.5.1 with an alpha level of 0.05. Results: Eighteen patients were categorized into two groups based upon results of tumor MMR testing and germline genetic testing: gLS/sLS (n = 6, 33.3%) or hMLH1 (n = 12, 66.7%). The hMLH1 patients had a higher mean BMI than gLS/sLS patients (40.9 vs 28.5, p = 0.003). Demographic and tumor characteristics were otherwise well-matched between groups. Median follow-up was 9.45 months with a response rate of 55.6%. When stratified by MLH1 hypermethylation status, response rate was 83.3% in gLS/sLS patients and 41.7% in hMLH1 patients, though this did not reach statistical significance (p = 0.152). Recurrence-free survival (RFS) was significantly decreased among hMLH1 patients (p = 0.031). Similarly, overall survival (OS) was significantly decreased for these patients (p = 0.037), as shown in Figure 1.