Inactivation Of MLH1 Research Articles

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.

P1.21-12 Microsatellite Instability Defines a Subset of Lung Cancers with Smoking Exposure, High Tumor Mutational Burden and MLH1 Inactivation

P1.21-12 Microsatellite Instability Defines a Subset of Lung Cancers with Smoking Exposure, High Tumor Mutational Burden and MLH1 Inactivation

Microsatellite Instability and Mismatch Repair Deficiency Define a Distinct Subset of Lung Cancers Characterized by Smoking Exposure, High Tumor Mutational Burden, and Recurrent Somatic MLH1 Inactivation

IntroductionMicrosatellite instability (MSI) and mismatch repair (MMR) deficiency represent a distinct oncogenic process and predict response to immune checkpoint inhibitors (ICIs). The clinicopathologic features of MSI-high (MSI-H) and MMR deficiency (MMR-D) in lung cancers remain poorly characterized. MethodsMSI status from 5171 patients with NSCLC and 315 patients with SCLC was analyzed from targeted next-generation sequencing data using two validated bioinformatic pipelines. ResultsMSI-H and MMR-D were identified in 21 patients with NSCLC (0.41%) and six patients with SCLC (1.9%). Notably, all patients with NSCLC had a positive smoking history, including 11 adenocarcinomas. Compared with microsatellite stable cases, MSI-H was associated with exceptionally high tumor mutational burden (37.4 versus 8.5 muts/Mb, p < 0.0001), MMR mutational signatures (43% versus 0%, p < 0.0001), and somatic biallelic alterations in MLH1 (52% versus 0%, p < 0.0001). Loss of MLH1 and PMS2 expression by immunohistochemistry was found in MLH1 altered and wild-type cases. Similarly, the majority of patients with MSI-H SCLC had evidence of MLH1 inactivation, including two with MLH1 promoter hypermethylation. A single patient with NSCLC with a somatic MSH2 mutation had Lynch syndrome as confirmed by the presence of a germline MSH2 mutation. Among patients with advanced MSI-H lung cancers treated with ICIs, durable clinical benefit was observed in three of eight patients with NSCLC and two of two patients with SCLC. In NSCLC, STK11, KEAP1, and JAK1 were mutated in nonresponders but wild type in responders. ConclusionsWe present a comprehensive clinicogenomic landscape of MSI-H lung cancers and reveal that MSI-H defines a rare subset of lung cancers associated with smoking, high tumor mutational burden, and MLH1 inactivation. Although durable clinical benefit to ICI was observed in some patients, the broad range of responses suggests that clinical activity may be modulated by co-mutational landscapes.

Targeting DNA mismatch repair pathway by CRISPR nanosystem for boosting checkpoint blockade cancer immunotherapy

Immune checkpoint blockade (ICB) therapy has exhibited great clinical benefits in multiple cancers types. However, most cancer patients such as colorectal cancers (CRC) show poor response to ICB. Here, we develop a magnetic nanoparticle (MNP)-mediated delivery of CRISPR/Cas9 system (LMPM) to target the DNA repair pathway and inactivate endogenous DNA mismatch repair (MMR)-related Mlh1 gene as a new strategy of immunotherapy. The genetically knockout of Mlh1 can be effectively realized by CRISPR/Cas9 edition with external magnetic field control. As a consequence, the inactivation of Mlh1 lead to an increased tumor mutation burden and enhancement of tumor immunogenicity in situ, which can elicit strong antitumor immunity. Both in vitro and in vivo studies demonstrate that deploying such a strategy of targeting inactivation of Mlh1 shows high inhibition efficacy in the established tumors. In this way, tumor infiltration of CD8+ T cells and ICB response are significantly improved compared with α-PD1 monotherapy. In short, it is certified that precision targeting of DNA MMR pathway can boost specific anti-tumor immune response and display the potent potential in versatile tumor immunotherapy therapeutic exploitation.

Abstract 1593: Genetic and pharmacological modulation of DNA mismatch repair promotes immune surveillance in murine colorectal cancer

Abstract Patients affected by colorectal cancer (CRC) with microsatellite instability (MSI), which is caused by DNA mismatch repair deficiency (MMRd), are eligible for therapies based on immune checkpoint inhibitors (CPI), while microsatellite stable (MSS) tumors are not. However, a subset of MSS CRCs contains variable fractions of MMRd cells. How the presence of MMRd cells in tumors classified as MSS impacts cancer immune surveillance is largely unknown. It is also unclear whether the pharmacological modulation of MMRd cells percentage in MMR heterogeneous tumor can occur and if this might result in the improvement of tumor immune control.To shed light on these aspects we studied isogenic mismatch repair proficient and deficient mouse tumor cells, generated by genetic inactivation of MLH1, a key component of the MMR machinery. We mixed MLH1+/+and MLH1-/-cells at different ratios, injecting the resulting heterogeneous populations in mice. In the presence of a competent immune system, the tumorigenic potential and immune surveillance of MSS/MSI (MLH1+/+/MLH1-/-) heterogeneous tumors was dependent upon the MMRd fraction. Tumor rejection was observed when at least 50% of the cells were MMRd, but tumor growth delay was also evident when as low as 20% of MMRd cells were present in the mixed population and this was paralleled by immune infiltration of the tumor. Molecular profiles of samples from MSS/MSI heterogeneous tumors that evaded immune control, showed enrichment of the MSS fraction. Treatment of MSS/MSI mixed populations with the antimetabolite 6-Thioguanine (6TG) greatly enriched the MMRd fraction and improved immune response. Overall, these results suggest that genetic and/or pharmacological modulation of the DNA mismatch repair machinery can foster immune surveillance of MMR heterogeneous tumors and modulate the cancer immune environment. Citation Format: Vito Amodio, Giovanni Germano, Benedetta Mussolin, Simona Lamba, Rosaria Chilà, Giuseppe Rospo, Caterina Marchiò, Silvia Marsoni, Gianluca Mauri, Federica Di Nicolantonio, Alberto Bardelli. Genetic and pharmacological modulation of DNA mismatch repair promotes immune surveillance in murine colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1593.

Histopathological and genetic features of mismatch repair-deficient high-grade prostate cancer.

Mismatch repair (MMR) deficiency is commonly caused by functional inactivation of MLH1, PMS2, MSH2 or MSH6. The morphological and molecular correlates of MMR deficiency have been extensively characterized in certain tumour types such as colorectal and endometrial adenocarcinoma. In contrast, the histological and molecular features of MMR-deficient prostate cancer remain incompletely described. In this study, we evaluated 19 MMR-deficient prostate cancers, including 11 cases without prior systemic treatment. All treatment-naive cases (11 of 11, 100%) were grade group 4-5 and had predominant cribriform and/or solid growth patterns. Solid components (any amount) and tumour infiltrating lymphocytes were 7 cases each (7 of 11, 64%). In 68 MMR-proficient grade group 5 prostate cancers, predominant cribriform or solid growth patterns, solid components (any amount) and tumour infiltrating lymphocytes were seen at significantly lower frequencies (31 of 68, 46%; 9 of 68, 13% and 6 of 62, 9%, respectively; P < 0.001 for all comparisons). Molecular evaluation of 19 cases demonstrated that MMR-deficiency was secondary to functional loss of MSH2/MSH6 and MLH1/PMS2 in 15 (79%) and 4 cases (21%), respectively. Definite or probable germline mutations were present in 4 cases (4 of 19, 21%). TMPRSS2::ERG rearrangements were identified in 2 cases (2 of 19, 11%). Recurrent cancer-relevant somatic mutations included (but were not limited to) ATM, TP53, FOXA1, RB1, BRCA2 and PTEN. MMR deficiency was most commonly secondary to inactivation of MSH2/MSH6 in this study. Importantly, MMR-deficient high-grade prostatic adenocarcinomas had morphological features that might be useful to identify selected cases for MMR immunohistochemistry.

Abstract PO-052: A multisystem approach doubles the number of patients referred for genetic testing and diagnosed with Lynch syndrome with equally success among ethnic/racial groups

Abstract Background: Lynch syndrome (LS) affects 1/279 individuals and accounts for 3-5% of all colorectal cancers (CRC). LS is due to germline mutations in mismatch repair (MMR) genes MLH1, MSH2, MSH6, or PMS2. Tumors exhibit MMR deficiency, expressed as loss of MMR protein expression on immunohistochemistry (IHC). A subset of sporadic tumors also has MMR deficiency due to inactivation of MLH1 via promoter methylation and about 2/3 have BRAFV600E mutations. Increased surveillance and chemoprevention reduce cancer incidence and mortality in LS. LS is largely underdiagnosed, particularly among minorities. Universal screening of CRC tumors through IHC is recommended in order to help identify patients for LS genetic testing. Nevertheless, even in programs that have implemented this, minority patients with MMR deficient tumors were found to be consistently under-referred mostly due to referral bias. We set up a strategy to increase LS and tackle disparities in cancer genetics (CG). Methods: This study reports on the analysis of universal CRC IHC testing for MMR expression, initially followed by BRAF V600E and later by MLH1 testing for loss of MLH1/PMS2 expression, from implementation (1/1012) until 1/1021. In 6/2015, a system was established where a designated Pathologist reviewed all results and a list of identified candidates for germline LS testing (abnormal MMR expression/wild type BRAF or methylation of MLH1) was sent to the CG program. After a two-week period, if no referral had been placed, a genetic counselor (GC) messaged the provider requesting a referral. The pathology side was fully automated in 9/2018 through the monitoring engine Repetitive Tasks Scheduling Engine (RTSE) that pulls data stored in the underlying relational database-Adaptive Server Enterprise, which is accessible to RTSE via a Java Database Connectivity API. This process automatically generates weekly emails that report all cases with suspicion criteria for LS to CG. Results: Since 1-2012, 1,433 patients with CRC underwent tumor testing and were included in this study: 78.5% non-Hispanic white (NHW); 12.2% African American (AA); 6.77% Hispanic (H). Prior to the interventions 25% of eligible patients were referred to CG and none among AA. Once the interventions were established, appropriate referrals prior to messaging the provider were 39.5% for NHW, 35.7% for AA, and 42.8% for H. As a result of the messages, referral rates increased to 80.5% (p=0.00) in NHW, 85.7% in AA (p=0.05), and 71.5% in H (ns). The total number of patients diagnosed with LS since universal testing started was 19: 2 prior to the interventions (0.8% of all CRC); 17 during the interventions (1.5% of all CRC). Conclusion: A systematic, automated, multi-system approach, that includes a targeted message to providers can double the number of appropriate referrals for genetic testing to rule out Lynch syndrome and resulted in almost twice the percentage of LS diagnosis with equal success among AA, H, and NHW. Citation Format: Vinit Singh, Amanda Ganzak, Peter Gershkovich, Joanna Gibson, Rosa M. Xicola, Xavier Llor. A multisystem approach doubles the number of patients referred for genetic testing and diagnosed with Lynch syndrome with equally success among ethnic/racial groups [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-052.

Clinicopathological and molecular characteristics of prostate cancer diagnosed in young men aged up to 45 years

AimsTo characterise and compare the poorly understood clinicopathological and molecular characteristics of prostatic adenocarcinoma (PCa) in very young patients.Methods and resultsWe compared the clinicopathological and molecular characteristics of PCa diagnosed in 90 patients aged ≤45 years with those of PCa diagnosed in 200 patients of typical screening age (i.e. 60–65 years). Patients diagnosed at a younger age had a higher frequency of a family history of PCa and lower prostate‐specific antigen (PSA) levels than those diagnosed at regular screening age. There were no statistically significant differences in clinical stage or pathological characteristics of the core biopsy specimens between the groups. Young patients had a higher frequency of Grade Group 1 disease at radical prostatectomy. A subset of 13 aggressive PCa cases from young patients underwent successful DNA‐based next‐generation sequencing. In all, 46.2% (6/13) had TMPRSS2 rearrangements and 23.1% (3/13) had relevant pathogenic variants in DNA damage repair genes, including a mismatch repair‐deficient case with biallelic inactivation of MLH1. No statistically significant differences were observed in PCa‐specific recurrence/progression between the younger and older patients, including after adjustment for clinical stage, PSA level, and Grade Group.ConclusionsIn this study, the clinicopathological and molecular features of PCa diagnosed in young patients were comparable to those of PCa diagnosed in patients of screening age. Early‐onset PCa cases were not enriched in any of the known molecular PCa subtypes in this small series.

Abstract 2366: Germline biallelic mutations in MCM8 are associated with early-onset Lynch-like syndrome

Abstract Purpose: The most frequent cause of hereditary colorectal cancer (CRC) is Lynch syndrome, accounting for 3% of all CRC cases. The underlying germline predisposition is a mutation in any of the four mismatch repair (MMR) genes (MLH1, MSH2, MSH6, PMS2), while microsatellite instability (MSI) is a hallmark of these tumors. However, CRC cases with MSI, no MLH1 somatic hypermethylation and absence of MMR germline mutation account for up to 60% of cases of MMR-defective tumors. They have been termed Lynch-like syndrome (LLS) and treatment management decisions are complicated due to unconfirmed suspicions of hereditary cancer. The aim of the present study was to investigate whether LLS cases in patients under the age of 40 carry potentially pathogenic germline variants in new genes for CRC predisposition causing a MMR-defective tumor phenotype. Methods: We performed whole-exome sequencing (WES) in the germline and tumoral DNA of 16 early-onset LLS patients. We filtered for recessive germline variants in genes involved in DNA repair. An exhaustive functional evaluation for 2 MCM8 genetic variants detected in one patient was performed. The CRISPR/Cas9 system was used to generate MCM8ko clones in a cell model (DLD-1). Site-directed mutagenesis produced the genetic variants for ectopic expression in the MCM8ko model. Functional characterization included the comet assay, which detected DNA damage induced by oxaliplatin in the transfected cells. MCM8ko cells were evaluated for MSI after 30, 60 and 90 days of culture, and mutational signatures were assessed with SigProfiler. Results: The MCM8 variants c.351_354delAAAG (p.Lys118GlufsTer5, p.K118fs) and c.414A&amp;gt;G (p.Ile138Met, p.I138M) were identified in one early-onset LLS patient with biallelic somatic inactivation of MLH1. Manual evaluation of WES data showed that germline MCM8 variants were in trans. In the comet assay, both MCM8 variants revealed higher sensitivity to oxaliplatin and more DNA damage compared to the wild-type control. One of the MCM8KO clones showed MSI after 30 days of sub-culturing. After 120 days, MCM8KO clones had acquired a significant contribution of the SBS20 mutational signature, which is associated with defective MMR DNA. Conclusions: We postulate that MCM8 is a new germline CRC predisposing gene in early-onset LLS, which may be involved in both MMR and double-strand break repair. We further provide evidence that in some LLS CRC cases, especially in young patients, the biallelic somatic MMR inactivation may be caused by new CRC germline predisposing genes. Additional mutational screening is warranted for such cases. Citation Format: Mariano Golubicki, Laia Bonjoch, José G. Acuña-Ochoa, Marcos Diaz-Gay, Jennifer Muñoz, Miriam Cuatrecasas, Teresa Ocaña, Juan Robbio, Enrique L. Roca, Antoni Castells, Fracesc Balaguer, Marina Antelo, Sergi Castellvi-Bel. Germline biallelic mutations in MCM8 are associated with early-onset Lynch-like syndrome [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2366.

Whole Exome Sequencing Identifies Candidate Genes Associated with Hereditary Predisposition to Uveal Melanoma

To identify susceptibility genes associated with hereditary predisposition to uveal melanoma (UM) in patients with no detectable germline BAP1 alterations. Retrospective case series from academic referral centers. Cohort of 154 UM patients with high risk of hereditary cancer defined as patients with 1 or more of the following: (1) familial UM, (2) young age (<35 years) at diagnosis, (3) personal history of other primary cancers, and (4) family history of 2 or more primary cancers with no detectable mutation or deletion in BAP1 gene. Whole exome sequencing, a cancer gene panel, or both were carried out. Probands included 27 patients with familial UM, 1 patient with bilateral UM, 1 patient with congenital UM, and 125 UM patients with strong personal or family histories, or both, of cancer. Functional validation of variants was carried out by immunohistochemistry, reverse-transcriptase polymerase chain reaction, and genotyping. Clinical characterization of UM patients with germline alterations in known cancer genes. We identified actionable pathogenic variants in 8 known hereditary cancer predisposition genes (PALB2, MLH1, MSH6, CHEK2, SMARCE1, ATM, BRCA1, and CTNNA1) in 9 patients, including 3 of 27 patients (11%) with familial UM and 6 of 127 patients (4.7%) with a high risk for cancer. Two patients showed pathogenic variants in CHEK2 and PALB2, whereas variants in the other genes each occurred in 1 patient. Biallelic inactivation of PALB2 and MLH1 was observed in tumors from the respective patients. The frequencies of pathogenic variants in PALB2, MLH1, and SMARCE1 in UM patients were significantly higher than the observed frequencies in noncancer controls (PALB2: P= 0.02; odds ratio, 8.9; 95% confidence interval, 1.5-30.6; MLH1: P= 0.04; odds ratio, 25.4; 95% confidence interval, 1.2-143; SMARCE1: P= 0.001; odds ratio, 2047; 95% confidence interval, 52-4.5e15, respectively). The study provided moderate evidence of gene and disease association of germline mutations in PALB2 and MLH1 with hereditary predisposition to UM. It also identified several other candidate susceptibility genes. The results suggest locus heterogeneity in predisposition to UM. Genetic testing for hereditary predisposition to cancer is warranted in UM patients with strong personal or family history of cancers, or both.

1411P - Methylation analysis of MLH1 using droplet digital PCR and methylation sensitive restriction enzyme

BackgroundLynch syndrome (LS) is caused by germline mutations in DNA mismatch repair (MMR) genes being MLH1 and MSH2 the most commonly mutated. By contrast MLH1 inactivation as the result of promoter methylation is strongly indicative of a sporadic cancer, providing LS exclusion criteria for 85% of high microsatellite instability (MSI-H) tumours. Here we present a cost effective strategy enabling to test methylation of MLH1 promoter without bisulfite conversion and with minimal DNA quantity requirement. MethodsAfter macrodissection from HE stained slides, DNA was extracted from FFPE tissue sections of colorectal carcinomas. A droplet digital PCR (ddPCR) was performed using two reactions mix. A 270-bp region of the MLH1 promoter (chr3:36993151-36993420) is amplified in the presence/absence of HinP1I, a methylation sensitive restriction enzyme, targeting 3 CpG islands. Analysis was made by the QuantaSoft™ (Bio-Rad) software which calculates amplicon concentrations between the 2 reactions to obtain a percentage of MLH1 methylation. Sensitivity of the technique was assigned to 5% of methylation with a minimal DNA concentration of 5ng. ResultsMethylation analysis by ddPCR was compared to pyrosequencing combined with bisulfite conversion for 65 samples and a 100% concordance was obtained. Moreover 10 samples were analysed by ddPCR and MethylLight RealTime-PCR with the same concordance. After validation, the technique was implemented in the clinical diagnosis and, in one year, out the 79 MMR-deficient colorectal carcinomas analysed, 60 (76 %) were MLH1-methylated tumours. ConclusionsThis ddPCR sequencing combining methylation sensitive restriction enzyme is a cost-effective strategy, requiring less technical turn around time and minimal DNA quantity as compared to standard analysis. Moreover, this technique could be further used for other promoter methylation analysis (such as MGMT) and on circulating tumoral DNA. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

527P - Screening strategy of Lynch syndrome for Chinese colorectal cancer patients with MLH1-immunoloss

Background: Inactivation of MLH1 due to promoter hypermethylation strongly suggests a sporadic origin, and nearly half sporadic colorectal cancer patients harbor BRAF mutation, providing exclusion criteria for Lynch syndrome (LS). However, there was little evidence from Chinese population. The aim of this study was to compare the utility of two tests, and explore the possibility of substituting BRAF immunohistochemical staining (IHC) for real-time PCR. Methods: We reviewed MMR expression status of consecutive patients who had undergone surgery for colorectal cancer between 2012.1.1 and 2014.12.30 in Chinese National Cancer Center. Among 317 patients who were identified as dMMR, 170 patients with MLH1 immunoloss were taken into final analysis. MLH1 methylation status was evaluated by MS-PCR(methylation – specific PCR) ,BRAF mutation was tested by IHC and real-time PCR. Results: 52.9%patients display MLH1 promoter hypermethylation. As for BRAF status, the mutation rate tested by IHC and real-time PCR was 14.1% and17.1%, respectively, and the concordance rate was 92.1%. BRAF mutation did better on ruling out patients whose relatives had CRC history. Although patients who had unmethylated CRCs had a notably stronger family history of CRC, there were still 13.3% patients in the hypermethylation group having family history of CRC, indicating a likelihood of LS.Table: 527PFeatureMLH1 methylated n = 90MLH1 unmethylated n = 80PBRAF mutation n = 24BRAF wild-type n = 146pMedian age62y50y64y54ySynchronic CRC3(3.3)8(10.0)0.0781(4.2)10(6.8)0.962FDR or SDR with LS-related tumor17(18.9)14(17.5)0.8153(12.5)28(19.2)0.617FDR or SDR with CRC12(13.3)32(40)0.000044(30.1)0.002FDR with CRC9(10.0)27(35.0)0.000036(24.7)0.016Revised Bethesda guidelines43(47.8)69(86.3)0.0008(33.3)104(71.2)0.000 Open table in a new tab Conclusions: The mutation rate of BRAF in Chinese population with MSI CRCs was significantly lower than that in western countries, leading to a decreased specificity. Thus, BRAF mutation alone was not efficient to be a negative predictor of LS. Due to the high specificity of MLH1 methylation test and the high concordance rate of IHC and real-time PCR for BRAF mutation, patients who have MLH1 hypermethylation, BRAF wild-type tested by IHC and family history of CRC should be recommended for germline mutation test additional to those with MLH1 unmethylated CRCs. Legal entity responsible for the study: National Cancer Center, China. Funding: Chinese Academy of Medical Science. Disclosure: All authors have declared no conflicts of interest.

PO-332 Genomic landscapes, neoantigen profiles and biological impact of MLH1 inactivation in cancer cells

IntroductionAlterations in DNA repair pathways are thought to fuel tumour progression. Mismatch Repair (MMR) deficient cancers show peculiar biological features such as an indolent progression and a resolute therapeutic response to checkpoint inhibitors. The genomic and biological bases of the peculiar clinical features are poorly understood. Further progress in this area is limited by the paucity of models to study the impact of MMR genes inactivation at the genomic and biological levels. To address this issue we developed a bioinformatic workflow to monitor the neoantigen repertoire induced by inactivation of the Mlh1 gene (a key player of the MMR machinery), in murine cell lines.Material and methodsWe inactivated Mlh1 throughout the CRISPR-Cas9 technology in CT26 (colon cancer), PDAC (pancreatic cancer) and TSA (breast cancer) murine cell lines. We performed whole exome sequencing (WES) at different time points and then we quantified the amount of mutations (SNVs and indels). We generated a pipeline that characterises the neoantigen repertoire, starting from annotated alterations and the HLA of specific murine strain. In parallel, we inoculated MMR-proficient and -deficient cells in immuno-compromised and -competent mice and monitored their growth.Results and discussionsIn all pre-clinical models analysed we found a massive increment in the number of non-synonymous alterations (up to 100% increase respect to basal population) after Mlh1 inactivation. Notably, analysis of MMR deficient mouse cells at different time points showed a renewal of mutational profile and consequently an accumulation of predicted neoantigens. We further characterised the SNVs and frameshifts acquired by Mlh1-knockout cells. In agreement with data in human tumours, the fraction of predicted neoantigens derived from frameshifts was higher than the SNV-derived neoantigens. When injected in immuno-compromised mice the Mlh1-knockout cells and their wild type counterpart showed comparable growth. On the contrary, MMR-deficient cells but not their control counterpart grew poorly in immuno-competent mice and responded promptly to treatment with checkpoint inhibitors.ConclusionWe find that Mlh1 gene inactivation drives dynamic neoantigen profiles, which can be monitored with an ad hoc bioinformatic pipeline. These analyses provide mechanistic support to understand why MMR deficient cells engage the immune system of the host, foster immune surveillance and tumour control.

Success of referral to genetic counseling after positive lynch syndrome screening test.

Lynch syndrome (LS) is a hereditary condition that increases one's risk of developing colorectal, endometrial, and other extracolonic cancers. MD Anderson Cancer Center at Cooper implemented a reflex screening protocol for DNA mismatch repair (dMMR) deficiency. Those with findings suspicious for LS were referred for genetic counseling (GC). Our goal was to assess compliance with GC and factors associated with successful follow-up. Immunohistochemistry (IHC) for the MMR proteins MSH2, MLH1, MSH6, and PMS2 was performed on all colorectal tumor resections from patients ≤70years old and all stage II cancers. Tumors with loss of MLH1/PMS2 were subsequently tested for BRAF mutation or MLH1 promoter methylation to identify tumors with likely epigenetic inactivation of MLH1. Patients with loss of MLH1/PMS2 without BRAF mutations or with absence of MLH1 promoter methylation and those with loss of MSH2/MSH6 were referred to GC. Compliance with GC was assessed. Between March 2014 and August 2016, 203 tumors were tested by IHC. Fifteen (7.4%) patients had abnormal MMR protein expression patterns in the absence of BRAF mutation or MLH1 promoter methylation suggestive of possible LS. GC compliance was 35.7% overall and 85.7% in those with family history of LS-associated cancers. Overall, GC compliance was relatively low in our study. Interestingly, patients with a strong family history of LS-associated neoplasms were more likely to pursue GC. In the future, assessing and addressing barriers to seeking GC will provide opportunities to improve patient care through increased identification of patients with cancer predisposition syndromes.

Exceptional Chemotherapy Response in Metastatic Colorectal Cancer Associated With Hyper-Indel-Hypermutated Cancer Genome and Comutation of POLD1 and MLH1.

A73-year-old woman with metastatic colon cancer experienced a complete response to chemotherapy with dose-intensified irinotecan that has been durable for 5 years. We sequenced her tumor and germ line DNA and looked for similar patterns in publicly available genomic data from patients with colorectal cancer. Tumor DNA was obtained from a biopsy before therapy, and germ line DNA was obtained from blood. Tumor and germline DNA were sequenced using a commercial panel with approximately 250 genes. Whole-genome amplification and exome sequencing were performed for POLE and POLD1. A POLD1 mutation was confirmed by Sanger sequencing. The somatic mutation and clinical annotation data files from the colon (n = 461) and rectal (n = 171) adenocarcinoma data sets were downloaded from The Cancer Genome Atlas data portal and analyzed for patterns of mutations and clinical outcomes in patients with POLE- and/or POLD1-mutated tumors. The pattern of alterations included APC biallelic inactivation and microsatellite instability high (MSI-H) phenotype, with somatic inactivation of MLH1 and hypermutation (estimated mutation rate > 200 per megabase). The extremely high mutation rate led us to investigate additional mechanisms for hypermutation, including loss of function of POLE. POLE was unaltered, but a related gene not typically associated with somatic mutation in colon cancer, POLD1, had a somatic mutation c.2171G>A[p.Gly724Glu]. Additionally, we noted that the high mutation rate was largely composed of dinucleotide deletions. A similar pattern of hypermutation (dinucleotide deletions, POLD1 mutations, MSI-H) was found in tumors from The Cancer Genome Atlas. POLD1 mutation with associated MSI-H and hyper-indel-hypermutated cancer genome characterizes a previously unrecognized variant of colon cancer that was found in this patient with an exceptional response to chemotherapy.

Hypermutation In Pancreatic Cancer

Pancreatic cancer is molecularly diverse, with few effective therapies. Increased mutation burden and defective DNA repair are associated with response to immune checkpoint inhibitors in several other cancer types. We interrogated 385 pancreatic cancer genomes to define hypermutation and its causes. Mutational signatures inferring defects in DNA repair were enriched in those with the highest mutation burdens. Mismatch repair deficiency was identified in 1% of tumors harboring different mechanisms of somatic inactivation of MLH1 and MSH2. Defining mutation load in individual pancreatic cancers and the optimal assay for patient selection may inform clinical trial design for immunotherapy in pancreatic cancer.

Patients with colorectal cancer associated with Lynch syndrome and MLH1 promoter hypermethylation have similar prognoses.

PurposeMismatch repair deficient (dMMR) colorectal cancer (CRC) is caused by Lynch syndrome (LS) in 3% and sporadic inactivation of MLH1 by hypermethylation (MLH1-hm) in 12% of CRC cases. It is not clear whether outcomes between LS-associated and MLH1-hm CRC differ. The objective of this study was to explore differences in clinical factors and outcomes in these two groups.MethodsPatients with dMMR CRC by immunohistochemistry staining treated at a single institution from 1998 to 2012 were included. MLH1-hm was established with BRAF mutational analysis or hypermethylation testing. Patients’ charts were accessed for information on pathology, germline MMR mutation testing and clinical course.ResultsA total of 143 patients had CRC associated with LS (37 pts, 26%) or MLH1-hm (106 pts, 74%). Patients with LS were younger, more often male, presented more often with stage III disease and had more metachronous disease than patients with MLH1-hm tumors. There was no difference in cancer-specific survival (CSS) between the groups while overall survival (OS) was longer in patients with LS but this difference was minimal after adjusting for age and stage at diagnosis.ConclusionCSS did not differ in LS-associated CRC compared to MLH1-hm CRC suggesting that they carry a similar prognosis.

Prognostic Impact of Deficient DNA Mismatch Repair and Mutations in KRAS, and BRAFV600E in Patients with Lymph Node-Positive Colon Cancer.

While tumor stage remains the key determinant of colorectal cancer (CRC) prognosis and treatment, there is considerable stage-independent variability in clinical outcome. Molecular markers hold promise for explaining variations in clinical behavior, and may identify patient subsets with differential efficacy and survival after adjuvant chemotherapy which is standard of care for patients with lymph node-positive, i.e., stage III, colon cancer. An increased understanding of the molecular evolution and progression of CRC has identified two major pathways of tumorigenesis that are characterized by chromosomal instability or microsatellite instability (MSI). MSI is a consequence of deficient DNA mismatch repair (MMR) that is generally due to epigenetic inactivation of MLH1 in tumors that often carry mutations in oncogenic BRAFV600E . Activating BRAFV600E and KRAS mutations are mutually exclusive and in this article, we review the current status of these mutations and MMR status as prognostic biomarkers in stage III colon cancers.

Promoter hypermethylation of CDKN2A, MGMT, MLH1, and DAPK genes in laryngeal squamous cell carcinoma and their associations with clinical profiles of the patients

Laryngeal squamous cell carcinoma (laryngeal SCC) is a frequently occurring cancer of the head and neck area. Epigenetic changes of tumor-related genes contribute to its genesis and progression. We assessed promoter methylation status of the selected genes (CDKN2A, MGMT, MLH1, and DAPK) using methylation-sensitive high resolution melting (MS-HRM) in 100 patients with laryngeal SCC and studied the correlations with clinical characteristics. The prevalence of promoter methylation in MGMT, CDKN2A, MLH1, and DAPK was 59 of 97 (60.8%), 46 of 97 (47.4%), 45 of 97 (46.4%), and 41 of 97 patients (42.3%), respectively. Significantly increased methylation of CDKN2A was observed in heavy smokers. Epigenetic inactivation of CDKN2A and MLH1 were found to be associated with lymph node involvement. An inverse correlation was present between MLH1 methylation and alcohol consumption. Our results strongly suggest that deregulation of p16-associated, and MLH1-associated pathways, because of promoter hypermethylation, is associated with increased cancer cell migration, tumor invasiveness, and, thus, aggressive phenotype.

Regulation of MLH1 mRNA and protein expression by promoter methylation in primary colorectal cancer: a descriptive and prognostic cancer marker study

In colorectal cancer MLH1 deficiency causes microsatellite instability, which is relevant for the patient's prognosis and treatment, and its putative heredity. Dysfunction of MLH1 is caused by sporadic gene promoter hypermethylation or by hereditary mutations as seen in Lynch Syndrome. The aim of this study was to determine in detail how DNA methylation regulates MLH1 expression and impacts clinical management. Colorectal cancer samples were collected from 210 patients. The laboratory methods used to study these samples included methylation specific multiplex ligation-dependent probe amplification (MS-MLPA), real-time quantitative PCR (qPCR), and immunohistochemistry (IHC). We found that the MLH1 mRNA and protein expression levels were highly related. MS-MLPA was successful in tumors from 195 patients. In these tumors, hypermethylation was observed in promoter regions A (n = 57), B (n = 30), C (n = 28), and D (n = 47), and in intron 1 (n = 25). The promoter region C and intron 1 methylation levels were found to be excellently suited for discriminating between low and high gene expression levels, whereas those of promoter regions A, B and D were less specific. Hypermethylation in any region (n = 77) served as an independent prognostic factor (hazard ratio 0.56, 95% confidence interval 0.36-0.89, p = 0.01). MLH1 inactivation through hypermethylation was found to be related to improved survival. Hypermethylation in promoter region C and intron 1 served as the most specific markers for this inactivation.