Mutations In DNA Mismatch Repair Genes Research Articles

MODL-03. THERAPEUTIC EFFICACY OF IMIPIRIDONES IN LYNCH SYNDROME-ASSOCIATED GLIOMAS

Abstract INTRODUCTION Lynch Syndrome, a hereditary cancer syndrome linked to DNA mismatch repair gene mutations (MMRD), predisposes individuals to various cancers, including high grade gliomas (Lynch-HGG). Effective treatments for Lynch-HGGs are scarce, and suitable pre-clinical models are needed to identify novel therapeutic combinations. METHODS In this study, we generated patient-derived cell line models of Lynch-HGGs (n=3), characterized them through Western blot analysis for MMRD gene markers, and conducted genome and transcriptomic analyses. Murine models were then generated via intracranial injection of cultured cells expressing mCherry-luciferase reporter genes. Drug screening based on transcriptomic analysis was performed to identify potential therapeutic agents. RESULTS Our findings showed loss of MMRD protein consistent with the primary mutation, and a constant loss of P53, a tumor suppressor protein. One cell line, 7316-3058, exhibited an absence of NF1 expression, another critical tumor suppressor protein. Transcriptomic analysis unveiled upregulation in cell cycle processes and SUMOylation of DNA damage response and repair proteins. In vitro treatment with imipiridone compounds (ONC201 and ONC206) demonstrated varying degrees of drug response, with 7316-3058 displaying high sensitivity to the targeted inhibitor. Unfortunately, this therapeutic efficacy did not translate to the in vivo murine model, suggesting underlying mechanisms that impede drug efficiency. CONCLUSION In summary, we developed effective pre-clinical models of Lynch-HGG that can be used to screen different drug combinations targeted to treat these tumors. Furthermore, despite promising in-vitro data, we demonstrated the limited efficacy of imipiridones in treating Lynch-HGG in vivo, emphasizing the challenges in translating in vitro findings to in vivo contexts. This study contributes valuable insights to the development of effective therapeutic strategies for Lynch Syndrome-associated gliomas.

The role of DNA mismatch repair in immunotherapy of human cancer.

DNA mismatch repair (MMR) is an important pathway which helps to maintain genomic stability. Mutations in DNA MMR genes are found to promote cancer initiation and foster tumor progression. Deficiency or inactivation of MMR results in microsatellite instability (MSI) which triggers neoantigen generation and impairs tumor growth. Immunotherapies targeting MMR can increase the burden of neoantigens in tumor cells. While MSI has been regarded as an important predictor of sensitivity and drug resistance for immunotherapy-based strategies. Different approaches targeting genomic instability have been demonstrated to be promising in malignancies derived from different tissues. Underlying MMR deficiency-associated immunogenicity is important for improving the therapeutic efficacy of immunotherapies. In this review we provide an overview of the MMR systems, their role in tumorigenesis, drug resistance, prognostic significance and potential targets for therapeutic treatment in human cancers, especially in hematological malignancies.

1007. Hypermutability in Clinical Strains of Klebsiella pneumoniae: Role of the V76G Mutation in MutH

Abstract Background Hypermutator (HM) bacteria exhibit high spontaneous mutation rates due to DNA mismatch repair (MMR) gene mutations, which may facilitate antibiotic resistance. HM is best described for chronic infections or colonization, in particular with P. aeruginosa. HM K. pneumoniae (KP) and carbapenem resistant Enterobacterales (CRE) are rarely studied. Methods Longitudinal isolates from 5 patients (pts) with long-term ST258 CRKP infections (median: 1.4 yr, 0.5-4.1 yr) underwent Illumina HiSeq whole genome sequencing. Strains from 1 pt were tested for HM and resistance. Mutant strains were created by complementation and CRISPR. Results In each pt, initial and recurrent isolates were genetically related (≤7 core genome (cg) SNP). In 1 pt, infection recurred 3.3 yrs after initial infection; baseline and recurrent (T0) isolates differed by 7-10 cgSNP. 4 ceftazidime-avibactam (CAZ) resistant isolates (T1-T4) were recovered ≥2 wks after treatment of T0 infection. Strains T1-T4 differed from T0 and each other by 109-214 and 58-137 cgSNP (Fig.1), respectively, and carried mutations in MMR genes (mutS -149∆C (∆pmutS); mutH V76G) and blaKPC3 (D179Y). T1-T4 mutations were enriched for genes involved in metabolism (adjusted p=1.46e-10), ABC transport (p=4.1e-7), 2-component systems (p=9.2e-5), signal transduction (p=6e-4), and transcription regulation (p=2.1e-4). mutS and mutH expression was 46-49% lower in T1-T4 than in T0. T1-T4 demonstrated rifampin mutational frequency >10-6.4, compared to < 10-7.3 for earlier strains. Upon passage in meropenem-vaborbactam (MV), colistin and gentamicin, T1-T4 developed resistance faster and higher MICs than T0 (Fig 2). MV resistance was associated with IS5 ompk36 promoter insertions or point, deletion or STOP mutations in ompK36 coding region. Complementation of T1-T4 with wild-type (WT) mutH restored phenotypes. Introduction of V76G to WT mutH in T0 established HM and in vitro passage resistance phenotypes. SNP matrix of 11 clinical isolates from a single patient with recurrent KPC-Kp infections The first 6 isolates were recovered within 6 months of transplant (Tinitial). The later 5 isolates were recovered ~40 months after intial GI colonization. Number of SNPs for each pariwise comparision on isolates are shown. Gray highlighted boxes shown SNP defferences between the 5 later strains. Serial passages of 4 clinical isolates. T1 and T4 harbored ΔpmutS. Ti=Tinitial (baseline) isolates Conclusion MMR mutations emerged in longitudinal CRKP, which conferred HM phenotypes and were associated with CAZ and other anti-CRE antibiotic resistance. mutH V76 is crucial in MMR. Long-term colonization or recurrent infections in face of antibiotic exposure might predispose CRKP strains to HM. Disclosures Cornelius J. Clancy, MD, Merck (Grant/Research Support)

Investigation of discrepant mismatch repair immunohistochemistry and microsatellite instability polymerase chain reaction test results for gynecologic cancers using next-generation sequencing

Gynecologic cancers are routinely screened for DNA mismatch repair (MMR) gene mutations using immunohistochemistry (IHC) and/or polymerase chain reaction (PCR) for microsatellite instability (MSI) to enable selection of immune checkpoint inhibitor therapy and screen for Lynch syndrome. The limited data that compareIHC and MSI in endometrial tumors have shown discordance rates of 5-10%. We reviewed MMR/MSI results in gynecologic cancers and used next-generation sequencing (NGS) to interrogate discrepancies. Of the 328 cases with both IHC and MSI results, 256 (78.0%) were microsatellite stable (MSS) with preserved MMR (pMMR), 64 (19.5%) cases were MSI-High (MSI-H) with MMR deficient (dMMR), 2 cases showed subclonal loss of MLH1 and PMS2 with MSI-H, and 6 cases were discordant. Overall, there was a 98.2% (322/328) IHC/MSI concordance. Discordant cases were retested and/or subject to NGS. Of the six discrepant cases, five showed dMMR with MSS and one showed pMMR with MSI-H. One dMMR/MSI-L case showed loss of PMS2 with a germline pathogenic mutation. The pMMR/MSI-H case was found to harbor pathogenic variants in MLH1 and MSH6. One of the two cases with subclonal populations demonstrated MSI-H in the dMMR area and MSS in the pMMR area. These results emphasize the importance of selecting the appropriate tumor tissue for both IHC and molecular testing and demonstrate that NGS can help resolve discrepant MMR and MSI results.

Pan-Cancer Analysis of the N6-Methyladenosine Eraser FTO as a Potential Prognostic and Immunological Biomarker

BackgroundFat mass and obesity-associated protein (FTO) is a critical N6-methyladenosine (m6A) demethylase that participates in tumorigenesis and is associated with the prognosis of patients in some cancers. However, the key roles of FTO in pan-cancer are still largely obscure.MethodsFTO expression levels in pan-cancer were estimated via the Genotype-Tissue Expression (GTEx), Cancer Cell Line Encyclopedia (CCLE), and The Cancer Genome Atlas (TCGA) databases. Univariate survival analysis was used to estimate the effects of FTO on prognosis. In addition, we used the Tumor Immune Evaluation Resource (TIMER) to assess the immune cell infiltration of FTO gene across cancers. The association of FTO expression with immune checkpoint genes expression, DNA mismatch repair (MMR) gene mutation, DNA methyltransferases, microsatellite instability (MSI), and tumor mutational burden (TMB) was investigated using Spearman’s correlation analysis. Moreover, Gene Set Enrichment Analysis (GSEA) was utilized to identify critical pathways in cancers. The STRING website was used to reveal the protein–protein interaction (PPI) network of FTO.ResultsFTO was aberrantly expressed across cancers and survival analysis demonstrated that its expression was associated with clinical prognosis of many cancer patients. Specifically, FTO expression was significantly associated with immune infiltrating cells in colon adenocarcinoma, kidney renal clear cell carcinoma, and liver hepatocellular carcinoma. In addition, FTO expression was significantly associated with immune checkpoint genes expression, MMR, DNA methyltransferases levels, TMB, and MSI in multiple cancers. Moreover, the GSEA unveiled that FTO was involved in the regulation of tumors and immune-related signaling pathways. In addition, several m6A related genes were implicated in the PPI network of FTO.ConclusionFTO was related to patients’ prognosis and tumor immune infiltrates in various cancers, and may serve as a novel and potential prognostic and immune biomarker in human pan-cancer.

SO-26 In-silico Lynch syndrome-related neoantigens prediction for a dendritic cell-based cancer prevention vaccine

Lynch syndrome (LS), caused by germline mutations in DNA mismatch-repair genes (MLH1, MSH2, MSH6, PMS2) predisposes patients to colorectal and endometrial cancer (CRC, EC), among other tumors. Although CCR prevention methods are effective, no preventive strategies exist for the majority of LS-related tumors. Ex-vivo generated and tumor-antigen-loaded dendritic cell (DC) vaccines are one of the recently featured antitumoral immunotherapies. However, their full therapeutic potential would likely be as a preventive approach in high-risk cancer patients. LS is a paradigmatic model for its limited and predictive mutational spectrum in repetitive DNA sequences termed microsatellites (MS). This study aims to identify the main frame-shift derived neopeptides (FSDN) that are shared among cancers from LS patients with the purpose of developing a DC-based neoepitope vaccine. A systematic search of coding MS and FSDN in LS tumors published in literature and in public databases was performed (Seltar Database; The Cancer Genome Atlas, TCGA), with application of epitope prediction pipelines and priorization of FSDN with the greatest coverage on the most frequent HLA-I and HLA-II haplotypes (pVACbind; pVACtools v2.0.1). 531 frame-shift peptide sequences derived from -1 (m1) and -2 (m2) nucleotide deletions from 269 coding microsatellites (cMS) of at least 8 bases were retrieved from SelTarbase and TCGA. Data in FASTA format was computed through the pVACbind epitope prediction pipeline with a labile HLA-binding affinity threshold (IC50 = < 5000 nM) to consider immunoediting events. We prioritized 98 epitopes, 53 HLA-I and 45 HLA-II restricted, from an original predicted number of 42828 HLA-I and 8350 HLA-II-restricted (m1 and m2) epitopes with a median coverage of 9,4 HLA-I alleles and 8,7 HLA-II alleles per epitope. Our predicted neoepitope set has an optimal coverage among LS patients in terms of HLA alleles, associated cancers and prevalence. These results are key to first perform ex vivo functional analysis to determine neoepitopes immunogenicity to later design a preventive antitumoral vaccine for LS.

P0728 - Risk of genitourinary malignancies in Lynch syndrome patients with DNA mismatch repair gene mutations in a large Canadian Familial Registry

P0728 - Risk of genitourinary malignancies in Lynch syndrome patients with DNA mismatch repair gene mutations in a large Canadian Familial Registry

Abstract 3534: The study of Lynch syndrome in a special population reveals a strong founder effect and an unusual mutational mechanism in familial adenomatous polyposis

Abstract Lynch syndrome (LS) is the leading cause of hereditary colorectal cancer (CRC). Germline mutations in DNA mismatch-repair (MMR) genes (MLH1, PMS2, MSH2, and MSH6) are known to cause LS in the heterozygous state. Biallelic mutations in these genes cause mismatch repair cancer syndrome (MMRCS), a very rare childhood cancer syndrome that predisposes to CRC among other cancer types. In this study, we aimed to characterize the mutational landscape of LS in a large CRC cohort (n=1,207) from a population with high rates of consanguinity. The overall contribution of germline mutations in LS gene was comparable to that observed in outbred populations (26/1207). Remarkably, however, we also observed the unusual homozygous occurrence of a founder PMS2 mutation. The homozygous individual presented with massive adenomatous polyposis of the colon at age 8 years, and mutation analysis of multiple polyps revealed the occurrence of multiple independent somatic APC mutations. Our study highlights a previously unrecognized role of LS genes in triggering APC-driven tumors, a phenomenon facilitated by consanguinity. Citation Format: Abdul K. Siraj, Sandeep K. Parvathareddy, Rong Bu, Tariq Masoodi, Khadija Alobaisi, Maha Alrasheed, Saeeda O. Ahmed, Zeeshan Qadri, Padmanaban Annaiyappanaidu, Allianah Benito, Dionne R. Rala, Fowzan Al-Kuraya, Khawla S. Al-Kuraya. The study of Lynch syndrome in a special population reveals a strong founder effect and an unusual mutational mechanism in familial adenomatous polyposis [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 3534.

MutEx: a multifaceted gateway for exploring integrative pan-cancer genomic data.

Somatic mutation and gene expression dysregulation are considered two major tumorigenesis factors. While independent investigations of either factor pervade, studies of associations between somatic mutations and gene expression changes have been sporadic and nonsystematic. Utilizing genomic data collected from 11315 subjects of 33 distinct cancer types, we constructed MutEx, a pan-cancer integrative genomic database. This database records the relationships among gene expression, somatic mutation and survival data for cancer patients. MutEx can be used to swiftly explore the relationship between these genomic/clinic features within and across cancer types and, more importantly, search for corroborating evidence for hypothesis inception. Our database also incorporated Gene Ontology and several pathway databases to enhance functional annotation, and elastic net and a gene expression composite score to aid in survival analysis. To demonstrate the usability of MutEx, we provide several application examples, including top somatic mutations associated with the most extensive expression dysregulation in breast cancer, differential mutational burden downstream of DNA mismatch repair gene mutations and composite gene expression score-based survival difference in breast cancer. MutEx can be accessed at http://www.innovebioinfo.com/Databases/Mutationdb_About.php.

Type 2 diabetes mellitus, blood cholesterol, triglyceride and colorectal cancer risk in Lynch syndrome

BackgroundType 2 diabetes mellitus and high total cholesterol and triglycerides are known to be associated with increased colorectal cancer risk for the general population. These associations are unknown for people with a germline DNA mismatch repair gene mutation (Lynch syndrome), who are at high risk of colorectal cancer.MethodsThis study included 2023 (56.4% female) carriers with a mismatch repair gene mutation (737 in MLH1, 928 in MSH2, 230 in MSH6, 106 in PMS2, 22 in EPCAM) recruited by the Colon Cancer Family Registry between 1998 and 2012. Weighted Cox regression was used to estimate the hazard ratios (HR) and 95% confidence intervals (CI) for the associations between self-reported type 2 diabetes, high cholesterol, triglyceride and colorectal cancer risk.Results Overall, 802 carriers were diagnosed with colorectal cancer at a median age of 42 years. A higher risk of colorectal cancer was observed in those with self-reported type-2 diabetes (HR 1.92; 95% CI, 1.03–3.58) and high cholesterol (HR 1.76; CI 1.23–2.52) compared with those without these conditions. There was no evidence of high triglyceride being associated with colorectal cancer risk.ConclusionFor people with Lynch syndrome, self-reported type-2 diabetes mellitus and high cholesterol were associated with increased colorectal cancer risk.

Clinical Features and Therapeutic Outcomes in Men with Advanced Prostate Cancer and DNA Mismatch Repair Gene Mutations

Mismatch repair (MMR) gene mutations are rare in prostate cancer, and their histological and clinical characteristics are largely unknown. We conducted a retrospective study to explore disease characteristics and treatment outcomes of men with metastatic prostate cancer harboring germline and/or somatic MMR mutations detected using clinical-grade genomic assays. Thirteen patients with a deleterious MMR gene mutation were identified. Median age was 64 yr, 75% had grade group 5 (Gleason sum 9 or 10), 23% had intraductal histology, 46% had metastatic disease at initial diagnosis, and 31% had visceral metastases. Most patients (46%) had MSH6 mutations, 73% demonstrated microsatellite instability, and median tumor mutational load was 18/Mb (range, 3–165 mutations/Mb). Surprisingly, responses to standard hormonal therapies were very durable (median progression-free survival [PFS] of 67 mo to initial androgen deprivation and median PFS of 26 mo to abiraterone/enzalutamide). Two of four men receiving PD-1 inhibitors achieved a ≥50% prostate-specific antigen response at 12 wk, with a median PFS duration in these four men of 9 mo. Despite aggressive clinical and pathological features, patients with MMR-mutated advanced prostate cancer appear to have particular sensitivity to hormonal therapies, as well as anecdotal responses to PD-1 inhibitors. Certain histological features (grade group 5, intraductal carcinoma) should prompt evaluation for MMR deficiency. These data are only hypothesis generating. Patient summaryProstate cancers with mismatch repair gene mutations have aggressive clinical and pathological features; however, these are very sensitive to standard and novel hormonal therapies, and also demonstrate anecdotal sensitivity to PD-1 inhibitors such as pembrolizumab.

Molecular changes preceding endometrial and ovarian cancer: a study of consecutive endometrial specimens from Lynch syndrome surveillance

Molecular alterations preceding endometrial and ovarian cancer and the sequence of events are unknown. Consecutive specimens from lifelong surveillance for Lynch syndrome provides a natural setting to address such questions. To molecularly define the multistep gynecological tumorigenesis, DNA mismatch repair gene mutation carriers with endometrial or ovarian carcinoma or endometrial hyperplasia were identified from a nation-wide registry and endometrial biopsy specimens taken from these individuals during 20 years of screening were collected. A total of 213 endometrial and ovarian specimens from Lynch syndrome individuals and 197 histology-matched (non-serous) samples from sporadic cases were available for this investigation. The specimens were profiled for markers linked to endometrial and ovarian tumorigenesis, including ARID1A protein expression, mismatch repair status, and tumor suppressor gene promoter methylation. In Lynch syndrome-associated endometrial and ovarian carcinomas, ARID1A protein was lost in 61–100% and mismatch repair was deficient in 97–100%, compared to 0–17% and 14–44% in sporadic cases (P = 0.000). ARID1A loss appeared in complex hyperplasia and deficient mismatch repair and tumor suppressor gene promoter methylation in histologically normal endometrium. Despite quantitative differences between Lynch syndrome and sporadic cases, ARID1A expression, mismatch repair, and tumor suppressor gene promoter methylation divided endometrial samples from both patient groups into three categories of increasing abnormality, comprising normal endometrium and simple hyperplasia (I), complex hyperplasia with or without atypia (II), and endometrial cancer (III). Complex hyperplasias without vs. with atypia were molecularly indistinguishable. In conclusion, surveillance specimens from Lynch syndrome identify mismatch repair deficiency, tumor suppressor gene promoter methylation, and ARID1A loss as early changes in tumor development. Our findings are clinically relevant for the classification of endometrial hyperplasias and have potential implications in cancer prevention in Lynch syndrome and beyond.

The uptake of presymptomatic genetic testing in hereditary breast-ovarian cancer and Lynch syndrome: a systematic review of the literature and implications for clinical practice.

Following the identification in a proband of a germline BRCA1/BRCA2 mutation in hereditary breast-ovarian cancer (HBOC) or a DNA mismatch repair gene mutation in Lynch syndrome (LS) he or she will be asked to inform at-risk family members about the option for presymptomatic DNA testing. However, in clinical practice multiple factors may complicate the process of information sharing. We critically evaluated studies on the uptake of presymptomatic genetic testing in both syndromes. A search of relevant MeSH terms and key words in PubMed, Embase and PsycINFO yielded 795 articles published between 2001 and 2017. Thirty of these publications included outcome measures relevant for the current study. Based on information provided by the proband (15 studies) the uptake of presymptomatic genetic testing ranged from 15 to 57% in HBOC, while one study in LS kindreds reported an uptake of 70%. Based on information provided by genetics centres (the remaining 15 studies) the uptake ranged from 21 to 44% in HBOC and from 41 to 94% in LS. However, when genetics centres contacted relatives directly a substantial number of additional family members could be tested. Proband-mediated provision of information to at-risk relatives is a standard procedure in hereditary breast-ovarian cancer and Lynch syndrome. However, the resulting uptake of presymptomatic testing is disappointing-an issue that is now urgent due to the increased use of genetic testing in clinical oncology. We propose that additional strategies should be introduced including the geneticist directly contacting relatives. The outcomes of these strategies should be carefully monitored and evaluated.

DNA Replication and associated repair pathways are involved in the mutagenesis of methylated cytosine

Transitions of cytosine to thymine in CpG dinucleotides are the most frequent type of mutations observed in cancer. This increased mutability is commonly explained by the presence of 5-methylcytosine (5mC) and its spontaneous hydrolytic deamination into thymine. Here, we describe observations that question whether spontaneous deamination alone causes the elevated mutagenicity of 5mC. Tumours with somatic mutations in DNA mismatch-repair genes or in the proofreading domain of DNA polymerase ε (Pol ε) exhibit more 5mC to T transitions than would be expected, given the kinetics of hydrolytic deamination. This enrichment is asymmetrical around replication origins with a preference for the leading strand template, in particular in methylated cytosines flanked by guanines (GCG). Notably, GCG to GTG mutations also exhibit strand asymmetry in mismatch-repair and Pol ε wild-type tumours. Together, these findings suggest that mis-incorporation of A opposite 5mC during replication of the leading strand might be a contributing factor in the mutagenesis of methylated cytosine.

Multidrug-Resistant Candida: Epidemiology, Molecular Mechanisms, and Treatment.

Invasive Candida infections remain an important cause of morbidity and mortality, especially in hospitalized and immunocompromised or critically ill patients. A limited number of antifungal agents from only a few drug classes are available to treat patients with these serious infections. Resistance can be either intrinsic or acquired. Resistance mechanisms are not exchanged between Candida; thus, acquired resistance either emerges in response to an antifungal selection pressure in the individual patient or, more rarely, occur due to horizontal transmission of resistant strains between patients. Although multidrug resistance is uncommon, increasing reports of multidrug resistance to the azoles, echinocandins, and polyenes have occurred in several Candida species, most notably Candida glabrata and more recently Candida auris. Drivers are overall antifungal use, subtherapeutic drug levels at sites of infection/colonization, drug sequestration in the biofilm matrix, and, in the setting of outbreaks, suboptimal infection control. Moreover, recent research suggests that DNA mismatch repair gene mutations may facilitate acquisition of resistance mutations in C. glabrata specifically. Diagnosis of antifungal-resistant Candida infections is critical to the successful management of patients with these infections. Reduction of unnecessary use of antifungals via antifungal stewardship is critical to limit multidrug resistance emergence.

A novel heterozygous germline deletion in MSH2 gene in a five generation Chinese family with Lynch syndrome.

Lynch syndrome (LS) is one of the most common familial forms of colorectal cancer predisposing syndrome with an autosomal dominant mode of inheritance. LS is caused by the germline mutations in DNA mismatch repair (MMR) genes including MSH2, MLH1, MSH6 and PMS2. Clinically, LS is characterized by high incidence of early-onset colorectal cancer as well as endometrial, small intestinal and urinary tract cancers, usually occur in the third to fourth decade of the life. Here we describe a five generation Chinese family with LS clinically diagnosed according to the Amsterdam II criteria. Immuno-histochemical staining of MSH2 and MSH6 shows only foci nuclear positive on the surface of the tumor with strong expression of MLH1 and PMS2 with diffuse immunoreactivity. In order to dig into the molecular basis of this LS pedigree, we collected the proband's blood sample, extracted the genomic DNA and applied the genetic screening. As a result, we identified a novel heterozygous deletion in MSH2 gene by targeted next generation sequencing, which is also proved to be co-segregated among other affected family members by following validation. To our knowledge, this novel heterozygous deletion (c.1676_1679 delTAAA) in MSH2 gene causes frameshift mutation (p.Asn560Lysfs*29) and leads to the formation of a truncated MSH2 protein which is confirmed to be a deleterious mutation according to the variant interpretation guidelines of American College of Medical Genetics and Genomics (ACMG). Identification of novel DNA mismatch repair (MMR) gene mutations can definitely benefit to the clinical diagnosis and management.

MLH1 Ile219Val Polymorphism in Argentinean Families with Suspected Lynch Syndrome.

Heredity is a major risk factor for colorectal cancer (CRC). Identification of individuals and families at increased risk allows for targeted surveillance, which has been shown to reduce morbidity and mortality from CRC (1, 2). Lynch syndrome is a multi-tumor syndrome with particularly high risks for colorectal, endometrial, and ovarian cancer (3–6). The syndrome is caused by germline DNA-mismatch repair (MMR) gene mutations with major contributions from MLH1 (MIM#120436) (42%), MSH2 (MIM#609309) (33%), MSH6 (MIM#600678) (18%), and PMS2 (MIM#600259) (8%). Only about one-third of the Lynch syndrome families fulfill the Amsterdam criteria (AC) (7–9). The cumulative incidence of any cancer at 70 years of age is 72% for MLH1 and MSH2 mutation carriers but lower in MSH6 (52%) and PMS2 (18%) mutation carriers. MSH6 and PMS2 carriers developed no cancers before 40 years of age (10). Mutation screening in a relatively large proportion of South American families with suspected Lynch syndrome has recently identified 99 disease-predisposing mutations in MLH1 and MSH2, which mutation spectrum is predominated by MLH1 (60%) and MSH2 (40%). Among the reported mutations, genetic hot-spot regions, new and potential founder mutations have been described in the South American population (11, 12). Several genome-wide association studies have identified single nucleotide polymorphisms (SNPs) in at least 15 independent loci associated with CRC risk (odds ratio ranging from 1.10 to 1.26 per risk allele) (13–15). Although there is no evidence that these SNPs associated with CRC in the general population are modifiers of the risk for MMR gene mutation carriers overall and therefore any evidence of proven clinical utility in Lynch syndrome (16). The MLH1 Ile219Val (rs1799977) is a common germline alteration, located in exon 8 at the nucleotide 655. This polymorphism has been described in a high frequency of the South American Lynch syndrome population, but no modifier effect of CRC risk and MMR disease-predisposing mutation carriers was observed (17). However, it has been reported to confer a twofold-increased risk of CRC development in sporadic Mexican patients (18). Other conditions that have been associated with this polymorphism include childhood acute lymphoblastic leukemia, breast cancer, radiation-induced rectal or bladder toxicity, and ulcerative colitis (19–23). It is unknown whether the MLH1 Ile219Val polymorphism has an effect on cancer risk and in the MMR capacity in Argentinean families with suspected Lynch syndrome. Thus, we aim to determine its frequency, its correlation with disease-predisposing MMR gene mutations, and to delineate the clinical characteristics from these families.

Multivitamin, calcium and folic acid supplements and the risk of colorectal cancer in Lynch syndrome.

People with a DNA mismatch repair (MMR) gene mutation have a substantially elevated risk of colorectal cancer (CRC) but the modifiers of this risk are not well established. We investigated the association between dietary supplement intake and CRC risk for carriers. This study included 1966 (56% female) carriers of an MMR gene mutation (719 MLH1, 931 MSH2, 211 MSH6 and 105 PMS2) who were recruited from the USA, Canada, Australia and New Zealand into the Colon Cancer Family Registry between 1997 and 2012. Information on lifestyle factors including supplement intake was collected at the time of recruitment. Using Cox proportional hazards regression weighted to correct for ascertainment bias, we estimated hazard ratios (HRs) and 95% confidence intervals (CIs) for associations between self-reported multivitamin, calcium and folic acid supplement intake and CRC risk. Of 744 carriers with CRC, 18%, 6% and 5% reported intake of multivitamin, calcium and folic acid supplements for at least 1 month, respectively, compared with 27%, 11% and 10% of 1222 carriers without CRC. After adjusting for identified confounding variables, a decreased CRC risk was associated with multivitam inintake for at least 3 years (HR 0.47, 95% CI 0.32-0.69) and calcium intake for at least 3 years(HR 0.42, 95% CI 0.23-0.74), compared with never users. There was no evidence of an association between folic acid supplement intake and CRC risk (P = 0.82). Intake of multivitamin and calcium supplements might be associated with a decreased risk of CRC for MMR gene mutation carriers.

Oligonucleotide-directed mutagenesis screen to identify pathogenic Lynch syndrome-associated MSH2 DNA mismatch repair gene variants

Single-stranded DNA oligonucleotides can achieve targeted base-pair substitution with modest efficiency but high precision. We show that "oligo targeting" can be used effectively to study missense mutations in DNA mismatch repair (MMR) genes. Inherited inactivating mutations in DNA MMR genes are causative for the cancer predisposition Lynch syndrome (LS). Although overtly deleterious mutations in MMR genes can clearly be ascribed as the cause of LS, the functional implications of missense mutations are often unclear. We developed a genetic screen to determine the pathogenicity of these variants of uncertain significance (VUS), focusing on mutator S homolog 2 (MSH2). VUS were introduced into the endogenous Msh2 gene of mouse embryonic stem cells by oligo targeting. Subsequent selection for MMR-deficient cells using the guanine analog 6-thioguanine allowed the detection of MMR-abrogating VUS. The screen was able to distinguish weak and strong pathogenic variants from polymorphisms and was used to investigate 59 Msh2 VUS. Nineteen of the 59 VUS were identified as pathogenic. Functional assays revealed that 14 of the 19 detected variants fully abrogated MMR activity and that five of the detected variants attenuated MMR activity. Implementation of the screen in clinical practice allows proper counseling of mutation carriers and treatment of their tumors.

Abstract IA44: Cancer prevention: Dendritic cell enhanced immune responses towards neoantigens in patients with Lynch syndrome

Abstract To date, dendritic cell (DC)-based immunotherapy is explored worldwide in clinical vaccination trials with cancer patients. Although during the past 15 years the concept of DC vaccination has been clearly proven and found safe, the number of patients that have long-term benefit is still limited. Therefore the development of bioassays that predict clinical outcome are essential to optimize cellular anticancer immunotherapy. We have developed a robust and simple skin test to evaluate the capacity of tumor-specific T cells to migrate, recognize their targets and exert effector functions. This bioassay detects skin infiltrating T lymphocytes (SKILs) with an elevated antineoplastic potential and hence identifies patients responding to immunotherapy once started. The identification of a biomarker already available before start of treatment would facilitate decision-making regarding (prophylactic) immunotherapy. We recently identified that the ratio between peri/intratumoral T cells in the primary tumor, is an excellent predictor of survival after DC-based immunotherapy in metastatic melanoma patients. Patients with a high density of T cells in primary tumors, hence putative candidates for DC vaccination, are Lynch syndrome patients. They have an up to 80% lifetime risk of colorectal cancer (CRC) due to a germline DNA mismatch-repair gene mutation (Lynch syndrome). MMR deficiency in tumor DNA causes shifts in the translational reading frame resulting in the expression of neoantigens. We exploited these neoantigens for vaccination of Lynch syndrome-associated CRC using DC. Patients were vaccinated with monocyte-derived DC loaded with MHC-class I-binding neoantigens (mutated Caspase-5 and TGF-βRII) and carcinoembryonic antigen (CEA). With our bioassay, we detected SKILs that were able to migrate to the antigen-challenged site, recognize their targets (neoantigens) and exert effector functions shown by high amounts of interferon-γ upon stimulation with neoantigen-loaded target cells. No severe adverse events were detected. Our data emphasize the potency of DC-based immunotherapy to enhance the hosts antitumor immunity. Furthermore, this study paved the way for vaccination aiming at cancer prevention in Lynch syndrome mutation carriers. We recently started a clinical study with the here developed dendritic cells in these lynch syndrome mutation carriers. To date, promising immunological results are noted. Citation Format: Jolanda de Vries, Gerty Schreibelt, Kalijn F. Bol, Harm Westdorp, Steve Boudewijns, Winald R. Gerritsen, Nicoline Hoogerbrugge, Carl G. Figdor. Cancer prevention: Dendritic cell enhanced immune responses towards neoantigens in patients with Lynch syndrome. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr IA44.