Mutations In Mismatch Repair Genes Research Articles

Prevalence of hypermutator isolates of Achromobacter spp. from cystic fibrosis patients

Bacteria colonising the lungs of cystic fibrosis (CF) patients encounter high selective pressures. Hypermutation facilitates adaptation to fluctuating environments, and hypermutator strains are frequently isolated from CF patients. We investigated the prevalence of hypermutator isolates of Achromobacter spp. among patients affiliated with the CF Centre in Aarhus, Denmark. By exposure to rifampicin, the mutation frequency was determined for 90 isolates of Achromobacter spp. cultured from 42 CF patients; 20 infections were categorised as chronic, 22 as intermittent. The genetic mechanisms of hypermutation were examined by comparing DNA repair gene sequences from hypermutator and normomutator isolates. Achromobacter spp. cultured from 11 patients were categorised as hypermutators, and this phenotype was exclusively associated with chronic infections. Isolates of the Danish epidemic strain (DES) of Achromobacter ruhlandii cultured from patients from both Danish CF centres showed elevated mutation frequencies. The hypermutator state of Achromobacter spp. was most commonly associated with nonsynonymous mutations in the DNA mismatch repair gene mutS; a single clone had developed a substitution in the S-adenosyl-L-methionine-dependent methyltransferase putatively involved in DNA repair mechanisms, but not previously linked to the hypermutator phenotype. Hypermutation is prevalent among clinical isolates of Achromobacter spp. and could be a key determinant for the extraordinary adaptation and persistence of DES.

Mismatch Repair Deficiency in Ovarian Carcinoma: Frequency, Causes, and Consequences.

Mismatch repair deficiency (MMRD) is involved in the initiation of both hereditary and sporadic tumors. MMRD has been extensively studied in colorectal cancer and endometrial cancer, but not so in other tumors, such as ovarian carcinoma. We have determined the expression of mismatch repair proteins in a large cohort of 502 early-stage epithelial ovarian carcinoma entailing all the 5 main subtypes: high-grade serous carcinoma, endometrioid ovarian carcinoma (EOC), clear cell carcinoma (CCC), mucinous carcinoma, and low-grade serous carcinoma. We studied the association of MMRD with clinicopathologic and immunohistochemical features, including tumor-infiltrating lymphocytes in EOC, the histologic type in which MMRD is most frequent. In addition, MLH1 promoter methylation status and massive parallel sequencing were used to evaluate the proportion of sporadic and Lynch syndrome-associated tumors, and the most frequently mutated genes in MMRD EOCs. MMRD occurred only in endometriosis-associated histologic types, and it was much more frequent in EOC (18%) than in CCC (2%). The most frequent immunohistochemical pattern was loss of MLH1/PMS2, and in this group, 80% of the cases were sporadic and secondary to MLH1 promoter hypermethylation. The presence of somatic mutations in mismatch repair genes was the other mechanism of MMRD in sporadic tumors. In this series, the minimum estimated frequency of Lynch syndrome was 35% and it was due to germline mutations in MLH1, MSH2, and MSH6. ARID1A, PTEN, KTM2B, and PIK3CA were the most common mutated genes in this series. Interestingly, possible actionable mutations in ERRB2 were found in 5 tumors, but no TP53 mutations were detected. MMRD was associated with younger age and increased tumor-infiltrating lymphocytes. Universal screening in EOC and mixed EOC/CCC is recommended for the high frequency of MMRD detected; however, for CCC, additional clinical and pathologic criteria should be evaluated to help select cases for analysis.

Pathological prognostic factors of pseudomyxoma peritonei: comprehensive clinicopathological analysis of 155 cases

Pseudomyxoma peritonei (PMP) is an extremely rare malignancy, characterized by extensive peritoneal implantation and colloidal ascites. This study was to explore the pathological prognostic factors of PMP. Specimens from 155 PMP patients were analyzed by H&E and immunohistochemistry. Parameters included primary tumor location, histological grade, lymph node metastasis, tumor emboli in the blood and lymph vessels, perineural invasion, Ki67 labeling index, p53, mismatch repair (MMR) gene mutations, MUC1, MUC2, MUC5AC, and MUC6. Clinicopathological and follow-up data were subjected to univariate and multivariate analyses. The patients included 63.2% (n = 98) low-grade mucinous carcinoma peritonei, 31.6% (n = 49) high-grade mucinous carcinoma peritonei and 5.2% (n = 8) high-grade mucinous carcinoma peritonei with signet ring cells. There were 9.7% (n = 15) with lymph node metastasis; 11.6% (n = 18) with angiolymphatic invasion; 6.3% (n = 8) with defective MMR (dMMR); 35.5% (n = 55) with Ki67 labeling index ≥ 50%; 36.1% (n = 56) with p53 mutation. For PMP from appendiceal origin (n = 140), univariate analysis identified 10 potential prognostic factors. But Multivariate analysis identified only histologic grade was the independent prognostic factor for OS. Mortality risk of high-grade peritoneal mucinous carcinoma or high-grade peritoneal mucinous carcinoma with signet ring cells was 7.056 times (P < .0001, 95% CI: 2.701-18.435) or 27.224 times (P < .0001, 95% CI: 6.207-119.408), respectively, higher than low-grade. For PMP from the appendiceal origin, histological grade could be the only independent prognostic factor.

Retained mismatch repair protein expression occurs in approximately 6% of microsatellite instability-high cancers and is associated with missense mutations in mismatch repair genes.

Immunohistochemistry for mismatch repair protein expression is widely used as a surrogate for microsatellite instability status- an important signature for immunotherapy and germline testing. There are no systematic analyses examining the sensitivity of immunohistochemistry for microsatellite instability-high status. Mismatch repair immunohistochemistry and microsatellite instability testing were performed routinely as clinically validated assays. We classified germline/somatic mutation types as truncating (nonsense, frameshift, in/del) versus missense and predicted pathogenicity of the latter. Discordant cases were compared to concordant groups: microsatellite instability-high/ mismatch repair-deficient for mutation comparison and microsatellite stable/ mismatch repair-proficient for immunohistochemical comparison. 32 of 443 (7%) microsatellite instability-high cases had immunohistochemistry. Four additional microsatellite instability-high research cases had discordant immunohistochemistry. Of 36 microsatellite instability-high cases with discordant immnohistochemistry, 30 were mismatch repair-proficient while 6 (5 MLH1 and 1 MSH2) retained expression of the defective mismatch repair protein and lost its partner. In microsatellite instability-high tumors with discordant immunohistochemistry, we observed an enrichment in deleterious missense mutations over truncating mutations, with nearly 70% (25/36) of cases having pathogenic germline or somatic missense mutations, as opposed to only 17% (6/36) in a matched microsatellite instability-high group with concordant immunohistochemistry (p=0.0007). In microsatellite instability-high cases with discordant immunohistochemistry and MLH1 or PMS2 abnormalities, less cells showed expression (p=0.015 and p=0.00095 respectively) compared to microsatellite stable/ mismatch repair-proficient cases. Tumor mutation burden, MSIsensor score, and truncating mismatch repair gene mutations were similar between microsatellite instability-high cases with concordant versus discordant immunohistochemical expression. Approximately 6% of microsatellite instability-high cases have retained mismatch repair protein expression and would be missed by immunohistochemistry-based testing, hindering patient access to immunotherapy. Another 1% of microsatellite instability-high cases show isolated loss of the defective gene’s dimerization partner, which may lead to germline testing of the wrong gene. These cases are enriched for pathogenic mismatch repair missense mutations.

Mismatch Repair Protein Deficiency/Microsatellite Instability Is Rare in Cholangiocarcinomas and Associated With Distinctive Morphologies.

Although germline mutations of mismatch repair (MMR) genes (Lynch syndrome) are not typically associated with cholangiocarcinomas, the US Food and Drug Administration recently approved the use of pembrolizumab in patients with advanced solid tumors at all sites that show MMR deficiency or associated high microsatellite instability. We analyzed 96 cases of intra- and extrahepatic cholangiocarcinomas for morphology using H&E and for MMR status using immunohistochemical staining. We submitted any results with MMR loss for microsatellite instability testing. We found that 6% of samples showed MMR deficiency. The best predictive factor was a nontypical infiltrating pattern of invasion (P < .0001). No patients with MMR deficiency had a history of a cancer typically associated with Lynch syndrome. Solid, mucinous, or signet-ring appearance of a cholangiocarcinoma should prompt MMR testing for immunotherapy options but should not necessarily raise concern about Lynch syndrome.

Variants of DNA mismatch repair genes derived from 33,998 Chinese individuals with and without cancer reveal their highly ethnic-specific nature

PurposeDNA mismatch repair (MMR) genes play important roles in maintaining genome stability. Mutations in MMR genes disrupt their mismatch repair function, cause genome instability and lead to increased risk of cancer in the mutation carriers as represented by Lynch Syndrome. Studies have identified a large number of MMR variants, mostly in the Caucasian population, whereas data from non-Caucasian populations remain poorly illustrated. With the population size of 1.4 billion, knowledge of MMR variants in the Chinese population can be valuable in understanding the roles of ethnic MMR variation and cancer and to further guide clinical applications in MMR-related cancer prevention and treatment in the Chinese population. In this study, we systematically analysed the MMR variants from the Chinese population. Experimental designWe performed a comprehensive MMR data mining and collected all the MMR variation data reported from 33,998 Chinese individuals consisting of 23,938 cancer and 10,060 non-cancer cases between January 1997 to May 2019. For the collected data, we performed standardisation following Human Genome Variation Society nomenclature and reannotated the MMR variant data following American College of Medical Genetics and Genomics guidelines and comparing with non-Chinese MMR data on various aspects. ResultsWe identified a total of 540 MMR variants in the Chinese population, including 194 in MLH1, 181 in MSH2, 59 in MSH6, 53 in PMS2 single-base/indel changes and 53 large deletions/duplications in MLH1, MSH2, MSH6 and PMS2, respectively. We determined that the pathogenic/likely pathogenic carrier rate in the Chinese population was 1.6%. Comparative analysis in variant spectrum, variant types, clinical classification and founder mutations showed substantial differences of MMR variation between Chinese and non-Chinese populations and the fact that over 90% of the variants were only present in the Chinese ethnicity reveals the highly ethnic-specific nature of the Chinese MMR variation . We also developed an open-access database, dbMMR-Chinese, to host all data (https://dbMMR-chinese.fhs.um.edu.mo). The rich MMR data from a large non-Caucasian population should be valuable to study MMR variation and its relationship with cancer and provide a valuable reference resource for MMR-related cancer prevention and treatment. ConclusionOur study provides the largest MMR data set from a single non-Caucasian population and reveals that MMR variation in the humans can be highly ethnic-specific.

Germline and somatic mutations of homologous recombination-associated genes in Japanese ovarian cancer patients

We explored the frequency of germline and somatic mutations in homologous recombination (HR)-associated genes in major histological types of ovarian cancer. We performed targeted sequencing to assess germline and somatic mutations of 16 HR-associated genes and 4 mismatch repair (MMR) genes among 207 ovarian cancer patients (50 high-grade serous carcinomas (HGSC), 99 clear cell carcinomas (CCC), 39 endometrioid carcinomas (EC), 13 mucinous carcinomas (MC), and 6 low-grade serous carcinomas (LGSC)). Germline or somatic mutations of HR-associated genes were detected in 44% of HGSC, 28% of CCC, 23% of EC, 16% of MC, and 17% of LGSC patients. The profile of HR-associated gene mutations was remarkably different among each histological type. Germline BRCA1/2 mutations were frequently detected in HGSC and were rarely observed in CCC, EC, and MC patients. ATM somatic mutation was more frequently detected in CCC (9%) and EC patients (18%) than in HGSC patients (4%). There was a positive correlation between MMR gene mutations and HR-associated gene mutations (p = 0.0072). Our findings might be useful in selection of ovarian cancer patients that should be treated with PARP inhibitors.

PATH-45. DIFFERENT TYPES OF HYPERMUTATOR PHENOTYPE OF GLIOBLASTOMA ACCORDING TO MUTATION PATTERNS OF MISMATCH REPAIR GENES

Abstract BACKGROUND Although precision medicine has been widely applied to routine care, only few molecular targeted drugs have been developed for glioma. Immune checkpoint inhibitors hold a potential promise to improve outcome of malignant glioma especially with the hypermutator phenotype. Mismatch repair deficiency could induce hypermutation during temozolomide (TMZ) treatment, but the mechanism of mutation acquisition is not well understood. METHODS We present the results of target sequencing of 57 longitudinal specimens from 27 individuals with glioblastoma (GBM). We used Ion AmpliSeq Cancer Hotspot Panel v2 (CHP) and/or Ion AmpliSeq Comprehensive Cancer Panel (CCP) for target sequencing. RESULTS Acquired mutations with G:C &gt;A:T transition at non-CpG sites were found in 70% of recurrent MGMT methylated samples, whereas only 8% in MGMT unmethylated cases (p&lt; 0.001). Two cases of hypermutator phenotype were identified in MGMT methylated, IDH wild type recurrent GBMs after TMZ chemotherapy. One case gained mutations in mismatch repair genes of MLH1, MSH2, MSH6, and PMS1. Most of the acquired mutations were G:C &gt;A:T mutations typical to TMZ-induced hypermutation. The recurrent tumor was highly aggressive with overall survival after recurrence of 3.7 months. The other case gained mutations in mismatch repair genes of MLH1 and PMS1 only. Most of the acquired mutations were insertion mutations. The prognosis after recurrence was much longer. CONCLUSIONS We identified two types of hypermutator phenotype according to mutation pattern of mismatch repair genes. It has been suggested that MSH2-MSH6 complex deficient tumor cannot repair TMZ-induced mismatch mutation, thereby causing hypermutation. Conversely, MLH1-PMS1 complex deficient tumor with intact MSH2-MSH6 complex can repair mismatch mutation but is susceptible to insertion mutation. Taken together, MSH6 mutation plays a key role in TMZ-induced hypermutation, while MLH1 and PMS1 mutations might cause insertion-based hypermutation. Larger and prospective studies are warranted to clarify the mechanism, outcome, and effectiveness of checkpoint inhibitors.

RARE-26. WHOLE GENOME SEQUENCING OF AN OSSEOUS METASTASIS DURING CHECKPOINT-CONTROLLED INTRACRANIAL GLIOBLASTOMA REVEALS NEW INSIGHTS INTO POTENTIAL MECHANISMS OF IMMUNE ESCAPE

Abstract Glioblastoma (GBM) has a devastating prognosis and recent advances in the treatment of a variety of cancer entities, e.g. through checkpoint inhibition, could so far not be translated into improved outcome in newly-diagnosed GBM. Characterizing rare cases of peripheral metastases which succeeded in overcoming immune control, can help to understand the mechanisms of immune escape. Here we describe the first reported case of a detailed genetic and immunological characterization of a peripheral bone metastasis from a GBM which was controlled intracranially by anti-PD1 checkpoint inhibition We performed whole genome sequencing (WGS) of the primary- and recurrent tumor, as well as the bone metastasis. Genomic data was analyzed for copy number variations and mutational profiles. In addition, immune monitoring with flow cytometric phenotyping and next-generation sequencing of the peripheral T-cell repertoire was used. A 70-year old patient developed multiple osseous metastases in the spine, while his IDHwt GBM recurrence was immunologically controlled with checkpoint inhibition. Biopsy confirmed peripheral GBM metastases. Immunophenotyping reflected the effective activation of the peripheral T-cell response, with, however, simultaneous upregulation of regulatory T-cells during disease progression. WGS sequencing demonstrated a distinct molecular profile of the GBM metastasis, with amplifications in chromosome 3 and 9, as well as genomic loss on chromosomes 4, 10 and 11. The peripheral metastasis was distinguished by mutations in mismatch repair genes, such as MSH4 and MLH1, associated with a hypermutated phenotype. Among the mutated genes we found potential candidates involved in immune escape of circulating tumor cells. This case represents a unique opportunity to analyze potential mechanisms of GBM-mediated immune escape during immune activation with anti-PD1 checkpoint therapy. It highlights the fact, that although an effective, disinhibited immune response can control the cranial GBM disease, hypermutated tumor clones can evade the tumor-specific T-cell response and disseminate to distant organs.

RARE-41. GLIOBLASTOMA MULTIFORME AND OTHER CNS TUMORS IN HEREDITARY MISMATCH REPAIR DEFICIENCIES

Abstract BACKGROUND Lynch syndrome (LS) and constitutional mismatch repair deficiency syndrome (CMMRD) are caused by germline mutations in mismatch repair (MMR) genes, namely MLH1, MSH2, MSH6, and PMS2. LS patients have an inherited mono-allelic MMR defect, whereas CMMRD patients have a bi-allelic defect. MMR is critical to maintaining genomic integrity during DNA replication and these germline defects are known to produce microsatellite instability (MSI). Both LS and CMMRD have reported cases of increased primary brain cancer, namely glioblastoma multiforme (GBM). Given the shared pathophysiology of these syndromes, the large number of cases and registry studies published, and recent treatments of CNS tumors in CMMRD, a literature review has been conducted. OBJECTIVES: Review the pathophysiology of germline MMR deficiency and how it relates to brain tumor formation. Review the risk of developing primary CNS tumors with various MMR defects. Review the current management of CNS tumors in LS and CMMRD. METHODS Literature review of the most recent understanding of pathophysiology, all literature on CNS tumors in LS and CMMRD, and the current management of GBM including recent literature on CMMRD. RESULTS The lifetime risk of developing a primary malignant brain tumor or other CNS tumor in the United States has been calculated at 0.62%. Based on registry data, current estimates of the lifetime risk of developing a primary CNS tumor in LS is between 1–8.7%. Of the 146 genetically confirmed cases of CMMRD, 58 cases of high-grade gliomas were identified (including GBM). Most patients with CMMRD carry homozygous germline mutations in PMS2 (approximately 60%). Although primary CNS tumors appears to be a less common extra-colonic manifestation in LS, it is relatively common amongst CMMRD patients. Acknowledgement: Henry Lynch

Current recommendations for surveillance, risk reduction and therapy in Lynch syndrome patients

Lynch syndrome (LS) is the most common hereditary colorectal cancer syndrome and accounts for ~3 % of all CRCs. This autosomal dominant disorder is caused by germline mutations in DNA mismatch repair genes (MLH1, MSH2, MSH6, PMS2, and EPCAM). One in 300 individuals of the general population are considered to be mutation carriers (300 000 individuals/Germany). Mutation carriers are at a high CRC risk of 15-46 % till the age of 75 years. LS also includes a variety of extracolonic malignancies such as endometrial, small bowel, gastric, urothelial, and other cancers. The German Consortium for Familial Intestinal Cancer consists of 14 university centers in Germany. The aim of the consortium is to develop and evaluate surveillance programs and to further translate the results in clinical care. We have revisited and updated the clinical management guidelines for LS patients in Germany. A surveillance colonoscopy should be performed every 12-24 months starting at the age of 25 years. At diagnosis of first colorectal cancer, an oncological resection is advised, an extended resection (colectomy with ileorectal anastomosis) has to be discussed with the patient. The lifetime risk for gastric cancer is 0.2-13 %. Gastric cancers detected during surveillance have a lower tumor stage compared to symptom-driven detection. The lifetime risk for small bowel cancer is 4-8 %. About half of small bowel cancer is located in the duodenum and occurs before the age of 35 years in 10 % of all cases. Accordingly, patients are advised to undergo an esophagogastroduodenoscopy every 12-36 months starting by the age of 25 years. LS colonic and extracolonic clinical management, surveillance and therapy are complex and several aspects remain unclear. In the future, surveillance and clinical management need to be more tailored to gene and gender. Future prospective trials are needed.

Germline mutations in cancer-predisposition genes in patients with biliary tract cancer.

The prevalence of germline mutations in patients with biliary tract carcinoma (BTC) remains unclear. Here, we investigated the prevalence and types of germline mutations in patients with BTC. We reviewed 269 patients with pathologically proven BTC and collected clinical characteristics, including medical and family histories. Additionally, we evaluated germline variants in 21 genes associated with hereditary predisposition for cancer by targeted sequencing in patients meeting ≥1 of the following criteria: 1) hereditary breast and/or ovarian cancer (HBOC) testing criteria modified for BTC, 2) Revised Bethesda Guidelines (RBGs) modified for BTC (modified RBG), 3) familial BTC criteria, or 4) young BTC criteria. Among the 269 patients, 80 met at least one criterion. Three pathogenic mutations in three patients were identified: two in BRCA2 and one in BRCA1. Among the 16 patients meeting modified HBOC testing criteria, 2 harbored germline BRCA2 mutations, and 1 harbored a germline BRCA1 mutation. However, no mutation in mismatch-repair genes were detected, despite 63 patients meeting modified RBG screening criteria and 18 qualifying as young BTC patients. We detected high prevalence of pathogenic germline mutations in BRCA1/2 and none in mismatch-repair genes in BTC patients following enrichment according to family or medical history in this study.

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.

2532 Lynch Syndrome With Lung Adenocarcinoma and Their Response to Nivolumab

INTRODUCTION: Lynch syndrome is caused by mutations in mismatch repair genes which cause microsatellite instability (MSI). Patients with Lynch syndrome are at high risk for developing synchronously and metasynchronously developing colorectal, uterine, ovarian, stomach, small bowel, pancreatic, kidney and brain cancers; however, the risk of lung cancer is same as that of general population. Programmed death-1 (PD-1) inhibitors such has nivolumab have shown good overall response rate in patients with mismatch repair-deficient non-colorectal cancers. CASE DESCRIPTION/METHODS: A 72-year-old female with 57-pack-year history of smoking was diagnosed with stage IV adenocarcinoma of the transverse colon in 2007. She underwent resection of the transverse colon followed by chemotherapy. In 2016, she was diagnosed with stage IV duodenal carcinoma with metastasis to the liver. A 14.4 mm right lower lobe nodule was also seen on computed tomography (CT) scan (Figure 1). This nodule was suspected to be metastatic from the duodenal carcinoma, however, biopsy of this nodule showed it to be poorly differentiated adenocarcinoma of the lung. Staining was positive for TTF-1 and CK7 confirming it to be a primary lung cancer. Since this was a stage 1 adenocarcinoma, testing for PD-L1 expression was not done. EGFR, ROS, ALK-1 and BRAF mutations were negative. The patient's mother had breast cancer, father had lung, stomach and liver cancer while her sister had lung cancer with metastases to the brain. Given this history, familial cancer syndrome was suspected and microsatellite instability was tested. Loss of MSH2 and MSH6 was detected via immunohistochemistry (IHC) in duodenal cancer, making it a high-frequency MSI (MSI-H) tumor. Immunotherapy with Nivolumab was started in April 2017 and CT scans 8 months later showed markedly decreased size of the duodenal mass and the hepatic metastases while the lung nodule was reduced to 9.7 mm (Figure 2). DISCUSSION: As per the results of CHECKMATE-142 trial, immunotherapy with Nivolumab has shown durable response and disease control in pre-treated patients with DNA mis-match repair deficient/MSI-H metastatic colorectal cancer. PD-L1 tends to be expressed at higher levels in MSI-H cancers than in microsatellite-stable cancers. Even though lung adenocarcinoma was not tested for PD-L1 expression, it was likely to be high given its response to Nivolumab. The findings of this case suggest that lung adenocarcinoma in patients with Lynch syndrome may be susceptible to immune check-point blockade.

1883P - Is there a role for next-generation sequencing (NGS) profiling on metastatic non-colorectal gastrointestinal carcinomas (MNCGIC) in developing countries? A single center experience

BackgroundMetastatic non-colorectal cancers have adverse prognosis and no target or immunotherapy is approved until now. NGS platforms are supposed to be useful tailoring systemic treatments and/or screening patients(pts) for early phase clinical trials. Although attractive, NGS-tailored therapies (NGS-TT) can have disappointing results in not approved indications outside clinical trials. In this analysis we evaluated the tumor genetic profiles including potential germline mutations, suggested therapies, available clinical trials, and the results for off-label NGS-TT. Methodswe performed a retrospective assessment of clinical and molecular characteristics, NGS-TT prescribed and responses in a cohort of MNCGIC evaluated through the 315 genes NGS platform between 2013-2019. We looked for potential germline mutations in mismatch-repair genes and BRCA1/2, as well as the TP53R337H founder mutation. Resultsamong 78 pts, the median age was 58.5y (20-79), with 51 (65%) males and 27 (35%) females. The most common sites were pancreas (41%), stomach (15.4%) and biliary tract (15,4%); 83% were ECOG 0/1, with up to 2 lines of therapy (60.25%). Mean number of altered genes was 4.41 (1-19) and tumor mutational burden (TMB) was assessed in 24 pts, with 79% TMB-low (mean 5.22 muts/Mb). Ten pts (12.85%) underwent off-label NGS-TT after discussion at multidisciplinary tumor boards; among 9 available for response evaluation, 7 experienced progression as best response. Clinical trials were suggested for 73 pts (93,6%), but only one patient (pt) was referred to it, since all trials where abroad. We also identified 3 cases for which germline sequencing would be of value (1pt with TP53 R337H mutation; 2 young gastric cancer pts: one with concurrent MSH6/BRCA2 mutations and another with MLH1truncation exon10 alteration). Conclusionsin our cohort, the adoption of NGS to tailor systemic treatment did not show an important impact for MNCGIC pts. Increase participation of developing country centers in clinical trials is strongly needed. Potentially germline mutations were present in this series and deserve further investigation. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureD. de Freitas: Honoraria (self): Roche. D.L.F. Jardim: Honoraria (self): Foundation Medicine. B. Gumz: Honoraria (self): Novartis. All other authors have declared no conflicts of interest.

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.

126 Association Between Sessile Serrated Adenoma and Extra-colonic Malignancies

INTRODUCTION: Sessile serrated adenomas (SSAs) are precursors for 20% of microsatellite unstable colon cancers with the CpG island methylator phenotype. They are the result of a BRAF mutation and promoter hypermethylation of tumor suppressor genes or mismatch repair genes. Eighty percent of SSA harbor BRAF V600E mutation with some having KRAS mutation. BRAF V600E mutation can lead to different kinds of malignancies. Therefore, we conducted a retrospective study to assess the association between SSAs and extra-colonic malignancies. METHODS: We performed a retrospective study of patients diagnosed with SSA on colonoscopies done in Kansas University Medical Center from 2016 to 2018. The diagnosis of SSA was confirmed by histologic exam interpreted by pathologists. The group of SSA patients was compared to patients diagnosed with non-SSA polyp (control group). The electronic records were reviewed for data extraction regarding demographics and extracolonic malignancies. RESULTS: 400 patients were diagnosed with SSA from 2016 to 2018 compared to 372 patients with tubular/villous adenomas. The majority of patients in SSA and non-SSA groups were Caucasians (92% and 84.5% respectively). Mean age in group of SSA was higher than control group (age 65 ± 11 vs 55 age ± 10). Underlying morbidity i.e., obese/overweight (43.75% vs 24.5%), and HTN (51.25% vs 21%) were more common in SSA group. The two groups demographics are shown in Table 1. 60% of patients with SSA, including 6.25% of patients with skin cancer and 5.5% of those with breast cancer, had family history of cancer in first degree relative. Only 2.75% of patients with SSA had history of different organ transplant. Thirteen extra-colonic malignancies were identified (Table 2). Patients with SSA had significantly higher rates of skin cancers including, non melanoma skin cancer (12%) and breast cancer (8.25%) compared to non-SSA patients (6.2%, and 3%, P-values &lt; 0.05 respectively). CONCLUSION: To our knowledge this is the largest study of the association between SSA and extra-colonic malignancies. We found a significant association between breast and skin cancer with SSA. This could be potentially explained by presence of BRAF V600E mutation in SSA which is a precursor for different types of tumors especially melanomas. We suggest prospective studies are needed to determine whether patients with SSA should have more expanded and different cancer screening tests. Until then we emphasize the importance of skin exams and screening mammograms for patients with SSA.

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.

Different responses to anti-programmed cell death protein 1 (PD-1) immunotherapy in a patient with Lynch syndrome and metachronous urothelial and colon cancer: A case report.

Lynch syndrome (LS), as a result of the germline mutations in DNA mismatch repair genes, is characterized by the increased risk of endometrium, colon, and urinary tract cancer. Individuals with this disorder may occasionally have multiple primary carcinomas. Regardless of tumor type, pembrolizumab was approved for the treatment of patients with unresectable or metastatic mismatch repair deficient tumors, which may be an optional therapeutic method for patients with LS with multiple primary carcinomas. This case study is of a MSH2-deficient patient with LS with metachronous urothelial and colon cancer, who received pembrolizumab treatment for 8 months. The responses of the two primary sites to immunotherapy differed. Based on the changes of tumor markers and tumor size illustrated by imageological examinations, no response was observed in the sigmoid colon lesion, whereas an immune-associated phenomenon known as pseudoprogression was detected in the ureteral lesion. Immunotherapy was innovatively applied to the patient with multiple primary carcinomas. This case proposes a novel concept in which immunotherapy may potentially control the cancer growth in patients with LS and multiple primary carcinomas. However, further large-scale investigations are required. Furthermore, it raises a challenge to monitor the effectiveness of immunotherapy.

Functional interrogation of Lynch syndrome‐associatedMSH2missense variants via CRISPR‐Cas9 gene editing in human embryonic stem cells

Lynch syndrome (LS) predisposes patients to cancer and is caused by germline mutations in the DNA mismatch repair (MMR) genes. Identifying the deleterious mutation, such as a frameshift or nonsense mutation, is important for confirming an LS diagnosis. However, discovery of a missense variant is often inconclusive. The effects of these variants of uncertain significance (VUS) on disease pathogenesis are unclear, though understanding their impact on protein function can help determine their significance. Laboratory functional studies performed to date have been limited by their artificial nature. We report here an in-cellulo functional assay in which we engineered site-specific MSH2 VUS using clustered regularly interspaced short palindromic repeats-Cas9 gene editing in human embryonic stem cells. This approach introduces the variant into the endogenous MSH2 loci, while simultaneously eliminating the wild-type gene. We characterized the impact of the variants on cellular MMR functions including DNA damage response signaling and the repair of DNA microsatellites. We classified the MMR functional capability of eight of 10 VUS providing valuable information for determining their likelihood of being bona fide pathogenic LS variants. This human cell-based assay system for functional testing of MMR gene VUS will facilitate the identification of high-risk LS patients.