DNA Mismatch Repair Mutations Research Articles

A164 OPTIMIZING A PROVINCIAL SCREENING PROGRAM FOR DETECTION OF LYNCH SYNDROME

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

The prevalence of lynch syndrome (DNA mismatch repair protein deficiency) in patients with primary localized prostate cancer using immunohistochemistry screening

BackgroundProstate cancer is one of the most heritable human cancers. Lynch syndrome is an autosomal dominant inheritance caused by germline mutations in DNA mismatch repair (MMR) genes, which are also associated with an increased incidence of prostate cancer. However, prostate cancer has not been defined as a Lynch syndrome-associated cancer. The proportion of Lynch syndrome patients in primary prostate cancers is unclear. In this study, we investigated MMR protein loss using universal immunohistochemical screening to determine the prevalence of Lynch syndrome in patients with localized prostate cancer who underwent radical prostatectomy.MethodsOne hundred twenty-nine surgical specimens from radical prostatectomy performed at Toranomon Hospital between 2012 and 2015 were retrospectively tested using universal screening with immunohistochemistry staining for expression of the MMR proteins MLH1, PMS2, MSH2, and MSH6. For all suspected MMR-deficient patients, germline genetic tests focusing on MMR genes were performed.ResultsMMR protein loss was found in only one patient (0.8%) who showed dual MSH2/MSH6 loss. This patient showed a single nucleotide pathogenic germline mutation from c.1129 C to T (p.Gln377*) at exon 7 in the MSH2 gene. He was diagnosed with a primary prostate cancer at 66 years of age. He had a documented history of Lynch syndrome (Muir–Torre syndrome) with previous colon cancer, sebaceous tumor, and keratoacanthoma as well as subsequent bladder cancer, all of which also showed dual MSH2/MSH6 loss. He also had a strong family history of colorectal and other Lynch syndrome-associated cancers. The pathological stage was pT3aN0M0, and the pathological grade was Gleason 7(4 + 3) with tertiary pattern 5.ConclusionsIn this study, immunohistochemical screening of MMR proteins for Lynch syndrome was performed in a series of prostate cancer cases. The prevalence of Lynch syndrome in localized prostate cancer was 0.8%, which is low compared with other Lynch syndrome-associated cancers.

Abstract 3797: Fecal miRNA profiles and gut metagenome composition in Lynch syndrome: results from a mouse model study and human subjects

Abstract Bsacground: Lynch Syndrome (LS) is an inherited cancer syndrome associated with an increased lifetime risk of colorectal cancer (CRC) and characterised by germline mutations in DNA mismatch repair (MMR) genes. Abnormalities in the function of MMR genes lead to errors during DNA replication, including microsatellite instability. Despite regular colonoscopies, up to 15% of LS patients still develop CRC. Gut microbiome, bowel inflammation, the environment and host genetic and epigenetic factors are involved in CRC development. In this sense, the concomitant analysis of stool host microRNA (miRNA) profiles and gut microbiome composition may arise to the identification of specific fecal markers in LS explaining the molecular basis for the development of cancer in these patients. PURPOSE: In the present study, the fecal miRNome and microbiome were characterized in a time progression in an in vivo model using a mismatch repair deficient mice model that recapitulates human LS. The main aim was to evaluate the changes and crosstalk of miRNAs released in the stool by the host and the microbial population residing in the gut during the carcinogenesis process induced in animals. Findings in mice were compared and correlated to data obtained from a cohort of human subjects affected by LS. Methods: We used an in vivo model for LS based on a conditional knockout mouse with the tissue-specific inactivation of Msh2 (Msh2LoxP) in the intestinal mucosa, combining Msh2LoxP allele with the Villin-Cre transgene (VCMsh2LoxP). Stool from mice were collected at different time points (6, 9 and 12 months of age). Small RNA-sequencing (sRNA-seq) and shotgun metagenomics analyses were performed in stool from all samples. In concomitance, we collected 78 stool samples of clinically diagnosed LS subjects, at different follow up time. At the sampling, 36 resulted negative for colorectal adenomas/lesions, 12 were negative but with a previous history of lesions, 23 presented a neoplasia and 6 developed a lesion in the follow-up. Twenty-two stool samples from healthy subjects were used as controls. Results: In mice, preliminary results from sRNA-seq and metagenomics analyses showed several dysregulated miRNAs and differential microbial relative abundances, either between VCMsh2loxP and controls or at different time points. In humans, we identified 41 and 17 miRNAs respectively up- and downregulated in LS subjects who presented a lesion compared to those not having any lesion at follow-up. In the same comparison the metagenomic data showed significantly different microbial abundances, including those of Eubacterium ramulus. Conclusions: This is the first study characterizing the concomitant alterations in microbial composition and host miRNome overtime in relation to the onset of cancerous/precancerous lesions due to LS. Citation Format: Giulia Francescato, Giulio Ferrero, Marc Beltrà, Sonia Tarallo, Giulia Piaggeschi, Carla Di Battista, Antonio Francavilla, Carlijn Bruggeling, Fabio Penna, Barbara Pardini, Paola Costelli, Annemarie Boleij, Alessio Naccarati. Fecal miRNA profiles and gut metagenome composition in Lynch syndrome: results from a mouse model study and human subjects. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3797.

Universal screening for Lynch syndrome in operated colorectal cancer by immunohistochemistry: a cohort of patients in Liaoning province, China.

Lynch syndrome (LS) is the most common hereditary colorectal cancer syndrome worldwide. Due to the decreasing family size in Liaoning province. The Bethesda and Amsterdam II criteria have lower sensitivity and specificity and are not suitable for the local population. Immunohistochemistry screening for mutations in DNA mismatch repair (MMR) in newly diagnosed colorectal cancer can improve the detection rate of LS. All newly diagnosed colorectal cancer patients who underwent surgery between January 2018 and June 2020 at Cancer Hospital of China Medical University and Shengjing Hospital of China Medical University from Liaoning China were included retrospectively, and the ratio of universal LS screening by immunohistochemistry, MMR protein deficiency (dMMR) ratio, MLH1 loss, MSH2 loss, MSH6 loss, and PMS2 loss was analyzed. The clinicopathological characteristics of patients with pMMR and dMMR were analyzed. A total of 7019 colorectal cancer patients underwent surgery and 4802 (68.41%) patients were screened by immunohistochemistry for MMR, 258 (5.37%) cases were reported to have a loss of MMR expression. In the dMMR group, a higher number of patients were under 50 years old, more tumors were located at the right colon, less patients have lymph node metastasis, more tumors were stage II, and histological types of mucinous carcinoma or signet ring carcinoma were more common, compared with the pMMR group. Only 2.71% dMMR patients meet Amsterdam criteria II, 2.71% of patients meet Revised Bethesda guidelines, and 17.83% meet Chinese LS criteria. Twenty-five dMMR patients were confirmed by next-generation sequencing and five families were confirmed as Lynch family. These data imply that universal screening for LS by immunohistochemistry may be effective in Liaoning province.

The role of the Pseudomonas aeruginosa hypermutator phenotype on the shift from acute to chronic virulence during respiratory infection.

Chronic respiratory infection (CRI) with Pseudomonas aeruginosa (Pa) presents many unique challenges that complicate treatment. One notable challenge is the hypermutator phenotype which is present in up to 60% of sampled CRI patient isolates. Hypermutation can be caused by deactivating mutations in DNA mismatch repair (MMR) genes including mutS, mutL, and uvrD. In vitro and in vivo studies have demonstrated hypermutator strains to be less virulent than wild-type Pa. However, patients colonized with hypermutators display poorer lung function and a higher incidence of treatment failure. Hypermutation and MMR-deficiency create increased genetic diversity and population heterogeneity due to elevated mutation rates. MMR-deficient strains demonstrate higher rates of mucoidy, a hallmark virulence determinant of Pa during CRI in cystic fibrosis patients. The mucoid phenotype results from simple sequence repeat mutations in the mucA gene made in the absence of functional MMR. Mutations in Pa are further increased in the absence of MMR, leading to microcolony biofilm formation, further lineage diversification, and population heterogeneity which enhance bacterial persistence and host immune evasion. Hypermutation facilitates the adaptation to the lung microenvironment, enabling survival among nutritional complexity and microaerobic or anaerobic conditions. Mutations in key acute-to-chronic virulence “switch” genes, such as retS, bfmS, and ampR, are also catalyzed by hypermutation. Consequently, strong positive selection for many loss-of-function pathoadaptive mutations is seen in hypermutators and enriched in genes such as lasR. This results in the characteristic loss of Pa acute infection virulence factors, including quorum sensing, flagellar motility, and type III secretion. Further study of the role of hypermutation on Pa chronic infection is needed to better inform treatment regimens against CRI with hypermutator strains.

DIPG-58. Therapeutic HDAC targeting in hypermutant CNS tumors

Abstract Diffuse intrinsic pontine glioma (DIPG) is a universally fatal tumor of the pons, most often characterized by mutations in genes encoding histone 3, that led to the classification diffuse midline glioma, H3 K27M-mutant (DMG). However, 15% of DIPG are histone wildtype (H3WT) and can have a hypermutant phenotype, characterized by mutations in DNA mismatch repair that also occur in pediatric high-grade glioma (pHGG). We previously published the preclinical efficacy of quisinostat in DMG. As hypermutant CNS tumors share transcriptional disruption with DMG, we investigated if HDAC inhibition was also therapeutic against hypermutant DIPG and pHGG. We tested PBT-24, a PMS2 mutant, treatment-naïve, patient-derived hypermutant DIPG model, and found similar quisinostat sensitivity compared to other DMG cultures in vitro (IC50 = ~30 nM), as well as significant efficacy in a xenograft flank model (60 day mean tumor size of 1109mm3 [vehicle] vs. 19mm3 [quisinostat-treated]). This in vivo effect was greater than the anti-tumor effect observed in a xenograft flank non-hypermutant DMG model PBT-09 (60 day mean tumor size of 1006mm3 [vehicle] vs. 244mm3 [quisinostat-treated]). To validate the effect of hypermutation on sensitivity to quisinostat, we generated PMS2 knock-out (KO) and MSH2 KO isogenic cell lines using the H3WT model VUMC-DIPG-10. In a xenograft flank model, we observed increased quisinostat sensitivity in the KO models (20 day mean tumor volume of 547mm3 [parental], 396mm3 [PMS2 KO], and 150mm3 [MSH2 KO]). RNA sequencing of quisinostat-treated PBT-24 revealed an increase in acute inflammatory response genes which we are investigating as a mechanism of action for HDACi against hypermutant tumors. Ultimately, we demonstrate the in vivo efficacy of quisinostat against hypermutant DIPG that supports the investigation of HDAC inhibition as a treatment strategy across hypermutant CNS tumors.

PD58-01 PREVALENCE AND SPECTRUM OF GERMLINE MUTATIONS IN PATIENTS WITH UPPER TRACT UROTHELIAL CARCINOMA

You have accessJournal of UrologyCME1 May 2022PD58-01 PREVALENCE AND SPECTRUM OF GERMLINE MUTATIONS IN PATIENTS WITH UPPER TRACT UROTHELIAL CARCINOMA Hong Truong, Rania Sheikh, Yelena Kemel, Manuel Jesus Escano, Andrew Lenis, Peter Reisz, Alvin Goh, Eugene Cha, Bernard Bochner, Maria Carlo, Hikmat Al-Ahmadie, and Jonathan Coleman Hong TruongHong Truong More articles by this author , Rania SheikhRania Sheikh More articles by this author , Yelena KemelYelena Kemel More articles by this author , Manuel Jesus EscanoManuel Jesus Escano More articles by this author , Andrew LenisAndrew Lenis More articles by this author , Peter ReiszPeter Reisz More articles by this author , Alvin GohAlvin Goh More articles by this author , Eugene ChaEugene Cha More articles by this author , Bernard BochnerBernard Bochner More articles by this author , Maria CarloMaria Carlo More articles by this author , Hikmat Al-AhmadieHikmat Al-Ahmadie More articles by this author , and Jonathan ColemanJonathan Coleman More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002643.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Deleterious germline mutations confer high cancer risks and require enhanced cancer screening, yet the prevalence and spectrum of germline mutations among patients with upper tract urothelial carcinoma (UTUC) are unknown. We sought to determine the frequency of germline mutations in cancer susceptibility genes in patients with UTUC and identify clinical and pathologic factors associated with the presence of germline mutations in DNA mismatch repair (MMR) genes. METHODS: We retrospectively reviewed patients with UTUC who underwent germline sequencing with a targeted panel of >75 cancer-associated genes as part of an institutional protocol of matched tumor-germline sequencing initiative from 04/2015 to 04/2021. Prevalence and spectrum of pathogenic/likely pathogenic variants were determined. Clinicopathologic characteristics were assessed by mutation status. All statistical computations were performed using STATA version 12.1. RESULTS: Of 232 patients, 70% were male, 85% had high grade UTUC, 12% had bilateral tumors, and 11% had metastasis at diagnosis. Germline mutations were identified in 37 (16%) patients. 21 (9%) had mutations in Lynch syndrome (LS) associated MMR genes (13 MSH2, 4 MSH6, 4 MLH1) and 11 (5%) had mutations in moderate/high penetrance non-MMR genes (3 BRCA1, 3 BRCA2, 2 ATM, 1 BARD, 1 BRIP1, and 1 CHEK2). In 21 patients with MMR mutations, 3 (14%) had bilateral UTUC, 10 (48%) and 15 (71%) had personal and family history of LS-associated cancers, respectively. Only 10 (48%) of patients with MMR mutations met current NCCN guideline for genetic testing. UTUC tumor analyses of patients with MMR mutations revealed 15/16 (94%) had MMR-deficiency, and 12/18 (67%) had microsatellite instability. CONCLUSIONS: In our cohort of patients with UTUC identified to have pathogenic germline variants in MMR gene, half did not qualify for germline testing based on current genetic guideline for Lynch syndrome. Importantly, most patients with MMR mutations had microsatellite unstable or MMR-deficient UTUC tumors which would qualify patients for immunotherapy. Our findings support germline testing, especially for MMR genes, for all patients with UTUC. Source of Funding: NIH T32 Ruth L. Kirschstein National Research Service Award 2020 - 2022 © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e1011 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hong Truong More articles by this author Rania Sheikh More articles by this author Yelena Kemel More articles by this author Manuel Jesus Escano More articles by this author Andrew Lenis More articles by this author Peter Reisz More articles by this author Alvin Goh More articles by this author Eugene Cha More articles by this author Bernard Bochner More articles by this author Maria Carlo More articles by this author Hikmat Al-Ahmadie More articles by this author Jonathan Coleman More articles by this author Expand All Advertisement PDF DownloadLoading ...

Correlation of mismatch repair deficiency with clinicopathological features and programmed death-ligand 1 expression in thyroid carcinoma.

Mutations in DNA mismatch repair (MMR) genes associated with thyroid carcinoma (TC) have rarely been reported, especially in East Asian populations. We examined tumor tissue from a cohort of 241 patients diagnosed with TC between 2008 and 2020. MMR proteins were detected using tissue microarray-based immunohistochemistry in order to identify MMR-protein-deficient (MMR-D) and MMR-protein-intact (MMR-I) tumors. We retrospectively summarized the clinicopathologic characteristics of patients with MMR-D TC, measured the expression of PD-L1, and recorded overall survival (OS) and other clinical outcomes. In our cohort, there were 18 (7.5%) MMR-D (MLH1, MSH2, MSH6, and PMS2) patients, including 12 with papillary TC (PTC) (6.7%), 2 with poorly differentiated TC (PDTC) (4.7%), and 4 with anaplastic TC (ATC) (22.2%). Half of them (9/18) showed a specific deletion in MSH6, and 6 of them also carried variants in the MSH6 and PMS2 gene. Survival was significantly better in patients with MMR-D ATC than in those with MMR-I tumors (p = 0.033). Four of the 18 MMR-D patients (22%) were found to be PD-L1 positive. Their OS was much shorter than that of PD-L1-negative patients. MMR-D and PD-L1 positivity appear to be associated with clinicopathological characteristics and prognosis in TC. The results indicate that MMR status may have important prognostic significance in TC. Therefore, immune checkpoint inhibitors that target the PD-1/PD-L1 pathway may be a treatment option for TCs.

Lynch-like Syndrome: Potential Mechanisms and Management.

Simple SummaryLynch-like syndrome (LLS) is defined as colorectal cancer cases with microsatellite instability (MSI) and loss of expression of MLH1, MSH2, MSH6, or PMS2 by immunohistochemistry (IHC) in the absence of a germline mutation in these genes that cannot be explained by BRAF mutation or MLH1 hypermethylation. The application of the universal strategy for the diagnosis of Lynch syndrome (LS) in all CRCs is leading to an increase in the incidence of cases of LLS. It has been described that risk of cancer in relatives of LLS patients is in between of that found in Lynch syndrome families and sporadic cases. That makes LLS patients and their families a challenging group for which the origin of CRC is unknown, being a mixture between unidentified hereditary CRC and sporadic cases. The potential causes of LLS are discussed in this review, as well as methods for identification of truly hereditary cases.Lynch syndrome is an autosomal dominant disorder caused by germline mutations in DNA mismatch repair (MMR) system genes, such as MLH1, MSH2, MSH6, or PMS2. It is the most common hereditary colorectal cancer syndrome. Screening is regularly performed by using microsatellite instability (MSI) or immunohistochemistry for the MMR proteins in tumor samples. However, in a proportion of cases, MSI is found or MMR immunohistochemistry is impaired in the absence of a germline mutation in MMR genes, BRAF mutation, or MLH1 hypermethylation. These cases are defined as Lynch-like syndrome. Patients with Lynch-like syndrome represent a mixture of truly hereditary and sporadic cases, with a risk of colorectal cancer in first-degree relatives that is between the risk of Lynch syndrome in families and relatives of sporadic colon cancer cases. Although multiple approaches have been suggested to distinguish between hereditary and sporadic cases, a homogeneous testing protocol and consensus on the adequate classification of these patients is still lacking. For this reason, management of Lynch-like syndrome and prevention of cancer in these families is clinically challenging. This review explains the concept of Lynch-like syndrome, potential mechanisms for its development, and methods for adequately distinguishing between sporadic and hereditary cases of this entity.

Loss of MMR and TGFBR2 Increases the Susceptibility to Microbiota-Dependent Inflammation-Associated Colon Cancer.

Background and AimsMutations in DNA mismatch repair (MMR) genes are causative in Lynch syndrome and a significant proportion of sporadic colorectal cancers (CRCs). MMR-deficient (dMMR) CRCs display increased mutation rates, with mutations frequently accumulating at short repetitive DNA sequences throughout the genome (microsatellite instability). The TGFBR2 gene is one of the most frequently mutated genes in dMMR CRCs. Therefore, we generated an animal model to study how the loss of both TGFBR2 signaling impacts dMMR-driven intestinal tumorigenesis in vivo and explore the impact of the gut microbiota.MethodsWe generated VCMsh2/Tgfbr2 mice in which Msh2loxP and Tgfbr2loxP alleles are inactivated by Villin-Cre recombinase in the intestinal epithelium. VCMsh2/Tgfbr2 mice were analyzed for their rate of intestinal cancer development and for the mutational spectra and gene expression profiles of tumors. In addition, we assessed the impact of chemically induced chronic inflammation and gut microbiota composition on colorectal tumorigenesis.ResultsVCMsh2/Tgfbr2 mice developed small intestinal adenocarcinomas and CRCs with histopathological features highly similar to CRCs in Lynch syndrome patients. The CRCs in VCMsh2/Tgfbr2 mice were associated with the presence of colitis and displayed genetic and histological features that resembled inflammation-associated CRCs in human patients. The development of CRCs in VCMsh2/Tgfbr2 mice was strongly modulated by the gut microbiota composition, which in turn was impacted by the TGFBR2 status of the tumors.ConclusionsOur results demonstrate a synergistic interaction between MMR and TGFBR2 inactivation in inflammation-associated colon tumorigenesis and highlight the crucial impact of the gut microbiota on modulating the incidence of inflammation-associated CRCs.

32Do the risks of Lynch syndrome-related cancers depend on the parent-of-origin of the mutation?

Abstract Background Individuals who carry pathogenic mutations in DNA mismatch repair (MMR) genes have high risks of cancer, and small studies have suggested that these risks depend on the sex of the parent from whom the mutation was inherited. We have conducted the first large study of such a parent-of-origin effect (POE). Methods Our study was based on all MMR gene mutation carriers and their relatives in the Colon Cancer Family Registry, comprising 18,226 people. The POE was estimated as a hazard ratio (HR) using a segregation analysis approach that adjusted for ascertainment. HR = 1 corresponds to no POE and HR > 1 corresponds to higher risks for maternal mutations. Results For all MMR genes combined, the estimated POE HRs were 1.02 (95% confidence interval (CI) 0.75-1.39, p = 0.9) for male colorectal cancer, 1.12 (95% CI 0.81-1.54, p = 0.5) for female colorectal cancer and 0.84 (95% CI 0.52-1.36, p = 0.5) for endometrial cancer. Separate results for each MMR gene were similar. Conclusions Despite being well-powered, our study did not find any evidence that cancer risks for MMR gene mutation carriers depend on the parent-of-origin of the mutation. Based on current evidence, we don’t recommend that POEs be incorporated into the clinical guidelines or advice for such carriers. Key messages MMR gene mutations inherited from the maternal and paternal side confer similar risks of developing colorectal and endometrial cancer.

Abstract 803: Molecular diagnosis and immunotherapy of a rare lung carcinoma patient associated with PMS2 c.1144+1G>A mutation-driven Lynch syndrome

Abstract Background: Lynch syndrome (LS) is an autosomal dominant genetic disease caused by mutations in DNA mismatch repair (MMR) genes, leading to an increased risk of colorectal cancer and other malignancies. Tumors with MMR-deficiency often have microsatellite instability (MSI) and high mutational burden (TMB) which predict high response rate to immunotherapy. Therefore, accurate diagnosis of LS can create valuable opportunities for the clinical management of a broad spectrum of cancers. Methods: We conducted next-generation sequencing to assess both somatic and germline mutations of the proband. LS diagnosis was confirmed by mismatch repair protein immunochemistry and germline genetic testing results. Furthermore, we applied Sanger sequencing to screen the candidate variant in his family members. Results: Our proband is a 62-year-old Chinese diagnosed with invasive lung adenocarcinoma (pT1aN2M0, stage IIIA) in the right inferior lobe. Based on the near completely loss of PMS2 in cancer cells by MMR IHC, as well as instability of three MSI markers (NR21, BAT-26 and MONO-27), we suspected LS and performed a germline NGS panel analysis which revealed a G>A nucleotide substitution (c.1144+1G>A) located at the +1 position of intron 10 of the PMS2 gene. We also found that the proband has an oncogenic KRAS p.G13D somatic mutation and his young daughter with the same PMS2 variant has no cancer yet. To characterize the pathogenicity of this PMS2 germline variant, we conducted a comprehensive genetic analysis according to the ACMG/Sherloc guidelines. In silico analysis with three splice prediction programs suggested that the PMS2 c.1144+1G>A variant will generate aberrant splicing transcripts. The population data in the gnomAD web portal showed that the allele frequency of PMS2 c.1144+1G>A variant is 2/282496 without homozygotes, falling into the pathogenic range defined by the Sherloc guideline. Importantly, in a family with germline PMS2 c.1144+2T>A variant, which disrupts the same splice donor site as c.1144+1G>A, the cosegregation between this variant and LS-related cancers was clearly seen. Based on all these results, we classified the PMS2 c.1144+1G>A variant as likely pathogenic, and disruption of this specific PMS2 splicing site results in LS. Combination chemotherapy with cisplatin (75 mg/m2), pemetrexed (500 mg/m2), and nivolumab (200 mg) was administered after surgery every 3 weeks for a total of 4 cycles to the patient. He achieved 16 months DFS. We then provided an up-to-date genetic counseling to the patient's daughter with the same PMS2 variant. Conclusion: Our findings suggest that accurate LS diagnosis with modern pathology approaches including MSI/MMR-d testing and NGS sequencing offers extracolonic cancer patients the opportunity to reduce cancer-related morbidity and mortality through innovative treatments such as immunotherapy. Citation Format: Quanli Han, Xiaomo Li, Tonghui Ma, Qi Wang, Zhi Cui. Molecular diagnosis and immunotherapy of a rare lung carcinoma patient associated with PMS2 c.1144+1G>A mutation-driven Lynch syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 803.

Abstract 472: Tumor mutational burden reveals tumor-specific patterns with intra-patient stability from multiple longitudinal tissue biopsies from 3,402 patients

Abstract Tumor mutational burden (TMB) continues to evolve as a predictive biomarker of immunotherapy response. The recent pan-tumor approval of pembrolizumab for solid tumors with high TMB (TMB-H), defined as ≥10 mutations/megabase (mut/Mb), offers a promising treatment option, especially in advanced tumors. Here, we sought to investigate genomic profiles in cancer patients with multiple longitudinal tissue biopsies to assess the prevalence and variability of TMB, and its relationship with other biomarkers. We utilized pan-tumor comprehensive genomic profiling data from 246,806 patients, tested as part of routine clinical care (FoundationOne). High prevalence (>30%) of TMB-H was observed in skin, lung and bladder tumors. In contrast, gastrointestinal (GI) tumors exhibited low prevalence of TMB-H (8.8%), with frequent co-occurrence of TMB-H with high microsatellite instability (TMB-H/MSI). Exclusive TMB-H represented only 4% of GI cases, with a median TMB of 12.2 mut/Mb, compared to a much higher median of 43.5 mut/Mb in the TMB-H/MSI subgroup. Uterus/endometrial tumors also displayed co-occurring TMB-H/MSI, however, unlike GI, exclusive TMB-H cases had a higher median of 16.3 mut/Mb. 3,402 patients with multiple longitudinal tissue biopsies across 38 tumor types were identified (median collection time difference, days (d): 472). Overall, largely stable patterns of TMB and MSI were observed. Only 234 cases (7%) showed a change in TMB status, of which 73% had a diagnostic status change from TMB-low to TMB-H. There was no significant correlation between change in TMB and time between tests. However, gliomas were a notable outlier with significant changes in TMB status. Breast, colorectal and ovarian tumors also showed evidence for a statistically significant TMB change (P < 0.05), primarily in larger time points (>3years). MSI tumors and cases with a DNA mismatch repair mutation signature (MMR+) showed a higher TMB change than MS-stable/MMR- tumors respectively, albeit limited by cohort size. 4.6% of MS-stable tumors shifted from TMB-low to TMB-H (median time difference: 658d), with a median TMB change of 7.5 mut/Mb. Of note, genomic loss of heterozygosity (gLOH)-high ovarian tumors showed a higher TMB change than gLOH-low tumors (P < 0.05). These observed TMB changes, although rare, may, in part, result from intervening therapies or evolving mutational processes in these cases. TMB is a continuous variable biomarker. Identifying a cutoff for clinical and analytical validation can be a challenge, particularly given the impact of underlying tumor heterogeneity on the diagnostic score. Our data suggest that TMB at the cutoff of 10 mut/Mb is stable across tumor types and potentially across treatment modalities. These results also shed light on the co-occurrence of TMB and MSI, particularly in GI tumors, which may further inform treatment decisions for patients. Citation Format: Smruthy Sivakumar, Ethan S. Sokol, Garrett M. Frampton, David Fabrizio, Brian Alexander, Priti S. Hegde, Axel Grothey. Tumor mutational burden reveals tumor-specific patterns with intra-patient stability from multiple longitudinal tissue biopsies from 3,402 patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 472.

Case Report: A Frameshift Mutation in MSH2 Exon 2 in a Kidney Recipient With Muir–Torre Syndrome

Muir–Torre syndrome (MTS), a rare subtype of Lynch syndrome, is mostly autosomal dominant, which is caused by germline mutations in DNA mismatch repair (MMR) genes, the resulting microsatellite instability (MSI) of which increases the risk of developing sebaceous and other visceral tumors. Several reports have showed an association between immunosuppressive agents and the progression of latent MTS. In this report, we described a 41-year-old man with a history of kidney transplantation, having a rapid growth of the nodule on the anterior chest under immunosuppressive therapy, which was histologically proved to be sebaceous carcinoma. Systemic evaluation for visceral malignancies revealed sigmoid adenocarcinoma. These findings were consistent with the clinical diagnosis of MTS. Histological findings showed an absence of MMR proteins, including MSH2 and MSH6 both in the sebaceous carcinoma and sigmoid adenocarcinoma on immunohistochemical (IHC) analysis. A frame-shift mutation of c.229_230delAG (p. Ser77fs) in the MSH2 exon 2 in the lesion was detected by next-generation sequencing (NGS) analysis. This case report not only reveals a new site of MSH2 mutation in this family of East Asian descent but also highlights the importance of adequate diagnosis for Muir–Torre syndrome, as well as further prevention of the development of latent visceral tumors in kidney transplant recipients.

RNA-Seq Reveals Differences in Expressed Tumor Mutation Burden in Colorectal and Endometrial Cancers with and without Defective DNA-Mismatch Repair

Tumor mutation burden (TMB) is an emerging biomarker of immunotherapy response. RNA sequencing in FFPE tissue samples was used for determining TMB in microsatellite-stable (MSS) and microsatellite instability-high (MSI-H) tumors in patients with colorectal or endometrial cancer. Tissue from tumors and paired normal tissue from 46 MSI-H and 12 MSS cases were included. Of the MSI-H tumors, 29 had defective DNA mismatch-repair mutations, and 17 had MLH1 promoter hypermethylation. TMB was measured using the expressed somatic nucleotide variants (eTMB). A method of accurate measurement of eTMB was developed that removes FFPE-derived artifacts by leveraging mutation signatures. There was a significant difference in the median eTMB values observed between MSI-H and MSS cases: 27.3 versus 6.7 mutations/megabase (mut/Mb) (P=3.5×10-9). Among tumors with defective DNA-mismatch repair, those with mismatch-repair mutations had a significantly higher median eTMB than those with hypermethylation: 28.1 versus 17.5 mut/Mb (P=0.037). Multivariate analysis showed that MSI status, tumor type (endometrial or colorectal), and age were significantly associated with eTMB. Additionally, using whole-exome sequencing in a subset of these patients, it was determined that DNA TMB correlated well with eTMB (Spearman correlation coefficient, 0.83). These results demonstrate thatRNA sequencing can be used for measuring eTMB in FFPE tumor specimens.

Prevalence of mutations in BRCA and MMR genes in patients affected with hereditary endometrial cancer

Endometrial cancer (EC) is the fifth most common cancer in women from developed countries, accounting for 4.8% of new cases and 2.1% of deaths. The genetic basis for the familial risk of endometrial cancer has not been completely defined. Mostly, hereditary EC is part of two syndromes as Lynch syndrome (LS) and Hereditary Breast and Ovarian Cancer syndrome (HBOC). LS is the prototypical hereditary cancer syndrome in EC and accounts for 2–6% of all endometrial cancers. This disease is caused by autosomal dominant mutations in DNA mismatch repair (MMR) genes. Patients carrying a germline mutation in one of the MMR genes have a cumulative lifetime risk to develop EC of 20–70%. HBOC is an autosomal dominantly inherited disease, which mostly predisposes to breast and ovarian cancers, but it can be also associated with other malignancies. HBOC results from germline mutations in BRCA1/2 genes. The aim of this study was to determine the mutational status of a cohort of 40 EC patients, 19 belonging to families with LS and 21 to HBOC. Mutation analysis of MLH1, MSH2, BRCA1 and BRCA2 genes showed pathogenic variants in 17/40 (42.5%) patients. Out of 19 patients belonging to LS families, 8 (42.1%) showed a pathogenic variant. Out of 21 patients belonging to HBOC families, 9 (42.8%) showed a pathogenic variant. 1/21 (4.8%) patient report 1 variant of unknown significance (UV), c.599 C > T (p.T200I), in BRCA2. Moreover, in 1/21 (4.8%) patient we identified a novel missense variant in BRCA2, c.9541A > T (p.Met3181Leu). Mutational analysis was extended to family members, both healthy and cancer affected, of mutated patients; all the tested relatives affected with cancer displayed the pathogenic variant. Our data suggest that patients with hereditary EC have a high percentage of mutations in the LS and HBOC main susceptibility genes; therefore, the surveillance for EC, already indicated in LS patients, should also be recommended for patients with HBOC.

Clinicopathological features and types of microsatellite instability in 1394 patients with colorectal cancer

To explore the clinicopathological features and types of genic mutations in DNA mismatch repair (MMR) in colorectal cancer (CRC). Immunohistochemistry was used to determine the expression of MMR proteins in 1394 patients with CRC, and PCR-capillary electrophoresis (PCR-CE) was used to detect microsatellite instability (MSI) in 106 cases of defective MMR (dMMR), 46 cases of proficient MMR (pMMR) with heterogeneous expression and 147 randomly selected cases of pMMR. The relationship between the expressions of MMR proteins and the clinicopathological features of the patients was evaluated. The consistency between the results of immunohistochemistry and PCR-CE was assessed. Immunohistochemical staining showed an incidence of dMMR of 7.6% in the patients. The main type of dMMR was co-deletion of MLH1 and PMS2, accounting for 55.7% of the total dMMR cases. The deletion of MMR proteins was significantly correlated with the patients' age, tumor location, tumor size, gross type, histological type, degree of differentiation, lymph node status and TNM stage (P < 0.05), but not with gender (P > 0.05). The total accordance rate of immunohistochemistry and PCR-CE was 98.7% in these patients. The main type of dMMR is co-deletion of MLH1 and PMS2 in patients with colorectal cancer. dMMR colorectal cancer has typical clinicopathological features and a lower incidence in China than in Western countries. The results of immunohistochemistry and PCR-CE are highly consistent for detecting dMMR in colorectal cancer patients.

Characteristics of Adrenocortical Carcinoma Associated With Lynch Syndrome.

Lynch syndrome (LS) is the most common inherited colorectal and endometrial cancer syndrome, caused by germline mutations in DNA mismatch repair (MMR) genes. It is also characterized by an increased risk of other tumors with lower prevalence, such as adrenal cortical carcinoma (ACC), an endocrine tumor with an incidence of <2 cases/million individuals/year. Most ACC developed during childhood are associated with hereditary syndromes. In adults, this association is not as well established as in children. Previous studies showed a 3.2% prevalence of LS among patients with ACC. The objective of this study is to determine the prevalence of ACC in a Spanish LS cohort and their molecular and histological characteristics. This retrospective study includes 634 patients from 220 LS families registered between 1999 and 2018. During the follow-up period, 3 patients were diagnosed with ACC (0.47%); all were carriers of a MSH2 germline mutation. The 3 ACC patients presented loss of expression of MSH2 and MSH6 proteins. One tumor analysis showed loss of heterozygosity of the MSH2 wildtype allele. Our findings support previous data that considered ACC as a LS spectrum tumor. MMR protein immunohistochemistry screening could be an efficient strategy to detect LS in patients with ACC.

Abstract 17: Ex-vivo assessment of the effect of Simvastatin in intestinal epithelium of Lynch Syndrome patients

Abstract Background: Lynch Syndrome (LS) is a hereditary condition secondary to germline mutations in DNA mismatch repair (MMR) genes, which predisposes patients to develop different tumor types but mainly colorectal and endometrial cancers. Due to the genetic predisposition, chemopreventive strategies become critical in these individuals. One such chemopreventive compound, statin, a potent pharmacologic inhibitor of cholesterol biosynthesis, is known to exert other pleotropic effects such as modulation of cell proliferation, apoptosis, and inflammation, mainly due to inhibition of the mevalonate pathway. Although, chemoprevention studies in the past were performed in cancer stem cell models the effects have not been previously assessed in the physiologic and MMR-deficient stem cell niche. Here, we have successfully established ex-vivo cultures of crypt-villus structures (termed ‘mini-guts' or organoids) in order to interrogate the chemopreventive effects of statins in Lynch Syndrome patients. Methods: Fresh endoscopic biopsy samples from a total of six LS patients followed at The University of Texas MD Anderson Cancer Center were obtained and ex-vivo organoid cultures were established. Each organoid line (passage &amp;lt; P6) was treated with two different doses of Simvastatin (5 and 10 µM) or DMSO (control) for 48 hours. Total RNA was isolated from organoids treated with control and Simvastatin 10 µM and RNASeq analysis was performed using Illumina HiSeq 4000. Validation of several critical genes was performed by qRT-PCR assay. Results: RNAseq analysis accounted for a total of 4,386 genes dysregulated in organoids treated with Simvastatin as compared to control. Stem cell markers, LGR5 and ASCL2, and differentiation markers, KRT20 (enterocytes) and MUC2 (goblet), were found to be induced by simvastatin. Gene Set Enrichment Analysis (GSEA) using Hallmark pathway gene sets showed significant enrichment for E2F target genes apart from canonical cholesterol homeostasis pathway. Ingenuity pathways analysis (IPA) revealed a list of top 10 genes which are upregulated/downregulated and implicated in cancer pathway. Validation of selected E2F Hallmark pathway genes (E2F1, TOP2A, Survivin, and KI-67) and IPA cancer pathway genes (KLF2, KLF6, EZH2, p57, and p21) showed a significant and dose dependent modulation upon treatment with Simvastatin. Conclusions: Treatment with Simvastatin exhibited a profound effect on key genes involved in maintenance of stem cell niche in patient-derived organoids from LS. Simvastatin had a significant effect on E2F target genes implicated in regulation of cell cycle and proliferation via modulation of Ki-67, TOP2A, and Survivin. Along with increase in cyclin-dependent kinase inhibitor p21, there was significant induction of p57, a candidate tumor suppressor gene. Hence, Simvastatin, while it maintains the stem niche, exerts its anti-proliferative effects by specifically modulating genes involved in cell cycle and apoptosis. Citation Format: Prashant Bommi, Wenhui Wu, Laura Reyes, Kyera Evans, Selvi Thirumurthi, Patrick Lynch, Krishna Sinha, Eduardo Vilar. Ex-vivo assessment of the effect of Simvastatin in intestinal epithelium of Lynch Syndrome patients [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 17.

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.