MSH2 Mutations Research Articles

The effect of DNA polymorphisms and natural variation on crossover hotspot activity in Arabidopsis hybrids

In hybrid organisms, genetically divergent homologous chromosomes pair and recombine during meiosis; however, the effect of specific types of polymorphisms on crossover is poorly understood. Here, to analyze this in Arabidopsis, we develop the seed-typing method that enables the massively parallel fine-mapping of crossovers by sequencing. We show that structural variants, observed in one of the generated intervals, do not change crossover frequency unless they are located directly within crossover hotspots. Both natural and Cas9-induced deletions result in lower hotspot activity but are not compensated by increases in immediately adjacent hotspots. To examine the effect of single nucleotide polymorphisms on crossover formation, we analyze hotspot activity in mismatch detection-deficient msh2 mutants. Surprisingly, polymorphic hotspots show reduced activity in msh2. In lines where only the hotspot-containing interval is heterozygous, crossover numbers increase above those in the inbred (homozygous). We conclude that MSH2 shapes crossover distribution by stimulating hotspot activity at polymorphic regions.

Inherited mutations in Chinese patients with upper tract urothelial carcinoma

Upper tract urothelial carcinoma (UTUC) accounts for 10% of urothelial carcinomas (UCs) and has a substantial hereditary component. However, the majority of our knowledge of germline spectrum comes from bladder cancer (BCa) data in White populations. Here, we sequence 309 Chinese UTUC cases and identify 71 germline pathogenic/likely pathogenic (P/LP) mutations in 62 patients (20.1%). Compared with White cases, we observe disparities and similarities in inherited mutational profiles. Association analysis reveals that germline P/LP mutations in MSH2, BRCA2, BRCA1, and BRIP1 significantly increase UTUC risk in Chinese populations. Furthermore, germline P/LP mutation in homologous recombination genes indicates poor prognosis for non-metastatic UTUC. Finally, we perform paired sequencing and observe significant correlations between germline mutation patterns and tumor subtypes. This study highlights the importance of genetic testing in patients with UTUC and calls for germline data from various ethnicities to better understand this disease.

Синдром Мьюир-Торре – предиктор рака желудка (клиническое наблюдение)

Muir-Torre syndrome is a hereditary autosomal dominant syndrome that belongs to HNPCC (Lynch syndrome), being its subspecies. The disease is associated with a high risk of developing colorectal cancer, as well as other cancers of the gastrointestinal tract, genitourinary tract and skin lesions in the form of tumors of the sebaceous glands and keratoacanthus. Visceral cancer in the syndrome is characterized by multiple localization and a low degree of malignancy. Mutations in the genes MSH2 and MLH1 - loci 2p22–p21 and 3p21.3 in the DNA repair system were detected in patients with Muir-Torre syndrome, which leads to its microsatellite instability. Skin tumors may precede visceral cancer, may develop in parallel with the malignant process of internal organs. The importance of timely diagnosis of skin neoplasms (sebaceous gland adenomas and keratoacanthus) as a marker of Muir-Torre syndrome is emphasized. The article presents a clinical case of Muir-Torre syndrome in a 50-year-old patient, which was manifested by a combination of gastric cancer, multiple adenomas of the sebaceous glands and solitary keratoacanthoma. The first manifestations of the syndrome were adenomas of the sebaceous glands of the facial skin, mistakenly regarded by dermatologists as a papular form of rosacea. After 4 years, the patient underwent surgery for a keratoacanthoma of the shin, a couple of months later, the patient was diagnosed with stage IIA gastric cancer (T3N0M0), a ring-cell variant. Skin rashes were regarded as hereditary paraneoplastic Muir–Torre syndrome. In order to detect oncological diseases early, it is necessary to improve the qualifications of doctors in the clinic and diagnosis of hereditary paraneoplastic dermatoses.

Clinicopathological and Dermoscopic Baselines in Patients with Lynch Syndrome.

Despite the fact that Lynch Syndrome (LS) patients may also develop extra-colonic malignancies, research evaluating the association between LS and skin cancers is currently very limited. We performed a monocentric clinical and dermoscopic study involving 42 LS patients which referred to the Dermatology Unit for cutaneous screenings. In total, 22 patients showed a mutation in MLH1 and 17 patients a MSH2 mutation. Out of the entire cohort, 83% of LS patients showed brown hairs and 78% brown eyes, and the most frequent phototypes were III and II (respectively, 71.5% and 21%). A positive medical history for an internal malignancy was present in 36% of patients, with colon cancer as the most frequent malignancy in 60% of cases. A total of 853 cutaneous lesions have been analyzed: 47% of patients showed a total number of nevi > 10. The main observed dermoscopic features were a uniform reticular pattern (77% of patients), a mixed pattern (9% of patients) and a uniform dermal pattern (14% of patients). Eruptive cherry angiomas were present in 24% of cases, eruptive seborrheic keratosis in 26% and viral warts in 7% of cases; basal cell carcinoma was detected in 7% of cases. We have not found specific associations with specific skin manifestations, and the clinical and dermoscopic appearance of the pigmented lesions reflected the features present in the general population. To date, there are currently no guidelines for skin screening in LS patients. According to our study, there is insufficient evidence to ensure increased surveillance in LS patients; further studies with larger samples of patients are needed to better investigate dermatological and dermoscopic features in LS carriers.

Case report: Undifferentiated sarcoma with multiple tumors involved in Lynch syndrome: Unexpected favorable outcome to sintilimab combined with chemotherapy

Patients with Lynch syndrome are at an increased risk of developing simultaneous or metachronous tumors, while sarcomas have been occasionally reported. Sarcomas are generally not considered part of the common Lynch syndrome tumor spectrum. However, more and more studies and case reports suggested that sarcoma could be a rare clinical manifestation of Lynch syndrome, leading to new treatment strategies for sarcoma. We report the case of a 74-year-old male patient with Lynch syndrome who had rectal mucinous adenocarcinoma and prostate adenocarcinoma and then developed undifferentiated sarcoma of the left neck two years later. Mismatch repair deficiency (dMMR) was confirmed by immunohistochemical staining for the mismatch repair proteins MSH2, MSH6, MLH1 and PMS2. The result of polymerase chain reaction (PCR) microsatellite instability (MSI) testing of sarcoma showed high-level microsatellite instability (MSI-H). Additionally, a pathogenic germline mutation in MSH2 (c.2459-12A>G) was detected by next-generation sequencing (NGS). Taking into account HE morphology, immunohistochemical phenotype, MSI status, NGS result, medical history and germline MSH2 gene mutation, the pathological diagnosis of left neck biopsy tissue was Lynch syndrome related undifferentiated sarcoma with epithelioid morphology. The patient has been receiving immunotherapy (sintilimab) combined with chemotherapy (tegafur, gimeracil and oteracil potassium capsules) and currently has stable disease. We also reviewed the literature to understand the association between sarcoma and Lynch syndrome. Sarcoma may now be considered a rare clinical manifestation of Lynch syndrome. Attention and awareness about the association between Lynch syndrome and sarcoma need to be increased. Therefore, timely detection of MMR proteins and validation at the gene level for suspicious patients are the keys to avoiding missed or delayed diagnosis and to identifying patients suited for immunotherapy, which may also help to provide appropriate genetic counseling and follow-up management for patients.

PATH-12. DIFFUSE HEMISPHERIC GLIOMA, H3 G34-MUTANT: A COMPREHENSIVE SEQUENCING AND HIGH-RESOLUTION GENOME-WIDE COPY NUMBER ANALYSIS

Abstract Diffuse hemispheric glioma, H3 G34-mutant (DHG_H3G34) is a novel tumor type in the 2021 WHO CNS classification. We describe a comprehensive sequencing and high-resolution genome-wide copy number analysis of a series of cases clinically tested by a single laboratory (2018-2022). Cases included tumors from 47 unique patients (1 reportedly recurrent) that had an H3-3A G34 mutation detected using an 187-gene mutation and fusion targeted neuro-oncology NGS panel (n=47). A subset (n=18) of cases was also tested Oncoscan chromosomal microarray. Median age at testing was 20 years (range, 12-50). H3-3A G34 mutations included G34R (n=44; 94%), G34V (n=2) and a novel G34E variant considered to be likely clinically relevant (n=1). All DHG_H3G34 were hemispheric tumors, and one tumor was multifocal with midline involvement. Concurrent mutations recurrently involved ATRX (n=38), TERT promoter (n=3; mutually exclusive with ATRX mutations), TP53 (n=43), PDGFRA (n=26), PTEN (n=5), NF1 (n=4, all patients over 20 years), PIK3CA (n=4) and CDKN2A (n=3). The single recurrent tumor also had an MSH6 mutation, and in addition to 3 other cases, had increased number of sequence variants suggestive of a high tumor mutational burden. A single case also showed an FGFR3::FAM184B (exon 17::exon 2) fusion. All cases with available chromosomal microarray data had unbalanced genomes with multiple chromosomal gains/losses, and regions of copy-neutral loss of heterozygosity. The most frequent recurrent (at least 50% of cases) abnormalities were losses involving 3q, 4q, 10q, 13q and 18q, and 17p copy-neutral loss of heterozygosity encompassing TP53 (all but one case had concomitant TP53 mutation), a pattern reminiscent of IDH-mutant astrocytomas. Oncogene amplifications (PDGFRA and PIK3CA) were detected in 3 (of 18) cases. CDKN2A/B deletion was observed in 8 cases (2 homozygous). In conclusion, we describe additional sequence and copy-number abnormalities in DHG_H3G34, expanding the spectrum of genetic changes associated with this novel tumor type.

EPCO-02. GENOME-WIDE DNA METHYLATION PROFILING AND GENETIC CHARACTERIZATION OF LONG-TERM SURVIVAL IDH WILD-TYPE GLIOBLASTOMA PATIENTS

Abstract Glioblastoma multiforme (GBM) has a dismal outcome of approximately 12 months. Less than 5% of patients (long-term survivors-LTS) survives more than 5-years, including IDH-mutant gliomas. Nevertheless, the molecular fingerprint of LTS remains largely uncharted. DNA methylation (DNAm) is an epigenetic modification, altered in cancer and used to classify brain tumors. In this multicentric study sponsored by Alleanza Contro il Cancro (ACC) network, we aimed to molecularly characterize LTS vs standard survival GBM (CTR) by DNAm/Copy Number Variation (CNV) and mutation profiling. We analyzed 51 IDHwt GBM samples (27LTS/24CTR) by Illumina EPIC beadChip and AmpliSeq Comprehensive Cancer Panel. We ran methylation data through the brain tumor classifiers v11b4 and v12.5, performed multidimensional scaling analysis and investigated differentially methylated regions (DMR) correlating them with survival. According to v11b4, cases were classified as: 43 GBM IDHwt (22LTS/21CTR), 27 with a calibrated score >0.84 (16LTS); Plexus Tumor with low scores (2LTS/3CTR); 3 LTS samples did not match. A diagnostic refinement was observed by the v12.5, with 48 cases classifying as GBM IDHwt (25LTS/23CTR) and 35 (19LTS) with optimal score. The LTS group was more heterogeneous, with 4 cases matching in the pedHGG subtypes, including 2 cases in young adults, one with high tumor mutational burden and MSH6 mutation. No specific DNAm genomic profile was observed for LTS patients but we identified 18 significant DMRs, 4 associated with survival probability. At CNV analysis, most cases showed classical alterations (Chr7 gain with or w/o EGFR amplification, 9p21 loss, Chr10 loss) while pedHGG were associated with PDGFRA amplification, 1q gain, 13q loss. NGS identified at least 1 pathogenic variant in 40 cases (22CTR/18LTS) and showed a higher prevalence of mutation negative cases among LTS. No significant difference between the 2 groups was observed in terms of prevalence of mutated genes or number of VUS per sample.

Descriptive Statistics for Patients with Glioblastoma Associated with Germline Mismatch Repair Gene Mutation

<h3>Purpose/Objective(s)</h3> Hereditary Nonpolyposis Colorectal Cancer (HNPCC), also known as Lynch Syndrome, is a genetic mismatch repair mutation associated with increased risk for development of glioblastoma (GBM). There is a paucity of data describing the clinical outcomes of GBM patients with HNPCC. We aim to describe tumor characteristics, treatment patterns, and outcomes. <h3>Materials/Methods</h3> IRB approved retrospective review of consecutive patients with a diagnosis of both HNPCC and GBM at a single institution. <h3>Results</h3> Nine patients with a diagnosis of HNPCC and GBM were treated at a single institution from 2001-2022. Six patients were male. Median age at GBM diagnosis was 61 (range 21-84). Median follow up from the time of GBM diagnosis was 7.83 months. Eight patients had prior malignancies: n=7 with colon adenocarcinoma, n=1 with Burkitt lymphoma. Two patients had active synchronous cancers with GBM. The most common presenting symptoms were facial droop (n=3) and cognitive changes (n=2). Others include headache, gait change, visual field defect, and seizures with n=1 each. All patients underwent surgery: n=2 gross total resection, n=4 subtotal resection, and n=3 biopsy alone. There were 3 temporal lobe, 3 parietal lobe, 2 frontal lobe, and 1 frontoparietal lobe tumors. Two patients had multifocal disease at diagnosis. One patient had a giant cell variant GBM. Seven patients had MSH6 mutations and 2 had MSH2 mutations. Two patients progressed rapidly after surgery and were unable to receive RT. Seven patients received adjuvant radiation therapy (RT): n=5 received concurrent TMZ- 1 with concurrent bevacizumab, 5-fluorouracil, and leucovorin due to synchronous metastatic colon cancer, 1 received no chemotherapy. The most common dose and fractionation schedules were 60 Gy in 30 fractions (fx) (n=3), 40.05 Gy in 15 fx (n=3), and n=1 received 30 Gy in 10 fx due to poor performance status and large tumor volume. One patient received proton therapy for active MS, n=8 received IMRT. Two patients initiated tumor treating fields following RT completion. The median time to progression after RT was 3.35 months, and all progressed within the radiation field. Median survival from diagnosis was 6.5 months (range 1.2-15.5). No patients received immune checkpoint inhibitors. One patient was alive at the time of data collection. Seven died due to GBM progression, and n=1 died from progression of a synchronous malignancy. <h3>Conclusion</h3> In this small series, patients with mismatch repair mutation associated glioblastoma had especially poor outcomes and a rapidly progressive disease course.

Preliminary study on the molecular features of mutation in multiple primary oral cancer by whole exome sequencing.

Multiple primary cancers (MPCs) refer to cancers that occur simultaneously or metachronously in the same individual. The incidence of MPC has increased recently, as the survival time of malignant tumor patients has been greatly prolonged. It is difficult to differentiate MPC from primary cancers (PCs) in the same anatomical region from the clinical manifestation alone. However, their biological behaviors appear to be distinct. In this study, we show that the prognosis of multiple primary oral cancers (MP-OCs) is worse than primary oral cancers (P-OCs). To better understand the molecular mechanisms of MP-OC, we used whole exome sequencing (WES) to analyze samples from 9 patients with MP-OC and 21 patients with P-OC. We found more somatic mutations in MP-OC than in P-OC. MP-OC had more complicated mutation signatures, which were associated with age-related and Apolipoprotein B mRNA Editing Catalytic Polypeptide-like (APOBEC) activity-related signatures. Tumor mutational burden (TMB) and mutant-allele tumor heterogeneity (MATH) of MP-OC trended higher compared to P-OC. KEGG and GO analysis showed the differential pathways of MP-OC versus P-OC. In addition, MP-OC took amplification, not loss, as the main pattern of copy number variation (CNV), while P-OC took both. Lastly, we did not find significantly different mutant germline genes, but MSH-6 mutation may be a potential MP-OC driver. In short, our preliminary results show that MP-OC and P-OC have different molecular characteristics.

146P Miniature molecules and the Mammoth treatment changes in endometrial cancer

The ESGO/ESTRO/ESP guidelines for endometrial cancer published in November 2020 in the International Journal of Gynaecological Cancer gave us a trailblazing guide to steer our wheels in the precisely carved direction for the treatment of endometrial cancer, the commander being the molecular markers(TP53, MSH6, PMS2 and POLE mutation). Since the complete molecular profiling is not feasible in a low middle income country like India we started getting the tissues of endometrial carcinoma tested for TP53 mutation because its mutation can label even a low risk disease to high risk disease, worsening the treatment outcomes.

Fertility-sparing treatment for endometrial cancer and atypical endometrial hyperplasia in patients with Lynch Syndrome: Molecular diagnosis after immunohistochemistry of MMR proteins.

IntroductionLynch Syndrome (LS) represents the hereditary condition that is most frequently associated with endometrial cancer (EC). The aim of this study is to assess the presence of Lynch Syndrome (LS) in young women with mismatch repair (MMR)-deficient atypical endometrial hyperplasia (AEH) and non-myoinvasive FIGO G1 endometrioid EC and its possible impact on the outcome of conservative treatment.MethodsSix MMR-deficient cases identified from a previous cohort of 69 conservatively treated patients were selected to be screened for germline mutations in MMR genes. In each patient, the outcomes of conservative treatment for AEH and EEC, including response, relapse, progression, and pregnancy, were assessed.ResultsFive out of 6 patients underwent genetic test for LS. Three out of these 5 patients showed a positive genetic test. Patient 1 showed the c.942 + 2 T>A heterozygous variant of MSH2 mutation; after 12 months of complete response, she had relapse and progression of disease. Patient 4 showed the c.2459-1G>C variant of MSH2 mutation; after complete response, she failed to achieve pregnancy; she had relapse after 24 months and underwent hysterectomy. Patient 6 showed the c.803 + 1 heterozygous variant of PMS2 mutation; she had relapse of disease after 18 months from the first complete response and then underwent hysterectomy.ConclusionsIn this series, 3 out of 6 women with MMR-deficiency had LS. None of the patients achieved pregnancy, and those who responded to treatment had subsequent relapse of disease. Patients undergoing fertility-sparing treatment for atypical endometrial hyperplasia and endometrial cancer should perform MMR immunohistochemical analysis in order to screen LS.

Novel MSH6 mutation predicted metastasis in eyelid and periocular squamous cell carcinoma.

Our previous research revealed the relative local aggressiveness of eyelid and periocular squamous cell carcinoma (EPSCC), but its distinct genetic characteristics involved remain unknown. We conducted this study based on next-generation sequencing to identify the genetic distinctiveness of EPSCC and damaging mutations for possible aetiology and poor prognosis. We performed sequencing using a 556-gene panel (SmartOnco) in 48 EPSCCs. Cox hazards model was applied to explore mutated genes that increase the risk of metastasis and death. Pathogenesis of the mutations was predicted by sequence alignment algorithms. The most commonly mutated genes were KMT2C (N = 17, 35%), LRP1B (N = 14, 29%), KMT2D (N = 12, 25%), PTCH1 (N = 10, 21%) and TP53 (N = 10, 21%). DNA mismatch repair (MMR) genes (42%) like MSH6 (19%) and MLH3 (12%) were among the most frequently mutated genes. Cell cycle regulators including TP53 (21%) and CDKN2A (10%) were less frequently mutated than in other squamous cell carcinomas (SCCs). Ultraviolet exposure, MMR deficiency and ageing were the main aetiology. Of note, KMT2C has a deleterious mutation hotspot. Patients burdened with MSH6 mutation has a higher risk of overall metastasis (P = 0.045, HR = 5.165) and nodal metastasis (P = 0.022, HR = 14.038). Moreover, a hotspot mutation MSH6E52A brought an even higher risk of nodal metastasis (P = 0.011, HR = 18.745). EPSCCs displayed a unique mutation profile from cutaneous SCCs and mucosal SCCs. We have identified novel damaging mutations in epigenetic regulators like KMT2C boosted early onset of EPSCCs in addition to UVR, ageing or MMR deficiency. And malfunction of MMR genes worsened prognosis.

Identification of Lynch Syndrome in Patients with Endometrial Cancer Based on a Germline Next Generation Sequencing Multigene Panel Test.

Simple SummaryLynch Syndrome (LS) is a hereditary cancer syndrome caused by an autosomal dominant mutation in one of the DNA mismatch repair (MMR) genes. Understanding the clinical traits of endometrial cancer, which is one of the most frequent as well as the earliest diagnosed cancer types of LS-associated cancers, has the potential to offer tailored screening programs and lifesaving interventions. Whereas most large-scale studies of LS-associated endometrial cancers are based on cohorts in Western countries, studies from a predominantly Korean population are relatively few. We conducted a multi-center, retrospective study of patients with endometrial cancer who underwent germline multigene panel testing that included MMR genes. Based on the analysis of 204 patients with endometrial cancer who underwent germline multigene panel testing, we investigated the prevalence of LS and non-LS mutation and the relative contribution of each MMR gene mutation that, in composite, led to the clinical phenotype of LS-associated endometrial cancer patients in a predominantly Korean population.We aimed to investigate the prevalence and relative contributions of LS and non-LS mutations in patients with endometrial cancer in Korea. We retrospectively reviewed the medical records of 204 patients diagnosed with endometrial cancer who underwent a germline next generation sequencing multigene panel test covering MLH1, MSH2, MSH6, PMS2, and EPCAM at three tertiary centers. Thirty patients (14.7%) with pathogenic mutations (12 MLH1; 6 MSH2; 10 MSH6; 2 PMS2) and 20 patients (9.8%) with 22 unclassified variants (8 MLH1; 8 MSH2; 2 MSH6; 3 PMS2; 1 EPCAM) were identified. After excluding four close relatives of a proband, the prevalence of LS was 13.0% (26/200). Patients with LS were more likely than those with sporadic cancer to be younger at diagnosis (48 vs. 53 years, p = 0.045) and meet the Amsterdam II criteria (66.7 vs. 3.5%, p < 0.001). Non-endometrioid histology was more prevalent in patients with MSH6 or PMS2 mutations (41.7%) than those with MLH1 or MSH2 mutations (5.6%, p = 0.026). In this pre-selected cohort of endometrial cancer patients who underwent next generation sequencing, the prevalence of LS was 13%, thus supporting the use of gene panel testing for endometrial cancer patients.

A Human MSH6 Germline Variant Associated With Systemic Lupus Erythematosus Induces Lupus-like Disease in Mice.

ObjectiveTo determine if single‐nucleotide polymorphisms (SNPs) in DNA repair genes are enriched in individuals with systemic lupus erythematosus (SLE) and if they are sufficient to confer a disease phenotype in a mouse model.MethodsHuman exome chip data of 2499 patients with SLE and 1230 healthy controls were analyzed to determine if variants in 10 different mismatch repair genes (MSH4, EXO1, MSH2, MSH6, MLH1, MSH3, POLH, PMS2, ML3, and APEX2) were enriched in individuals with SLE. A mouse model of the MSH6 SNP, which was found to be enriched in individuals with SLE, was created using CRISPR/Cas9 gene targeting. Wildtype mice and mice heterozygous and homozygous for the MSH6 variant were then monitored for 2 years for the development of autoimmune phenotypes, including the presence of high levels of antinuclear antibodies (ANA). Additionally, somatic hypermutation frequencies and spectra of the intronic region downstream of the VHJ558‐rearranged JH4 immunoglobulin gene was characterized from Peyer's patches.ResultsBased on the human exome chip data, the MSH6 variant (rs63750897, p.Ser503Cys) is enriched among patients with SLE versus controls after we corrected for ancestry (odds ratio = 8.39, P = 0.0398). Mice homozygous for the MSH6 variant (Msh6 S502C/S502C ) harbor significantly increased levels of ANA. Additionally, the Msh6 S502C/S502C mice display a significant increase in the infiltration of CD68+ cells (a marker for monocytes and macrophages) into the lung alveolar space as well as apoptotic cells. Furthermore, characterization of somatic hypermutation in these mice reveals an increase in the DNA polymerase η mutational signature.ConclusionAn MSH6 mutation that is enriched in humans diagnosed with lupus was identified. Mice harboring this Msh6 mutation develop increased autoantibodies and an inflammatory lung disease. These results suggest that the human MSH6 variant is linked to the development of SLE.

Abstract 5219: Frequency of germline mutations in Mexican children with cancer

Abstract Childhood cancer is a leading cause of death by disease in children from 5 -14 ages, for whom there are no prevention strategies. Due to early-age of diagnosis and short-time environmental factors exposition, increasing evidence suggests childhood cancer could have stronger association with germline alterations in predisposition cancer genes. It has been estimated that up to 30% all childhood cancer diagnosis may be caused by germline variants, but their frequency and distribution remain unclear. Elucidating the prevalence and role of germline mutations in the development of childhood cancer is crucial for understanding its bases, causes, cancer-risk reduction plan, surveillance, and treatment. Here, we present a pilot study on cancer children’s patients and their relatives. A total of 40 Mexican-mestizo children with any cancer diagnosis were recruited from several Institutions of Health in Mexico and a sample of saliva or blood was obtained. Whole Exome Sequencing was performed and bioinformatic analysis was carried out following GATK best practices for germline genetic variants. Additionally, we evaluated a risk selection tool for recognition of genetic predisposition in pediatric cancer patients. Pathogenic or likely-pathogenic genetic variants (mutations) were identified from the application of a filtering chain based on ACMG guidelines, and they were subsequently validated by Sanger sequencing. Germline mutations in MSH6, NF1, MUTYH, CDKN2A, CHEK2, DICER1, FANCA and SBDS genes were identified in 5/40 (12.5%) patients with cancer, only 3 of 5 index cases (60%) reported family history of cancer and all patients meet at least one criterion of the risk selection tool. Two patients were carriers of one mutation, while in three patients were identified 2 pathogenic variants. Additionally, we identified four nonsense candidate genetic variants on EWSR1, FANCL, EPCAM and HRAS genes and two pathogenic variants in non-cancer predisposition gene, ABCA4. Our results show for the first time a higher frequency (12.5%) of germline mutations in cancer predisposition genes in Mexican-mestizo children with cancer. The lack of family history in two carriers showed the need to establish standardized suspicion criteria for identification of children with higher risk of cancer who can benefitfrom genetic testing. Further characterization of additional genetic variants identified without clinical classification will be important to evaluate their real contribution, as well as the influence of environmental factors on de novo mutation promotion. Citation Format: Oscar Alonso Luna, Jorge Meléndez Zajgla, Marta Zapata Tarres, Luis Enrique Juárez Villegas, Elvia Cristina Mendoza Caamal, Elianeth Rey Helo, Socorro Aida Borges Yanez, Gabriela Elisa Mercado Celis, Rodrigo Barquera Lozano. Frequency of germline mutations in Mexican children with cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5219.

Abstract 6262: Emergence of tumor mismatch repair deficiency and increased mutational burden in blood and tissue of metastatic colorectal cancer patients treated with temozolomide

Abstract The majority of metastatic colorectal cancers (mCRC) are mismatch repair (MMR) proficient (MMRp) and unresponsive to immunotherapy, while MMR deficient (MMRd) tumors often respond to immune checkpoint blockade (ICB). We previously reported that treatment of CRC preclinical models with temozolomide (TMZ) leads to MMR deficiency, increased tumor mutational burden (TMB) and, sensitization to immunotherapy. To clinically translate these findings, we designed the ARETHUSA clinical trial whereby O6-Methylguanine-DNA-methyltransferase (MGMT) deficient, MMR proficient and KRAS mutant mCRC patients receive priming therapy with TMZ. Analysis of solid tissue biopsies and circulating tumor DNA (ctDNA) obtained after TMZ treatment revealed the emergence of TMZ mutational signature, alterations in MMR genes and increased TMB in 14 out of 16 patients. Genetic mutations induced by TMZ were dose-dependent and multiple alterations in the nucleotide context favored by the TMZ signature emerged in MMR genes such as the MSH6 T1219I variant which was detected in ctDNA and tissue of 13/14 (93%) of the cases. A subset of the patients whose tumors after TMZ priming displayed the MSH6 mutation, the TMZ mutational signature and increased TMB, achieved disease stabilization upon pembrolizumab treatment. Overall, we provide proof-of-concept that treatment of MGMT deficient/MMR proficient KRAS mutant mCRCs with TMZ can be tracked by mutational signature analysis and lead to inactivation of the MMR pathway, emergence of the TMZ mutational signature, TMB increase, and, in some cases, to disease stabilization during ICB. Citation Format: Giovanni Crisafulli, Andrea Sartore-Bianchi, Luca Lazzari, Filippo Pietrantonio, Alessio Amatu, Marco Macagno, Ludovic Barault, Andrea Cassingena, Alice Bartolini, Paolo Luraghi, Gianluca Mauri, Paolo Battuello, Nicola Personemi, Valeria Pessei, Pietro Paolo Vitiello, Federica Tosi, Laura Idotta, Emanuele Valtorta, Emanuela Bonoldi, Giovanni Germano, Federica Di Nicolantonio, Silvia Marsoni, Salvatore Siena, Alberto Bardelli. Emergence of tumor mismatch repair deficiency and increased mutational burden in blood and tissue of metastatic colorectal cancer patients treated with temozolomide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6262.

HGG-61.Landscape of cancer predisposition in pediatric high-grade glioma

Abstract Current estimates indicate that approximately 8–10% of cancer incidence in young patients can be attributed to constitutional alterations in genes linked to tumor initiation. The rarity and highly heterogeneous nature of high-grade glioma (HGG) in children and adolescents requires coordinated efforts to capture the diversity of subtypes in a normal-tumor matched sequencing cohort. Combining sequencing data of two registries based at the Hopp Children's Cancer Center (KiTZ) Heidelberg (INFORM and MNP2.0) allowed us to investigate the landscape of constitutional alterations across 350 children with high-grade gliomas. Both cohorts independently showed a surprisingly high proportion of patients (17%) with constitutional pathogenic alteration variants in cancer predisposition genes (n=40/233 and 23/128). Our results confirm a high frequency of alterations affecting genes of the DNA mismatch repair (MMR) pathway in 44% of IDH-mutant tumors (n=12/27). Additionally, we observed an almost exclusive correlation of MSH6 constitutional variation and somatic mutation in the IDH1 gene (n=7/8). A hypermutator phenotype defined by a tumor mutational burden &amp;gt;10 mutations/mb) was linked in 14/17 patients to a constitutional alteration in one of the following MMR genes: MLH1, MSH2, MSH6 or PMS2, with IDH-wildtype cases mostly classified as ‘RTK1 subtype’ by DNA methylation analysis. Constitutional alterations in TP53 were found in 5% of the total cohort. Furthermore, these tumors occurred exclusively in the group of H3-/IDH-wildtype glioblastoma (12%), and 8/18 tumors belong to the pedHGG_MYCN subgroup (conversely, this represents 40% of all ‘MYCN subtype’ patients, a highly significant enrichment over other subtypes (p&amp;lt;0.001). The overall high percentage of constitutional alterations in pediatric HGG warrants human genetics counselling for all affected patients and their families. Additionally, the strong correlation of MSH6 and IDH mutations as well as Li-Fraumeni-Syndrome and MYCN positive signature should be further explored as this might open the avenue for much needed new therapeutic approaches.

Comparative analysis of microsatellite instability-high (MSI-H) BRAF V600E-mutated versus MSI-H BRAFwild type colorectal cancers (CRC), including tumor microenvironment (TME), associated genomic alterations, and immunometabolomic biomarkers.

3066 Background: The BRAFV600E mutation is associated with the hypermethylator phenotype CIMP, which can also lead to the MSI-H phenotype. BRAFV600E mutation and MSI-H/dMMR status seem to be biologically intertwined; however, the impact of coexisting BRAFV600E mutations on the TME and immunometabolomic features of MSI-H/dMMR CRC tumors is not well characterized. Methods: A retrospective review of deidentified records of patients with MSI-H/dMMR CRC tumors was conducted using next-generation sequencing data (Tempus |xT assay: DNA-seq of 595-648 genes at 500x coverage, and full transcriptome RNA-seq). Several immune markers of tumor immunogenicity in BRAF wild-type ( BRAFwt) vs. V600E-mutated (BRAFV600E) tumors were assessed, including tumor mutational burden (TMB), neoantigen tumor burden (NTB, ScanNeo), PD-L1 expression, immune infiltration, and canonical immuno-metabolomic pathways (82 geneset signatures). Results: A total of 459 MSI-H/dMMR CRC tumors were analyzed, of which 123 (27%) tumors harbored BRAFV600Emutations, and 336 (73%) were BRAFwt. MSI-H/dMMR BRAFV600E tumors were more frequently identified in females (69% vs. 55%; P= 0.01), non-Hispanic or non-Latino (100% vs. 73%; P =0.001), and older patients (median age: 76 yrs vs. 62 yrs; P&lt; 0.001). Compared to BRAFWT, BRAFV600E tumors exhibited significantly higher TMB (Median: 49 mut/MB vs. 36 mut/MB; P &lt; 0.001) and were more frequently associated with TMB-High status (&gt; 10 mut/MB; 100% vs. 95%; P = 0.008); however, no significant differences were observed with tumor NTB, immune score, or T cell infiltration (CD4 or CD8). NK cell infiltration was higher in the BRAFV600Ecohort (&lt; 0.001). When compared to BRAFWT tumors , BRAFV600E tumors harbored a greater frequency of mutations in MSH6(42% vs. 20%), B2M (33% vs. 16%), BRCA2 (31% vs. 12%), ATM (23% vs. 12%), and TP53 (30% vs. 19%) but a lower frequency of MSH2 (3.3% vs. 11%), all P&lt; 0.05. Pathway enrichment analysis identified 10 significantly altered signaling pathways, most of which related to stromal/immune cell signaling and metabolism. Five were upregulated among BRAFV600E tumors: glycerophospholipid, galactose, cyclin-dependent cell signaling; Nucleotide, and TH1 inflammation. Five pathways were downregulated (Wnt, Notch, TH17 inflammation, amino sugar, and cancer stem cell signaling). Conclusions: MSI-H/dMMR BRAFV600E CRCs undergo broad metabolic reprogramming (e.g., glycerophospholipidgalactose, nucleotide). A rise in lipid metabolism, particularly with NK inflammation, suggests that BRAFV600Emutated tumors may be associated with a non-classical immune component. BRAFV600E and BRAFwt CRCs exhibited similar NTB and T cell infiltration, suggesting that both are likely to benefit from immune checkpoint inhibitors.

Clinical characteristics of patients with PMS2 mutations.

10610 Background: Lynch syndrome is a hereditary cancer predisposition syndrome caused by mutations in mismatch repair genes, MLH1, MSH2, MSH6, and PMS2. The cancer risks and clinical presentation of PMS2 associated Lynch syndrome is not well defined. This study outlines the characteristics of patients with PMS2 mutations identified in a large academic center. Methods: Patients with a pathogenic or likely pathogenic PMS2 variant identified between June 1, 2009 and Dec 31, 2021 were selected from a database at the Nancy and James Grosfeld Cancer Genetics Center at Beaumont Health. Data on demographics, cancer type and molecular testing were retrospectively analyzed. Results: A total of 92 patients from 61 families were found to carry a pathogenic or likely pathogenic variant in PMS2. The mean age at testing was 54 (19 to 95). A majority (50) of the family were Caucasian, while the rest were African American (2), Middle Eastern (2), Multiracial (8). The most common variant was c.137G &gt; T (p.Ser46Ile), seen in twelve of 61 families. Forty-two patients (45.6%) had a personal history of cancer, with a mean age at diagnosis of 63. Of these forty-two patients, four had multiple malignancies, including one patient with six separate cancers. The most common malignancies were breast (16), colon (12), followed by uterine cancer (8), pancreatic (4), prostate (3) and other cancers. The mean age of diagnosis of breast cancer was 56 (41 to 78), colon cancers was 57 (32 to 75), uterine cancer 59 (50 to 72). Tumor IHC was performed in thirteen cases and 11 demonstrated loss of PMS2 protein expression. MSI was performed in 8 cases and 5 were unstable and 3 were stable. Conclusions: This study reveals the the clinical spectrum of cancers in PMS2 related Lynch syndrome. The most common cancers were breast, colon and uterine cancer, with older age of diagnosis. There was evidence of a more severe phenotype, exemplified by a patient with six cancers, suggesting higher penetrance. Further research is needed to better characterize the clinical presentation of PMS2 related Lynch syndrome.

Genomic landscape of SMARCA4-deficient lung tumors by clinical RNA sequencing.

3118 Background: SMARCA4-deficient lung cancer is an undifferentiated lung cancer subtype associated with poor prognosis and morphological features that make them challenging to distinguish from sarcoma. These tumors are known to be resistant to standard of care surgery, radiation, and chemotherapy. Recent case reports suggest that these tumors might be resistant to immunotherapy. An assessment of the genomic and transcriptomic features of SMARCA4-deficient thoracic tumors may identify potential novel targets and treatment strategies for this new WHO lung cancer classification. Methods: We retrospectively analyzed de-identified NGS data from 8,484 thoracic formalin-fixed, paraffin-embedded tumor biopsies from lung cancer patients sequenced using the Tempus|xT solid tumor assay (DNA-seq of 595-648 genes at 500x coverage; whole-exome capture RNA-seq). Tumor-normal match sequencing was performed for all tumors, enabling the detection of incidental germline alterations across 46 genes. SMARCA4-deficiency was defined as tumors with a pathogenic or likely pathogenic SMARCA4 single nucleotide variant, insertion/deletion, or copy number alteration. Statistical significance was determined using Fisher's exact test and Wilcoxon rank-sum tests. Results: SMARCA4-deficiency was detected in 370 (4.4%) tumors, of which over 80% were stage III or IV. SMARCA4-deficient tumors included more male patients (63% vs 49%, p &lt; 0.001) and younger age at diagnosis (median 64 vs 68 years, p &lt; 0.001). There were more patients with high tumor mutational burden (TMB-H, ≥10 mutations per megabase) (34% vs 15%, p &lt; 0.001), and fewer patients with positive PD-L1 immunohistochemical staining (44% vs 54%, p = 0.009) compared to SMARCA4 wild-type tumors. Microsatellite instability status occurred at similar low frequencies across SMARCA4-deficient vs wild-type tumors (0.8% vs 0.5%, p = 0.5). SMARCA4-deficient tumors showed enrichment for somatic mutations in TP53 (71% vs 47%, q &lt; 0.001), STK11 (22% vs 6.8%, q &lt; 0.001), KEAP1 (15% vs 4.2%, q &lt; 0.001), and CDKN2A (15% vs 5.9%, q &lt; 0.001) compared to wild-type. Tumor normal-match sequencing identified incidental germline mutations in MUTYH (2.2%), ATM (1.1%), ATP7B (0.5%), and MSH6 (0.5%) for SMARCA4-deficient tumors. RNA-sequencing analysis confirmed reduced transcriptional expression of SMARCA4 (p &lt; 0.001), CD274 (PD-L1; p &lt; 0.001), TNFRSF18 (p &lt; 0.001), and TNFRSF4 (p = 0.035) in deficient tumors vs wild-type. Furthermore, SMARCA4-deficient tumors revealed reduced infiltration of CD4+ T cells (19% vs 22%, p &lt; 0.001). Conclusions: This study reveals the unique genomic and transcriptional characteristics of SMARCA4-deficient lung tumors. Further studies are needed to assess the impact of immunotherapies and targeted therapies among this patient population.