Mismatch Repair Mutations Research Articles

INNV-06. ADULT GLIOBLASTOMA WITH MISMATCH REPAIR DEFICIENCY FORMS A DISTINCT HIGH-RISK MOLECULAR SUBGROUP FOR WHICH TREATMENT WITH IPILIMUMAB-NIVOLUMAB SHOWS SURVIVAL ADVANTAGE

Abstract Glioblastoma is the most frequent and malignant primary brain tumor. Although the survival is generally dismal for glioblastoma patients, risk stratification and the identification of high-risk subgroups is important for prompt and aggressive management. The G1-G7 molecular subgroup classification based on the MAPK pathway activation has offered for the first time a non-redundant, all-inclusive classification of adult glioblastoma. Five patients from the large, 220-patient prospective cohort showed germline mutations in mismatch repair (MMR) genes and significantly worse median survival (3.25 months post-surgery) than those from other subgroups or from the rest of the cohort adjusted for age. These tumors were assigned to a new subgroup, G3/MMR, a spin-off from the major G3/NF1 subgroup, as they generally show genomic alterations leading to RAS activation. An integrated clinical, histological, immunohistochemical and molecular analysis of the G3/MMR tumors showed distinct characteristics as compared to other glioblastomas, including those with iatrogenic high tumor mutation burden (TMB), warranting a separate subgroup. Among these, prior history of cancer, midline location or multifocality, presence of multinucleated giant cells, positive p53 and MMR immunohistochemistry, and specific molecular characteristics, including high TMB, alert to this high-risk G3/MMR subgroup. High TMB constitutes the criterium for treatment with immune checkpoint inhibitors in solid cancers. The FDA-approved first-line therapy for advanced non-small cell lung cancer and high-TMB colorectal cancer is with the dual ipilimumab-nivolumab regimen. One of the five G3/MMR subgroup patients received the dual ipilimumab-nivolumab regimen and survived much longer than the rest of the G3/MMR patients, setting a proof-of-principle example for the treatment of these very aggressive G3/MMR glioblastomas.

BIOM-64. EXPLOITING CELL-FREE DNA TO DISSECT MECHANISMS OF RESISTANCE TO TEMOZOLOMIDE IN GLIOBLASTOMA CELLS

Abstract Temozolomide (TMZ) is a cytotoxic alkylating agent that serves as a cornerstone in glioblastoma (GBM) treatment, yet is notorious for eliciting hypermutation in recurrent tumors that have developed drug resistance by downregulating mismatch repair (MMR). We reasoned that cell-free DNA (cfDNA), a non-invasive measure for detecting tumor-derived biomarkers, could be used to temporally examine the mechanism of resistance to TMZ. We therefore designed an in-vitro experiment using the patient-derived GBM43 cell line. Over five days, batches of cells were treated with either TMZ or vehicle. Cell counts and cfDNA were collected every 24 hours. We observed an associated increase in both dead cell counts and cfDNA yield in treated cells compared to untreated cells. In TMZ-treated samples, cfDNA showed a periodic fragmentation pattern consistent with internucleosomal cleavage and TMZ-induced apoptosis. Intriguingly, higher numbers of variants were found in the cfDNA of untreated samples when compared to treated cfDNA, possibly reflecting TMZ-induced selection of resistant subclones in treated samples. This was further supported by the finding that the average variant allele frequency (VAF) increased over time in TMZ-treated samples, consistent with purifying selection. Conversely, cfDNA from vehicle-treated samples demonstrated a decrease in the average VAF over time, consistent with clonal diversification. Although we did not observe an enrichment of the TMZ-associated mutational signature SBS11, we did find higher levels of defective MMR mutation signature SBS44 in treated samples, potentially indicating the selection of subclones with defective MMR mechanisms. Overall, our preliminary findings show that cfDNA can be used to resolve mechanisms of TMZ resistance over time. We are addressing open questions by performing longer treatment times, testing other patient-derived cell lines, and performing murine xenotransplantation experiments to test TMZ-associated hypermutation in vivo. Our work will contribute to our understanding of TMZ resistance, and importantly push the development of cfDNA-based biomarkers for measuring TMZ-sensitivity in patients.

EXTH-23. NEW DNA-CROSSLINKING CHEMOTHERAPY IS EFFECTIVE AGAINST POST-TEMOZOLOMIDE MISMATCH REPAIR-DEFICIENT PATIENT-DERIVED HYPERMUTANT GLIOMAS

Abstract The DNA mismatch repair (MMR) pathway triggers glioblastoma (GBM) apoptosis when base mismatches induced by the alkylating agent temozolomide (TMZ) cannot be repaired. GBMs often develop hypermutation and resistance to TMZ through acquired inactivation of DNA MMR enzymes (MSH2, MSH6, MLH1, PMS2). A newly developed therapeutic, KL-50, fluoro-ethylates DNA bases, leading to DNA inter-strand cross-links. This form of DNA damage triggers MMR-independent apoptosis. Thus, KL-50 can target MMR-deficient tumor cells. Previous work showed that KL-50 is effective against GBM cell lines that were rendered TMZ-resistant through shRNA knockdown of MMR genes (PMID 35901163). In those studies, KL-50 was most effective when used in combination with an MGMT enzyme inhibitor, or in MGMT-deficient cells. Here, we extend those findings with a preclinical trial of KL-50 in GBM patient-derived xenografts (PDX), including PDX models of post-TMZ GBM that acquired MMR mutations and hypermutation through serial TMZ exposure (PMID 31852831). Among TMZ-naïve PDX, KL-50 extended survival of engrafted mice by 24 days for GBM6 (partially MGMT-deficient), and by 38 days for GBM12 PDX (fully MGMT-deficient) compared to vehicle (p<0.0001, Log-Rank). Combining KL-50 with 4.0 Gy radiation further extended survival of GBM12 mice (9 days, p=0.02), but not GBM6 mice. With engraftments of GBM6R-m185, a post-TMZ, MMR-deficient derivative of GBM6, 14/14 (100%) of KL-50 treated mice are still alive 60 days post-engraftment compared to 0/13 (0%) of vehicle control mice (median survival 37 days, p<0.0001, experiment ongoing). Complementary in vitro studies showed that GBM6R-m185 cells are more sensitive to KL-50 than TMZ at matched concentrations of 50µM (p=0.001, one-way ANOVA) and 100µM (p=0.047). These data demonstrate the potential of KL-50 in treating post-TMZ MMR-deficient recurrent gliomas.

Assessing Influence of Mismatch Repair Mutations on Survival in Patients After Resection of Pancreatic Ductal and Periampullary Adenocarcinoma.

Background: Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths in the United States. Previous studies have indicated that microsatellite instability and deficient mismatch repair (MMR) may be associated with improved survival in patients with pancreatic cancer. Here, we aim to investigate the impact of deficient MMR (dMMR) status on oncologic outcomes in patients after resection of PDAC and periampullary adenocarcinoma. Methods: This is a single-institution, retrospective study based on a prospectively maintained database. Pancreatic ductal adenocarcinoma (N = 342) and periampullary adenocarcinoma patients (N = 76) who underwent pancreatic resection surgery between 2016 and 2021 were included. Immunohistochemistry staining results of MMR proteins and next-generation sequencing data were recorded. Cancer-type dependent Cox regression analyses were performed to assess overall and disease-free survival, which was complemented with a 1:2 propensity-score matching for each of the cancer types in order to compare oncologic outcomes. Results: A total of 418 pancreatic cancer patients were included in the analysis. Fifteen patients (3.5%) were diagnosed as dMMR (PDAC N = 7 and periampullary adenocarcinoma N = 8). Cox regression modeling of dMMR status interaction with TNM staging and cancer type revealed that dMMR status strongly improves overall survival (p < 0.05). After propensity-score matching, Cox regression identified dMMR status as a significant marker of improved overall survival (HR = 0.27, 95%CI 0.09-0.88, p = 0.029). Conclusions: Overall, our findings suggest that dMMR status is associated with markedly improved survival outcomes in patients after resection of pancreatic and periampullary cancer. Future large-scale studies are needed to further validate this finding.

Solitary Loss of PMS2 in a patient with Colon carcinoma

Abstract Introduction/Objective In the United States, there are about 150,000 new cases of Colorectal cancer (CRC) and about 50,000 CRC related deaths each year. Hereditary nonpolyposis colorectal cancer (Lynch Syndrome) accounts for 2% - 4% of CRC. It is characterized by germline mutations in mismatch repair (MMR) genes - MLH1, MSH2, MSH6 and PMS2. Patients with Lynch syndrome have up to an 80% lifetime risk (LTR) of CRC and up to 60% LTR of endometrial cancer. Lynch syndrome patients also have an associated LTR of ovarian cancer (38%), upper urologic tract cancer (18%), gastric cancer (13%), small bowel cancer (6%), hepatobiliary tract cancer (4%), and brain tumors (3% - usually glioblastoma). Methods/Case Report A 76-year-old man with history of moderately differentiated colorectal adenosarcoma involving the transverse colon and a recently diagnosed grade group 5 (Gleason 4 + 5) prostate adenocarcinoma, currently being managed at an outside hospital. The case was sent to UAB for DNA MMR/microsatellite instability (MSI) status evaluation testing. Immunostaining showed loss of PMS2 and intact expression of MLH1, MSH2 and MSH6. These results were confirmed by the IdyIIa MSI assay which detected microsatellite instability in 5 of 7 markers and negative for BRAF V600E variant confirming microsatellite instable tumor. Results (if a Case Study enter NA) NA Conclusion Solitary loss of PMS2 in colonic adenocarcinoma is uncommon and may be caused by an underlying germline mutation of MLH-1 or PMS2, thus could represent Lynch Syndrome. Our patient’s age doesn’t favor Lynch syndrome. Next step would be to perform MLH-1 promoter methylation to confirm sporadic nature of carcinoma. Somatic methylation of the MLH1 promoter occurs in approximately 80% of CRC with defective MMR. Prostate cancer is not currently considered a part of Lynch syndrome.

Human DNA polymerase ε is a source of C>T mutations at CpG dinucleotides

C-to-T transitions in CpG dinucleotides are the most prevalent mutations in human cancers and genetic diseases. These mutations have been attributed to deamination of 5-methylcytosine (5mC), an epigenetic modification found on CpGs. We recently linked CpG>TpG mutations to replication and hypothesized that errors introduced by polymerase ε (Pol ε) may represent an alternative source of mutations. Here we present a new method called polymerase error rate sequencing (PER-seq) to measure the error spectrum of DNA polymerases in isolation. We find that the most common human cancer-associated Pol ε mutant (P286R) produces an excess of CpG>TpG errors, phenocopying the mutation spectrum of tumors carrying this mutation and deficiencies in mismatch repair. Notably, we also discover that wild-type Pol ε has a sevenfold higher error rate when replicating 5mCpG compared to C in other contexts. Together, our results from PER-seq and human cancers demonstrate that replication errors are a major contributor to CpG>TpG mutagenesis in replicating cells, fundamentally changing our understanding of this important disease-causing mutational mechanism.

Exploring the Relationship Between Gene Expression and Low-Frequency Somatic Mutations in Arabidopsis with Duplex Sequencing.

Intragenomic mutation rates can vary dramatically due to transcription-associated mutagenesis or transcription-coupled repair, which vary based on local epigenomic modifications that are nonuniformly distributed across genomes. One feature associated with decreased mutation is higher expression level, which depends on environmental cues. To understand the magnitude of expression-dependent mutation rate variation, we perturbed expression through a heat treatment in Arabidopsis thaliana. We quantified gene expression to identify differentially expressed genes, which we then targeted for mutation detection using duplex sequencing. This approach provided a highly accurate measurement of the frequency of rare somatic mutations in vegetative plant tissues, which has been a recent source of uncertainty. Somatic mutations in plants may be useful for understanding drivers of DNA damage and repair in the germline since plants experience late germline segregation and both somatic and germline cells share common repair machinery. We included mutant lines lacking mismatch repair (MMR) and base excision repair (BER) capabilities to understand how repair mechanisms may drive biased mutation accumulation. We found wild-type (WT) and BER mutant mutation frequencies to be very low (mean variant frequency 1.8 × 10-8 and 2.6 × 10-8, respectively), while MMR mutant frequencies were significantly elevated (1.13 × 10-6). Interestingly, in the MMR mutant lines, there was no difference in the somatic mutation frequencies between temperature treatments or between highly versus lowly expressed genes. The extremely low somatic variant frequencies in WT plants indicate that larger datasets will be needed to address fundamental evolutionary questions about whether environmental change leads to gene-specific changes in mutation rate.

Lynch Syndrome-associated Genomic Variants.

Lynch Syndrome, a hereditary disorder characterized by germline mutations in mismatch repair (MMR) genes, is a major contributor to colorectal cancers. It has also been identified in endometrial cancer. Despite the established role of MMR deficiency in tumorigenesis, the specific genomic alterations driving Lynch syndrome-associated endometrial cancer, and their overlap with colorectal cancer, remain incompletely understood. This study aims to fill this gap by performing a detailed comparative analysis of germline and somatic mutations in endometrial cancer within the context of Lynch syndrome. We conducted whole exome sequencing on matched germline and somatic DNA from 13 patients diagnosed with Lynch syndrome-associated endometrial cancer. High-depth sequencing was performed, followed by rigorous bioinformatics analysis to identify and annotate variants, focusing on their potential pathogenicity and relevance to both endometrial and colorectal cancer. Our analysis revealed 1,118 germline and 14,051 somatic variants, with 493 variants common to both. Recurrent pathogenic mutations in MLH1, MSH2, and MSH6 were confirmed, highlighting their critical role in Lynch syndrome. Notably, frequent somatic mutations in the PIK3CA and PTEN genes were identified, implicating the PI3K/AKT/mTOR pathway as a key oncogenic driver in these cancers. Additionally, novel somatic mutations in genes related to the extracellular matrix such as FBN1 and SPARC were uncovered, suggesting a possible unique role in endometrial tumor progression. This study provides new insights into the molecular basis of Lynch syndrome-associated endometrial cancer, emphasizing the overlap in oncogenic pathways with colorectal cancer. The discovery of shared and unique genetic mutations highlights the importance of developing combined treatment strategies and suggests that targeting these specific mutations could improve therapy for patients with Lynch syndrome-associated cancers.

Lynch syndrome-associated and sporadic microsatellite unstable colorectal cancers: different patterns of clonal evolution yield highly similar tumours.

Microsatellite unstable colorectal cancer (MSI-CRC) can arise through germline mutations in mismatch repair (MMR) genes in individuals with Lynch syndrome (LS), or sporadically through promoter methylation of the MMR gene MLH1. Despite the different origins of hereditary and sporadic MSI tumours, their genomic features have not been extensively compared. A prominent feature of MMR-deficient genomes is the occurrence of many indels in short repeat sequences, an understudied mutation type due to the technical challenges of variant calling in these regions. In this study, we performed whole genome sequencing and RNA-sequencing on 29 sporadic and 14 hereditary MSI-CRCs. We compared the tumour groups by analysing genome-wide mutation densities, microsatellite repeat indels, recurrent protein-coding variants, signatures of single base, doublet base, and indel mutations, and changes in gene expression. We show that the mutational landscapes of hereditary and sporadic MSI-CRCs, including mutational signatures and mutation densities genome-wide and in microsatellites, are highly similar. Only a low number of differentially expressed genes were found, enriched to interferon-γ regulated immune response pathways. Analysis of the variance in allelic fractions of somatic variants in each tumour group revealed higher clonal heterogeneity in sporadic MSI-CRCs. Our results suggest that the differing molecular origins of MMR deficiency in hereditary and sporadic MSI-CRCs do not result in substantial differences in the mutational landscapes of these tumours. The divergent patterns of clonal evolution between the tumour groups may have clinical implications, as high clonal heterogeneity has been associated with decreased tumour immunosurveillance and reduced responsiveness to immunotherapy.

Enhanced Commendable Sensitivity and Specificity for MSI in Colorectal Cancer by a New PCR-HRM Based 8-Loci MSI Assay.

This study evaluated the performance of the PCR-HRM assay by comparing it with immunohistochemistry (IHC) for mismatch repair (MMR) proteins and the PCR capillary electrophoresis (PCR-CE) methods. A total of 224 patients with colorectal cancer participated in the study, with nearly half having mismatch repair deficiency (dMMR) tissues and the remainder possessing pMMR tissues. There was a 97.77% concordance between the PCR-HRM assay and IHC, and a 97.56% concordance between PCR-HRM and the PCR-CE assay. In comparison with IHC for dMMR proteins, the PCR-HRM demonstrated a sensitivity of 96.36% and a specificity of 99.12%. When juxtaposed with the PCR-CE assay, its sensitivity was 98.96% and specificity stood at 96.33%. The mutations observed in the microsatellite loci were uniformly distributed across all eight loci. Discrepant outcomes were more frequent in instances of MLH1 and PMS2 deficiency. Furthermore, the germline mutation status of MLH1, MSH2, PMS2, and MSH6 in 62 patients was ascertained using next-generation sequencing. All patients displaying MMR gene pathogenic mutations (N = 14) were identified as MSI-H by PCR-HRM, whereas those with MSS tissues (N = 43) did not exhibit MMR gene pathogenic mutations. Thus, the PCR-HRM method proficiently pinpoints tumors with verified germline MMR mutations, indicative of Lynch syndrome. Conclusively, the PCR-HRM assay emerges as a swift and congruent diagnostic tool for microsatellite instability, boasting commendable sensitivity and specificity in colorectal cancer.

Identification of DNA Repair-related Genes as Biomarkers Reflecting MSI, TMB, and TIL in Colorectal Cancer.

There are no established biomarkers for immune checkpoint inhibitors (ICI) in colorectal cancer (CRC) with microsatellite stability (MSS) or proficient mismatch repair (pMMR). Therefore, this study aimed to identify biomarkers for ICI benefit in patients with pMMR by analyzing the down-regulated DNA repair-related genes involved in highly immunogenic and immune responses, and comparing their expression levels and clinical features. Mismatch repair (MMR), tumor-infiltrating lymphocytes (TIL), and tumor mutation burden (TMB) were evaluated in 13 CRC cases and mRNA expression levels of 95 DNA repair-related genes were measured. DNA repair-related genes with reduced mRNA expression in the high immunogenicity and high immune response groups were identified. Then, the mRNA expression levels of the identified DNA repair-related genes were measured in 135 patients with CRC. Hierarchical cluster analysis was performed using the mRNA expression levels to compare the clinicopathological characteristics of each cluster. ATR, LIG4, and RAD52 mRNA levels were significantly down-regulated in the high immunogenicity group. GADD45B, SMUG1, and XRCC6 mRNA levels were significantly down-regulated in the high immune response group. Cases in the cluster with reduced mRNA expression of the six genes were pMMR cases. CD8 mRNA expression level was higher in this cluster than in the other clusters. Decreased mRNA expression levels of ATR, LIG4, RAD52, GADD45B, SMUG1, and XRCC6 genes were associated with high cytotoxic T cell and TMB levels, suggesting that these genes could serve as biomarkers for ICI efficacy in pMMR cases.

Homopolymer switches mediate adaptive mutability in mismatch repair-deficient colorectal cancer

Mismatch repair (MMR)-deficient cancer evolves through the stepwise erosion of coding homopolymers in target genes. Curiously, the MMR genes MutS homolog 6 (MSH6) and MutS homolog 3 (MSH3) also contain coding homopolymers, and these are frequent mutational targets in MMR-deficient cancers. The impact of incremental MMR mutations on MMR-deficient cancer evolution is unknown. Here we show that microsatellite instability modulates DNA repair by toggling hypermutable mononucleotide homopolymer runs in MSH6 and MSH3 through stochastic frameshift switching. Spontaneous mutation and reversion modulate subclonal mutation rate, mutation bias and HLA and neoantigen diversity. Patient-derived organoids corroborate these observations and show that MMR homopolymer sequences drift back into reading frame in the absence of immune selection, suggesting a fitness cost of elevated mutation rates. Combined experimental and simulation studies demonstrate that subclonal immune selection favors incremental MMR mutations. Overall, our data demonstrate that MMR-deficient colorectal cancers fuel intratumor heterogeneity by adapting subclonal mutation rate and diversity to immune selection.

HGG-26. MLH1, MSH2, MSH6, AND PMS2 IMMUNOHISTOCHEMISTRY REPRESENTS A HIGHLY SENSITIVE SCREENING METHOD FOR MISMATCH REPAIR DEFICIENCY SYNDROMES IN PEDIATRIC HIGH-GRADE GLIOMA

Abstract BACKGROUND Pediatric central nervous system (CNS) tumors can occur as first manifestations of cancer predisposition syndromes (CPS) resulting from pathogenic variants in DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6, and PMS2. Early detection of MMR deficiencies (MMRD) may warrant the therapeutic use of checkpoint inhibitors and enable genetic consulting for the patient’s families. METHODS We prospectively screened 128 pediatric high-grade gliomas (pedHGG) for MMR protein expression by immunohistochemistry (IHC) as part of the HIT-HGG-2013 clinical trial. We compared IHC results to independently collected patient information on CPS including MMRD to test the method’s efficiency in screening for Lynch syndrome and Constitutional MMRD syndrome CMMRD. RESULTS From 128 successfully tested tumors, ten cases (10/128, 7.8%), showed loss of expression of at least one of the MMR proteins. In seven of the ten affected patients (7/128, 5.5%), genetic testing uncovered heterozygous (6/10) or homozygous (1/10) pathogenic germline variants in MMR genes (MLH1, MSH2 or MSH6) confirming the diagnosis of Lynch syndrome respectively CMMRD. In three cases (3/128 2.3%), MMR alterations were restricted to tumor tissue. The group of diffuse midline gliomas, H3 K27-altered, (67/128, 52.3%) did not show MMR alterations, while either somatic or germline MMR mutations were detected in two of eight patients with diffuse hemispheric glioma, H3 G34-mutant. In 88 patients (88/128, 68.8%) either clinical or molecular-genetic information on CPS could be provided. All ten patients with signs of MMRD were successfully detected by IHC from tumor tissue. CONCLUSIONS IHC for MMR proteins represents a highly sensitive screening method for MMRD in pedHGG. Considering the occurrence of at least 11.5% (7/61) of MMRD in pediatric patients with diffuse high-grade glioma excluding DMG, MMRD IHC should be part of routine diagnostics as cost-effective, broadly available and robust screening method.

Molecular alterations in claudin 18 suppressed and non-suppressed gastric adenocarcinomas to guide targeted therapies

ABSTRACT Background Gastric adenocarcinoma represents an aggressive type of cancer and an important cause of cancer mortality. Progress in gastric cancer therapeutics has resulted from a better understanding of the molecular pathogenesis of the disease and introduction of targeted therapies, but most gastric cancer patients still rely on non-targeted chemotherapy as the mainstay of treatment for advanced disease. Methods An analysis of publicly available series from The Cancer Genome Atlas (TCGA) gastric cancer cohort was undertaken to delineate the clinical and genomic landscape of gastric cancers with suppressed expression of claudin 18 compared with cancers with non-suppressed claudin 18. Claudin 18 suppressed cancers were defined as having an mRNA expression z-score relative to normal samples (log RNA Seq V2) of less than −1. Claudin 18 non-suppressed cancers were defined as having an mRNA expression z-score relative to normal samples (log RNA Seq V2) above 0.5. Results Gastric cancers with claudin 18 mRNA suppression represented 7.7% of the gastric adenocarcinomas of TCGA cohort, while non-suppressed cancers represented 46.6% of the cases. The two groups did not differ in clinical and genomic characteristics, such as mean age, histology, grade, and stage. The mutation landscape of claudin 18 suppressed cases included high mutation rates of TP53, of genes of the WNT/β-catenin pathway and of ubiquitin ligase FBXW7. Moreover, a subset of both claudin 18 suppressed and non-suppressed cancers displayed mutations in Mismatch Repair (MMR) associated genes or a high tumor mutation burden (TMB). At the mRNA expression level, claudin 18 suppressed gastric cancers showed up-regulation of EMT core transcription factor Snail 2 and down-regulation of genes of HLA cluster. The survival of gastric cancer patients with claudin 18 mRNA suppression was not significantly different compared with patients with non-suppressed claudin 18. Conclusion Sub-sets of gastric cancers with claudin 18 mRNA suppression displayed characteristics of potential therapeutic interest, such as mutations in WNT and PI3K pathways and MMR defects. These may guide the development of alternative targeted therapies, in this sub-set of gastric cancers which are not candidates for claudin 18 targeting therapies.

The Validity of Immunohistochemistry in Detecting Microsatellite Instability in Pediatric Solid Neoplasms

Abstract Background: The DNA mismatch repair (MMR) is the biological pathway that plays a key role in maintaining genomic stability during DNA replication and recombination. The value of MMR pathway is under investigation in pediatrics’ solid tumors. Aims: In this research work, we investigated the proteins involved in the oncogenesis of pediatric solid neoplasms and detect these proteins in a representative cohort sample of Saudi pediatric cases under the bioinformatic networking technique. We also described the MLH1, BRAF, p53, proliferating cell nuclear antigen, and PMS2 along with MSH2-MSH6 antibodies to be a diagnostic immunohistochemistry (IHC) panel for identifying MMR mutations. This research will open the new doors for advanced research on proteins involved in the oncogenesis of pediatric solid neoplasms. The hypotheses were tested on a sample of solid malignancies and IHC results were reported. Material and Methods: The study was conducted in different institutions in Saudi Arabia. The inclusion criteria required enrolling biopsies of solid neoplasms or resected solid malignant neoplasms presented to the laboratories in the participating institutions of all pediatric patients (aging from 0 to 14 years). The specimens were examined microscopically utilizing Hematoxylin and Eosin stain as well as the utilization of MMR proteins immunohistochemistry (IHC), and PNCA. Results: The qualitative assessment showed that IHC diagnosis yielded positive results with ≥80% of positive cells (intact) for MMR proteins (MSH2, MSH6, PMS2, and MLH1). The PCNA protein was absent only in vaginal germ cell tumor and metastatic medulloblastoma. Conclusion: In our sample, we have found that there is an intact MMR proteins expression. Also, the IHC technique presents accuracy and ability as a diagnostic technique for identifying the different types of pediatric cancers. The MMR protein panel accompanied with PCNA panels holds additional value, as it helps reduce dependency solely on MMR protein expressions.

Abstract 5615: Functional analysis of variants of uncertain significance in the MSH6 mismatch repair gene

Abstract Lynch syndrome (LS) is a hereditary condition that increases patients’ lifetime risk of cancer, primarily colorectal cancer. LS is caused by germline mutations in mismatch repair (MMR) genes, MLH1, MSH2, MSH6, and PMS2. Identification and classification of these mutations is important in LS diagnosis for guiding treatment plans and preventative care for patients’ and their families. However, the consequences of some variants in these genes are not immediately obvious, granting them classification as variants of uncertain significance (VUS). Laboratory functional analysis to determine whether these variants disrupt MMR function in human cells can provide important evidence for understanding the pathogenicity of VUS and reclassifying them. Half of known variants in the MMR gene MSH6 are VUS and, thus, the goal of my project is to provide evidence for a subset of these variants to help provide evidence for reclassification for these variants. We are calibrating and validating functional assays for MSH6 variants, using known MSH6 pathogenic and benign controls. We will then test MSH6 VUS identified in patients as reported in the InSiGHT database, ClinVar database, and current literature. We are using CRISPR gene editing to recreate these variants in the endogenous MSH6 loci in human embryonic stem cells (hESC). Using these edited hESC lines we are examining the impact of the variants on RNA and protein stability, repair of DNA microsatellites, which is a hallmark of normal MMR function, and induction of the MMR-dependent DNA damage response. With this compilation of data, we will convert the results into a quantitative Odds of Pathogenicity value. This Odds of Pathogenicity score can be used by expert variant interpretation committees to help readily reclassify these variants and provide a clearer diagnosis for these suspected Lynch syndrome patients. Citation Format: Elizabeth Szabo, Emily Blackburn, Alexander Radecki, Abhijit Rath, Christopher Heinen. Functional analysis of variants of uncertain significance in the MSH6 mismatch repair gene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5615.

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

Apparition of germline mutation c.1395-1397del of MUTYH in Algerian consanguineous family with colorectal cancer

MUTYH is a glycosylase that removes adenine opposite 8-oxoguanine (OG) during base-excision repair of DNA. Variants of MUTYH defective in functional activity lead to MUTYH-associated polyposis (MAP), autosomal recessive predisposition to colorectal cancer (CRC). MAP combines clinical features with other hereditary CRC syndromes, and exhibits phenotypic overlap, particularly with Lynch syndrome (LS). To determine the impact of MUTYH mutations in colorectal adenomas and cancer susceptibility, this prospective objective screens the MUTYH gene in seventeen Eastern Algerian families with clinically suspected LS but without mismatch repair (MMR) mutations. Methods: We examined the presence of the mutations in the probands and their relatives using direct sequencing of the entire coding region of the MUTYH gene. Results: The biallelic and monoallelic pathogenic MUTYH mutations, c.1395_1397del, were discovered in a consanguineous family with CRC and gastric cancer as a novel finding in our Eastern Algerian population. Conclusion: High rates of consanguinity in Algerian population increase the risk of CRC caused by biallelic mutations in the MUTYH gene. In our families, the LS and MAP phenotypes might coexist.

Outcomes with immunotherapy between Lynch syndrome vs non-Lynch syndrome microsatellite instability-high colorectal cancer.

175 Background: Alterations to mismatch repair (MMR) genes such as MLH1, MSH2, MSH6, and PMS2, can lead to microsatellite instability-high (MSI-H) tumors. MMR mutations can be inherited in Lynch syndrome (LS) but can also be a result of de novo alterations resulting in MSI-H malignancies. Treatment with immune checkpoint inhibitors (ICIs) have been shown to improve survival in such patients (pts) compared to systemic chemotherapy. However, there is a paucity of information in the literature with respect to outcomes of pts with germline vs somatic MMR mutations in MSI-H colorectal cancer (CRC). Furthermore, the presence of underlying BRAF mutation confers resistance to chemotherapy in pts with CRC, however data regarding outcomes with immunotherapy in such pts with MSI-H CRC is lacking. Methods: Pt records from the Mayo Clinic (AZ, MN, FL) between 2008-2023 denoted as MSI-H CRC, were included for retrospective review. Pt demographics, treatment courses, and genomic profiles were collected. Overall survival (OS) and progression-free survival (PFS) were estimated using the Kaplan-Meier method. Outcome differences between sub-groups were accessed with the log rank test and univariate analyses were assessed with Cox-regression. Results: A total of 81 pt records were identified (n = 18 LS, n = 63 non-LS). Stage IV disease was found in 28% of LS pts vs 59% of non-LS pts at diagnosis. Pembrolizumab was the most common ICI selected for metastatic CRC pts for both LS and non-LS pts (65% vs 94%) with median treatment duration for each group 11.7 vs 8.8 months. There were no differences in OS or PFS for any stage between LS vs non-LS. Median OS for all patients was 82 months. For all stage IV patients, median OS was 44 months, and median PFS was 19 months. Interestingly, for stage IV pts treated with immunotherapy, tumors that harbored BRAF V600E mutation (n = 21) vs BRAF wild type (n = 20) had a significantly lower median OS 19 vs 113 months (HR = 2.69, p = 0.043) as well as median PFS 12 vs 95 months (HR = 2.48, p = 0.041). Conclusions: In this study, we found that patient treated with ICIs have similar outcomes in the presence of germline vs. somatic MMR mutations in MSI-H CRC. The presence of an associated BRAF V600E mutation, which occurs in non-LS MSI-H CRC conferred worse outcomes.

Benchmarking mismatch repair testing for patients with cancer receiving immunotherapy.

179 Background: Mismatch repair deficiency (MMR-D) serves as a theranostic marker for directing immune checkpoint inhibitor (ICI) treatment across various cancer stages and tumor types. The College of American Pathologists endorses immunohistochemistry (IHC) as the primary approach for assessing mismatch repair (MMR) testing in colorectal (CRC) and endometrial cancer (EC) patients. An alternative method involves genetic evaluation of microsatellite instability (MSI). Previous studies have shown that resistance to immune checkpoint inhibitors (ICIs) may occur due to inaccurate MSI or MMR IHC status determination. Accurate assessment of MMR status is critical for treatment decision-making. Methods: We hypothesized that tumor-only next generation sequencing (NGS) for MMR mutation signature assessment for identification of MMR-D tumors is more sensitive than MMR IHC testing. Using a standardized, clinically validated, tumor-only NGS mutation signature pipeline in place in the Oncopanel assay at DFCI/BWH, we compared the concordance in MMR determination between NGS and IHC. We then evaluated a subset of ICI-treated patients to assess the clinical implications of these findings. Results: 1655 colorectal cancer (CRC) and endometrial carcinoma (EC) patients who had both NGS MSI (NGSd = deficient or NGSp = proficient) and MMR IHC (IHC- = absent or IHC+ = intact) assays performed were assessed. Discordant NGSd/IHC+ results were identified in 1% of CRC and 5% of EC cases that were IHC+. Importantly, patients with discordant NGSd/IHC+ (median OS: not reached [NR], 95% CI, 9.9 months-NR; median TTF: 44.4 months, 95% CI, 4.1-NR) and concordant NGSd/IHC- results, showed a similar overall survival and time to treatment failure with ICI treatment (p=non-significant) which contrasted with results for patients with concordant NGSp/IHC+ results (median OS: 14.9 months, 95% CI, 7.3-24.6, p-value 0.0025 and &lt; 0.0001; median TTF: 5.0 months, 95% CI, 3.2-7.3; p-value 0.0014 and &lt; 0.0001). Patients with EC or CRC and discordant NGSd/IHC+ results had better survival when treated with ICI-based regimens, compared to those who received non-ICI treatments (p= 0.054 and 0.017). Conclusions: In 2022, 151,030 new CRC and 65,950 new EC cases were diagnosed in the USA. Extrapolating our findings to this larger population, use of NGS mutational signature analysis instead of IHC would amount to about 1510 additional MMR-D CRC cases and 3297 additional MMR-D endometrial cases identified each year for whom first-line ICI therapy is preferred. Our findings support revisiting guideline recommendations for diagnostic testing of MMR to incorporate both IHC and targeted NGS tumor panel testing using sensitive and specific mutation signature calling algorithms. Larger prospective studies are warranted to confirm these findings in patients with mCRC and mEC treated with ICIs. Evaluation of this approach in other cancer types is also warranted.