Mismatch Repair Research Articles

MSH2-Mutated Lynch Syndrome With 9 Synchronous Colon and Rectum Adenocarcinomas: An Extremely Rare Case Report

Synchronous colorectal carcinoma is having more than 1 primary carcinoma detected in a single patient at the same time or within 6 months of tumor diagnosis. Metachronous colorectal carcinoma is the presence of more than 1 primary carcinoma detected consecutively in a single person after a set time interval. Patients with Lynch syndrome and Muir-Torre syndrome (a subset of Lynch syndrome) inherit a germline mutation in 1 of the mismatch repair (MMR) genes. Patients with synchronous colorectal carcinoma have a higher proportion of MMR-mutated cancers than patients with solitary colorectal carcinoma. Most studies in the literature indicate that patients with synchronous colorectal cancers typically have only 2 carcinomas. However, there have been reports of a single patient having up to 6 synchronous carcinomas in the large intestine. This report discusses a patient with 9 simultaneous colorectal cancers at the initial diagnosis, along with a history of bladder cancer, sebaceous adenoma, and duodenal adenoma, associated with a germline mutS homolog 2 ( MSH2) mutation. Additionally, the report explores various aspects of having synchronous colorectal cancers. More studies are needed to clarify the clinicopathologic and molecular landscape of these rare tumors and identify the best management and treatment strategies for these patients.

The impact of preoperative treatment on mismatch repair protein and HER2 expression in colorectal cancer: an analysis of paired samples.

Colorectal cancer (CRC) is a leading cause of cancer-related mortality, highlighting the necessity for multifaceted treatment strategies, including preoperative treatment (PT), which can enhance surgical outcomes and provide prognostic insights. This study aims to clarify the impact of PT-induced changes in mismatch repair (MMR) and human epidermal growth factor receptor 2 (HER2) expression, potentially informing tailored treatment strategies and improving clinical outcomes for CRC patients. This retrospective study analyzed 120 paired samples from CRC patients who underwent preoperative treatment, comparing pre- and post-treatment specimens. A control group of 60 untreated surgical specimens was also included. Immunohistochemistry assessed MMR proteins (MSH6, MSH2, MLH1, PMS2) and HER2 expression. MSI status was determined in samples with low MMR expression. Compared to pre-treatment samples, post-treatment samples exhibited lower levels of MSH6, MSH2, and total MMR expression, along with higher levels of HER2 expression. However, when compared to the untreated control group, there were no significant differences in the expression of MSH6, total MMR, and HER2. All samples that exhibited weak MMR expression and those that shifted to deficient mismatch repair (dMMR) status following treatment had stable microsatellite status. No clear clinicopathological characteristics or prognostic factors were found to be associated with changes in MMR and HER2 expression, except for the use of fluorouracil or capecitabine, which was related to changes in total MMR scores. ypTNM stage and TRG scores were identified as independent factors affecting disease progression in our study. PT is associated with a reduction in MMR expression, notably for the MSH2 protein, while it does not appear to influence HER2 expression.

Mainstreaming cancer genetics: feasibility of an advanced nurse practitioner-led service diagnosing Lynch syndrome from colorectal cancer in Ireland.

Colorectal cancer (CRC) is a common cancer in Ireland. Of all CRCs, 2-4% are attributable to Lynch Syndrome (LS), the most common CRC predisposition syndrome. LS is caused by constitutional pathogenic variants (PVs) affecting mismatch repair (MMR) genes with resultant MMR protein deficiency (dMMR). Screening of all CRCs with MMR immunohistochemistry (IHC) testing is advocated to increase the detection of LS. However, successful implementation requires appropriate downstream management. In Ireland the traditional pathway involves referral to cancer genetics services to assess eligibility for genetic testing. Cancer genetics services in Ireland face many challenges in providing uniform access to timely healthcare with current wait times for assessment in excess of 1 year. An increasingly adopted pathway is that of mainstreaming, whereby genetic testing is managed locally by a multidisciplinary team member. Our institution therefore implemented an Advanced Nurse Practitioner (ANP)-led service with responsibility for the LS Diagnostic Pathway and mainstream genetic testing. Data was extracted from a prospectively maintained database of all newly diagnosed CRC patients discussed at our institutions CRC multidisciplinary meeting (MDM) between January 1st, 2023, and May 31st, 2024. MMR IHC testing was performed in 97.9% of the 385 patients diagnosed with CRC. The median time from histological confirmation of CRC to the availability of the MMR IHC report was 6 days. All 51 patients (100%) who required sequential tumor testing underwent BRAF V600 ± MLH1 promoter methylation testing. Additionally, 100% of the 14 patients eligible for mainstream genetic testing were referred to the ANP-led genetics service. The median time from the initial MDM discussion to the initiation of genetic testing was 69 days, while the median time from testing to the availability of results was 19 days. Patients received their results within a median of 21 days. MMR IHC testing increases the detection of LS through identification of dMMR tumours. Successful downstream delivery of clinical services, however, requires appropriate subsequent management, in a resource-limited environment. Our institutional experience demonstrates the feasibility, efficiency, and effectiveness of an ANP-led mainstreaming model of care for hereditary colorectal cancer.

Mutant HTT protein decreases with CAG repeat expansion: implications for therapeutics and bioassays

Abstract Huntington’s disease is an inherited neurodegenerative disorder caused by a CAG repeat expansion that encodes a polyglutamine tract in the HTT protein. The mutant CAG repeat is unstable and expands in specific brain cells and peripheral tissues throughout life. Genes involved in the DNA mismatch repair pathways, known to act on expansion, have been identified as genetics modifiers, therefore, it is the rate of somatic CAG repeat expansion that drives the age of onset and rate of disease progression. In the context of an expanded CAG repeat, the HTT pre-mRNA can be alternatively processed to generate the HTT1a transcript, that encodes the aggregation prone and highly pathogenic HTT1a protein. This may be a mechanism through which somatic CAG repeat expansion exerts its pathogenic effects, as the longer the CAG repeat, the more HTT1a and HTT1a is produced. The allelic series of knock-in mouse models: HdhQ20, HdhQ50, HdhQ80, HdhQ111, CAG140 and zQ175 with polyglutamine expansions of 20, 50, 80, 111 140 and ∼190 can be used to model the molecular and cellular consequences of CAG repeat expansion within a single neuron. By western blot of cortical lysates, we found that mutant HTT levels decreased with increasing CAG repeat length; mutant HTT was only 23% and 10% of wild-type levels in CAG140 and zQ175 cortices, respectively. To identify the optimal bioassays for detecting the full-length HTT and HTT1a isoforms, we interrogated the pairwise combinations of seven well-characterized antibodies on both the ‘homogeneous time resolved fluorescence’ and ‘Meso Scale Discovery’ platforms. We tested 32 assays on each platform to detect ‘full-length mutant HTT’, HTT1a, ‘total mutant HTT’ (full-length HTT and HTT1a) and ‘total full-length HTT’ (mutant and wild type). None of these assays recapitulated the full-length mutant HTT levels as measured by western blot. We recommend using isoform- and species- specific assays that detect either full-length mutant HTT, HTT1a or wild-type HTT as opposed to those that detect more than one isoform simultaneously. Our finding that as the CAG repeat expands, full-length mutant HTT levels decrease, whilst HTT1a and HTT1a levels increase has implications for therapeutic strategies. If mutant HTT levels in cells containing (CAG)200 are only 10% of wild-type, HTT-lowering strategies targeting full-length HTT at sequences 3’ to intron 1 HTT will predominantly lower wild-type HTT, as mutant HTT levels in these cells are already depleted. These data support a therapeutic strategy that lowers HTT1a and depletes levels of the HTT1a protein.

The genetic landscape of Lynch syndrome in the Israeli population.

Deciphering the spectrum and founder disease-causing variants (DCVs) in specific populations can shape and facilitate the diagnostic process of Lynch Syndrome (LS). The aim of this report was to comprehensively update on the genetic landscape of LS in the ethnically diverse Israeli-Jewish population. The cohort included 1080 carriers from 588 families; some from underrepresented, understudied Israeli ethnic groups recruited from 8 genetic institutes and high-risk clinics throughout the country. Variant classification was performed according to the American College of Medical Genetics criteria. A total of 157 DCVs were identified, 12 are reported here for the first time, and 9 reclassified. MSH2 DCVs were identified in 286 families (49%). Most DCVs (125/157, 80%) were noted in one or two families only. Sixteen DCVs, each detected in ≥ 5 families, and accounted for LS in 378/588 (64%) families. Constitutional mismatch repair deficiency (CMMRD) was diagnosed in 7 families. Twenty-five carriers (2.3%) had an additional DCV or risk alleles in another cancer susceptibility gene. In conclusion, MMR gene variant distribution in Israel is diverse. MSH2 is most commonly mutated due to founder DCVs. Though the 16 prevalent LS-associated DCVs were frequently detected in our cohort, none of them is frequently reported in the general population. These data should facilitate variant interpretation, spouse and cascade testing.

Molecular classification and adjuvant treatment in endometrioid endometrial cancer with microcystic elongated and fragmented (MELF) invasion pattern.

To assess the characteristics, molecular classification, and role of adjuvant treatment in patients with endometrioid endometrial cancer (EEC) and microcystic elongated and fragmented (MELF) myometrial invasion pattern. This study included patients who were diagnosed with EEC with a MELF invasion pattern and underwent surgery from January 2019 to December 2023. We analyzed molecular classification, clinicopathological characteristics, and prognostic outcomes, including recurrence and adjuvant therapy. Out of 529 patients, 84 (15.9%) exhibited the MELF pattern, with 1 (1.2%) classified as POLE-mutation, 19 (22.6%) as mismatch repair deficient, 53 (63.1%) as no specific molecular profile, and 11 (13.1%) as p53-mutant subtype. Fifty (59.5%) patients with MELF invasion pattern received adjuvant chemotherapy (CT) ± external beam radiation therapy (EBRT), 19 (22.6%) received EBRT only, and 15 (17.9%) received no adjuvant treatment. Receiving adjuvant therapy was significantly associated with the risk level defined by the ESMO guideline for endometrial cancer (p = 0.002). With a median follow-up of 26 months (range: 1-59), the progression-free survival at 3-years for the MELF invasion patients was 91%. Seven patients with the MELF pattern experienced recurrence, of whom one was in stage IA (low risk, local recurrence) and did not receive any additional treatment, two were in stage IB (intermediate / high-intermediate risk, distant recurrence) and received EBRT only and the remaining four were in stage III to IV and had distant recurrence despite receiving adjuvant chemotherapy with or without EBRT. Among 43 intermediate- and high-intermediate-risk EEC patients, receiving CT ± EBRT was significantly associated with better DFS than without CT (p = 0.047). The presence of the MELF pattern in EEC should be incorporated into decision-making regarding adjuvant therapy. The use of adjuvant treatment should be tailored based on histology and molecular type.

A novel mouse model recapitulating the MMR-defective SCLC subtype uncovers an actionable sensitivity to immune checkpoint blockade.

Small cell lung cancer (SCLC) has an extremely poor prognosis. Despite high initial response rates to chemotherapy and modest survival improvements with the addition of immune checkpoint inhibitors (ICI), almost all patients experience relapse and fatal outcomes. Recent genomic insights uncovered extensive molecular heterogeneity in addition to the almost uniform loss of RB1 and TRP53. Additionally, defective DNA mismatch repair (MMR) has recently been described in some SCLC cases. Here, we generated a novel SCLC mouse model capturing MMR deficiency and assessed immunotherapy responses. We developed an MMR-deficient genetically engineered mouse model (GEMM) of SCLC by introducing a conditional Msh2 gene, crucial for maintaining MMR integrity, into the standard Rb1fl/fl;Trp53fl/fl (RP) model. Genomic characteristics and preclinical therapy responses were evaluated by focusing on overall survival and whole exome sequencing (WES) analyses. MMR-defective SCLC tumors (Rb1fl/fl;Trp53fl/fl;Msh2fl/fl (RPM)) developed later than tumors in MMR-proficient mice. However, the time from tumor manifestation to death of the affected animals was substantially shortened (median survival 55 days in RP vs. 46.5 days in RPM), indicating increased aggressiveness of MMR-defective tumors. RPM tumors exhibited MMR deficiency, high tumor mutational burden (TMB), and an elevated load of candidate neoantigens, compared to RP lesions (p = 0.0106), suggesting increased immunogenicity. Importantly, the overall survival of RPM animals was significantly improved when exposed to ICI. We propose a novel RPM mouse model as a suitable system to mimic MMR-defective SCLC and tumors with high TMB. We provide in vivo evidence that Msh2 deficiency enhances ICI sensitivity. These findings could contribute to stratifying SCLC patients to immunotherapy, thereby improving treatment outcomes.

Molecular profiling reveals novel therapeutic targets and clonal evolution in ovarian clear cell carcinoma.

Ovarian clear cell carcinoma (OCCC) has a disproportionately high incidence among women in East Asia. Patients diagnosed with OCCC tend to experience worse clinical outcomes than those with high-grade serous carcinoma (HGSC) at advanced stages. The unfavorable prognosis of OCCC can be partly attributed to its frequent resistance to conventional chemotherapy. Within a precision medicine framework, we sought to provide a comprehensive molecular characterization of OCCC using whole-exome sequencing to uncover potential molecular targets that may inform novel therapeutic strategies. We performed whole-exome sequencing analysis on tumor-normal paired samples from 102 OCCC patients. This comprehensive genomic characterization of a substantial cohort of OCCC specimens was coupled with an analysis of clonal progression. On analyzing 102 OCCC samples, ARID1A (67%) and PIK3CA (49%) emerged as the most frequently mutated driver genes. We identified tier 1 or 2 clinically actionable molecular targets in 40% of cases. This included DNA mismatch repair deficiency (n = 1), as well as BRCA2 (n = 1), PIK3CA (n = 36), KRASG12C (n = 1), and ATM (n = 4) mutations. Furthermore, 45% of OCCC samples displayed ARID1A biallelic loss. Interestingly, we identified previously unreported mutations in the 5' untranslated region of the TERT gene that harbored an adverse prognostic significance. Clock-like mutational processes and activated APOBECs were major drivers of somatic point mutations. Mutations arising from DNA mismatch repair deficiency were uncommon. Reconstruction of clonal evolution revealed that early genetic events likely driving tumorigenesis included mutations in the ARID1A, PIK3CA, TERT, KRAS, and TP53 genes. Our study provides a comprehensive characterization of the genomic landscape and clonal evolution in OCCC within a substantial cohort. These findings unveil potentially actionable molecular alterations that could be leveraged to develop targeted therapies.

Abstract I003: Inhibitors of DHX9 RNA helicase as synthetic lethal cancer therapeutics

Abstract Over 100 modifications to RNA are known to occur in human cells, where they play critical roles in many aspects of normal cellular physiology, such as cell fate decisions and terminal differentiation, through effects on RNA biology such as protein and nucleic acid interactions. Our survey of human RNA-modifying enzymes suggests many are cancer essential enzymes with striking synthetic lethal profiles, including the DHX9 helicase. DHX9 is a multifunctional DExH-box RNA helicase which can unwind regions of double-stranded DNA and RNA helices but has a greater propensity for secondary structures such as DNA/RNA hybrids (R-loops) and DNA/RNA G-quadruplexes. DHX9 interacts with and regulates many proteins, including key members of DNA damage repair pathways. We have found that DHX9 knockdown is synthetic lethal in microsatellite high (MSI-H) or defective mismatch repair (dMMR) tumor models. A suite of assays was developed to identify and optimize potent and selective inhibitors of the DHX9 helicase, which recapitulate our findings with genetic tools. Profiling of DHX9 inhibitors across a broad panel of cancer cell lines reveals that tumor cells with mutations in the DNA damage repair genes BRCA1 and/or BRCA2 are also responsive to DHX9 inhibitor treatment in vitro and in vivo. DHX9 inhibition leads to increased RNA/DNA secondary structures such as R-loops and G-quadruplexes, resulting in subsequent DNA damage and increased replication stress, leading to cell cycle arrest and apoptosis. These results suggest that DHX9 inhibitors are a novel treatment modality for patients with defective DNA damage repair pathways such as dMMR and/or BRCA mutations. Citation Format: Serena J Silver. Inhibitors of DHX9 RNA helicase as synthetic lethal cancer therapeutics [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr I003.

Action-At-A-Distance in DNA Mismatch Repair: Mechanistic Insights and Models for How DNA and Repair Proteins Facilitate Long-Range Communication

Many DNA metabolic pathways, including DNA repair, require the transmission of signals across long stretches of DNA or between DNA molecules. Solutions to this signaling challenge involve various mechanisms: protein factors can travel between these sites, loop DNA between sites, or form oligomers that bridge the spatial gaps. This review provides an overview of how these paradigms have been used to explain DNA mismatch repair, which involves several steps that require action-at-a-distance. Here, we describe these models in detail and how current data fit into these descriptions. We also outline regulation steps that remain unanswered in how the action is communicated across long distances along a DNA contour in DNA mismatch repair.

Prospective Screening of Cancer Syndromes in Patients with Mesenchymal Tumors

Background: The etiology of most mesenchymal tumors is unknown, and knowledge about syndromes with an increased risk of tumors in bone or soft tissue is sparse. Methods: We present a prospective germline analysis of 312 patients with tumors suspected of being sarcomas at a tertiary sarcoma center. Germline and tumor whole genome sequencing, tumor transcriptome, and methylome analyses were performed. Results: Germline pathogenic or likely pathogenic variants associated with an increased risk of tumors were detected in 24 patients (8%), of which 11 (4%) harbored a detectable second hit in the tumor. Second hits were confirmed in genes with (NF1, RB1, TP53, EXT2, and SDHC) and without (ATM, CDC73, MLH1, MSH6, POLG, and KCNQ1) known association with mesenchymal tumor predisposition. Sarcomas from two Lynch syndrome patients showed mismatch repair deficiency, predicting a treatment response to immune checkpoint inhibitors (Level 1 biomarker according to the FDA (Federal Drug Administration) and ESMO (European Society for Medical Oncology)). None of the three CHEK2 carriers had a second hit in the tumor, suggesting a weak link to sarcoma. Conclusions: We conclude that second-hit analyses can be used in standard of care to identify syndrome-related tumors. This approach can help distinguish true manifestations of tumor syndromes from unrelated germline findings and enhance the understanding of germline predisposition in soft tissue tumors. Prospective screening using germline whole genome sequencing should be considered when comprehensive somatic sequencing is introduced into clinical practice.

Clinicopathological Characteristics of Neutrophil-Rich Hepatocellular Carcinoma: An Uncommon Subtype of Primary Liver Cancer.

Introduction. Neutrophil-rich hepatocellular carcinoma (HCC) is an extremely uncommon subtype of HCC with an overall incidence of <1%. Neutrophil-rich HCC shows poor cellular differentiation and sarcomatoid transformation in most patients. There is prominent neutrophilic inflammatory cell infiltration in the tumor. These tumors are associated with poor prognosis, high rate of recurrence, and metastasis. Methods. Herein, we investigated 4 patients with neutrophil-rich HCC reported at our center. Clinical, radiological, and pathological findings were reviewed. Immunophenotypic characterization of the tumors were done. Granulocyte colony-stimulating factor (G-CSF), programmed cell death ligand 1 (PD-L1), and mismatch repair immunostains were performed in all 4 tumors. Results. We report 4 neutrophil-rich HCCs in 3 male patients and one female patient with an age range of 43 to 64 years. Three underwent living donor liver transplantation and one underwent right hepatectomy. Tumor measured 0.5 cm to 12 cm in maximum dimension. Histologically, tumors demonstrated moderate to marked cellular pleomorphism. Spindle cell transformation was noted in 3 tumors. Three tumors showed vascular invasion, and one tumor showed bile duct invasion. Immunopositivity for Hep Par-1, arginase-1, and glypican-3 was present in all tumors. Tumors also expressed stemness markers including KRT19 and EpCAM. Cytoplasmic positivity for G-CSF and immunoexpression of PD-L1 was demonstrated. We also report proficient mismatch repair by immunohistochemistry in all tumors. Conclusion. Neutrophil-rich HCC is an aggressive primary liver cancer which demonstrates stemness-related features. Programmed cell death ligand 1 expression in tumor cells suggests distinct immunogenic features and potential role of anti-PD-L1 therapies in inoperable disease.

EPCO-32. PROSPECTIVE GERMLINE SEQUENCING OF PATIENTS WITH GLIOMAS, GLIONEURONAL OR NEURONAL TUMORS

Abstract BACKGROUND Several genetic syndromes have been linked to the development of central nervous system (CNS) tumors; however, the prevalence and clinical significance of germline pathogenic variants in this population remain unclear. METHODS 2,367 patients with a glioma, glioneuronal, or neuronal tumor (WHO grade 1-4) ages 0.2-93.8 years underwent tumor and matched normal sequencing with MSK-IMPACT. Germline variants and copy-number variants affecting 90 well-established cancer predisposing genes were analyzed; the presence of biallelic inactivation was determined using FACETS. RESULTS Eleven percent harbored germline pathogenic mutations in high-, moderate-, and low-penetrance genes. High- and moderate-penetrance genes included CHEK2 (n=32, 1.3%), BRCA2 (n=10; 0.4%), TP53 (n=9; 0.4%), NF1 (n=6; 0.2%), and mismatch repair (MMR) genes (n=20, 0.9%). Low-penetrance genes included monoallelic MUTYH (n=42; 1.8%) and the APC I1307K variant (n=28, 1.2%). Biallelic inactivation was found in all tumors from patients with germline TP53 and NF1 mutations, with lower rates in tumors from patients with a germline MMR alteration (53%). Biallelic inactivation was rare among patients with a germline mutation in homologous recombination pathway (HRD) genes (27%), and in the aforementioned low-penetrance APC (15.3%) and MUTYH (10.2%) variants. All patients with biallelic inactivation of an MMR gene were hypermutated. Biallelic inactivation of MMR genes and TP53 were observed in IDH-mutant astrocytoma and IDH-WT gliomas, while biallelic inactivation of NF1 and somatic IDH1/2 mutation were mutually exclusive. When examining the age at diagnosis among patients with germline alterations, tumors with biallelic inactivation were diagnosed at a younger age compared to tumors with monoallelic alterations (37.7 vs 51.6 years, p=0.002). CONCLUSION Clinical germline sequencing identifies a germline mutation in a high proportion of patients with CNS tumors. Biallelic inactivation was most commonly identified in tumors from patients with germline TP53 or NF1 mutations and were less common in patients with a germline MMR alteration.

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&amp;lt;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&amp;lt;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.

IMMU-40. INVESTIGATING THE ROLE OF MUTATIONAL BURDEN IN TUMOR IMMUNOGENICITY USING A PRE-CLINICALMLH1-DEFICIENT GLIOBLASTOMA MODEL

Abstract BACKGROUND Although there is an established correlation between tumor mutational burden (TMB) and response to immune checkpoint inhibition (ICI) in many cancers, such an association has not been definitively shown in high-grade gliomas. Most studies of ICI therapy in gliomas, including glioblastoma (GBM), have failed to demonstrate efficacy, with the exception of hypermutated cases resulting from rare inherited defects in mismatch repair (MMR) genes. We sought to investigate the association of TMB to immune response using a syngeneic murine model with a spectrum of mutational burdens. METHODS We used CRISPR-Cas9 to knockout the MMR gene Mlh1 in the murine glioma model SB28, an ICI-resistant cell line with a very low mutational burden (108 non-synonymous mutations). RESULTS Single-cell sorted Mlh1 knockout SB28 clones revealed a two- to six-fold increase in TMB. Importantly, loss of Mlh1 also brought about global changes in immune- and inflammatory-related gene expression in vitro, without correlation to increasing TMB. While all clones had the same rate of in vitro proliferation, flank injection of a high TMB vs a low TMB SB28 Mlh1 knockout clone resulted in similar survival benefits compared to Mlh1 wildtype SB28. CONCLUSIONS These preliminary results suggest that, although disruption of the MMR system may result in heightened tumor immunogenicity, elevated TMB may not be the sole significant determinant. We are currently investigating tumor proliferation after flank injection in immunocompromised mice to confirm that slowed growth is immune-related, as well as clone-specific response to ICI treatment. We are also exploring the role of specific cytokines/chemokines in conferring this survival advantage and potential immune response. By generating a GBM model of varied mutational burdens and immune profiles and comparing to the baseline SB28 cell line, we can interrogate the relative significance of TMB in tumor immunogenicity and ICI response.

DNAR-13. LONG TERM FOLLOW-UP OF A LYNCH SYNDROME-ASSOCIATED GLIOBLASTOMA RESISTANT TO STANDARD-OF-CARE CHEMORADIATION

Abstract BACKGROUND Lynch syndrome is caused by pathogenic germline variants in the DNA mismatch repair (MMR) genes, predisposing individuals to malignancies, including glioblastoma (GBM). MMR-deficient tumors are resistant to monofunctional alkylators such as temozolomide but not to bifunctional alkylators such as the nitrosourea lomustine. Anti-programmed cell death protein 1 (PD1) immune checkpoint inhibitor (ICI) pembrolizumab represents another treatment option, which is approved by the United States Food and Drug Administration (FDA) for the treatment of mismatch repair deficient cancers. METHODS We present a patient with multifocal GBM and Lynch syndrome who experienced rapid progression following standard-of-care chemoradiation, followed by durable responses to lomustine and then to pembrolizumab. RESULTS A 58-year-old man with Lynch syndrome (MSH2 mutation) and prior history of colon cancer presented with seizures. Magnetic resonance imaging (MRI) brain identified right frontal and left parietal masses. Right frontal tumor was resected; pathology was consistent with GBM, IDH wildtype, MGMT-unmethylated. Both lesions were treated with radiation therapy (60 Gy, 30 fractions) with concurrent and adjuvant temozolomide. After two cycles of adjuvant temozolomide, his left parietal tumor progressed and was subtotally resected, followed by four cycles of lomustine (stopped due to myelotoxicity). He remained progression-free for 15 months. Surveillance MRI identified new enhancement involving the left frontal lobe, left periventricular white matter, and right internal auditory canal, concerning for leptomeningeal dissemination. MRI spine identified no additional lesions. Cerebrospinal fluid cytology was negative for tumor cells on two occasions. Patient was treated with pembrolizumab. He remains on treatment with stable disease four months after ICI initiation and 31 months after GBM diagnosis. CONCLUSIONS Patients with MMR-deficient GBM resist standard chemoradiation. Alternatives such as nitrosourea or ICI therapy may achieve durable responses. While data is insufficient to administer these agents as upfront therapy in this specific setting, both should be considered as options for recurrent disease.

CCRG-01. MYT1 BLOCKADE AS A NOVEL GLIOMA THERAPEUTICS THROUGH ABROGATIING OF THE G2/M CELL CYCLE CHECKPOINT

Abstract BACKGROUND The cell cycle is tightly regulated by checkpoints, which play a vital role in controlling its progression and timing. Cancer cells exploit the G2/M checkpoint, which serves as a resistance mechanism against genotoxic anticancer treatments, allowing for DNA repair prior to cell division. Manipulating cell cycle timing has emerged as a potential strategy to augment the effectiveness of DNA damage-based therapies. METHODS In this study, we conducted a forward genome-wide CRISPR/Cas9 screening with repeated exposure to the alkylating agent temozolomide (TMZ) to investigate the mechanisms underlying tumor cell survival under genotoxic stress. The screening hits were validated through in patient derived glioma cells and xenografts. RESULTS Our findings revealed that canonical DNA repair pathways, including the Ataxia–telangiectasia mutated (ATM)/Fanconi and mismatch repair, determine cell fate under genotoxic stress. Notably, we identified the critical role of PKMYT1, in ensuring cell survival. Depletion of PKMYT1 led to overwhelming TMZ-induced cytotoxicity in cancer cells. Isobologram analysis demonstrated potent drug synergy between alkylating agents and a Myt1 kinase inhibitor, RP-6306. Mechanistically, inhibiting Myt1 forced G2/M-arrested cells into an unscheduled transition to the mitotic phase without complete resolution of DNA damage. This forced entry into mitosis, along with persistent DNA damage, resulted in severe mitotic abnormalities. Ultimately, these aberrations led to mitotic exit with substantial apoptosis. Preclinical animal studies demonstrated that the combination regimen involving TMZ and RP-6306 prolonged the overall survival of glioma-bearing mice.

IMMU-14. TRANS-SPECIES ANALYSIS OF REPLICATION-REPAIR DEFICIENT (RRD) MEDULLOBLASTOMA AND RESPONSE TO IMMUNE CHECKPOINT INHIBITION: AN IRRDC REPORT

Abstract BACKGROUND Replication-repair deficiency (RRD) stemming from mismatch repair and polymerase proofreading gene defects (MMRD/PPD) lead to hypermutant childhood cancers, most frequently brain tumors. While medulloblastoma is reported, the clinical, genomic and immune landscape remains unknown. METHODS We analysed the genome, methylome, transcriptome (bulk, single-nuclei) and immune-microenvironment of the largest cohort of RRD-medulloblastoma patients enrolled by the International RRD Consortium and correlated these to clinical outcomes. RRD mice-models were used to preclinically assess response to immunotherapy. Results: RESULTS RRD-medulloblastoma (n=43) were enriched for anaplasia (61%) and localised disease (77%). Methylation/nano-string-based subgrouping failed to classify 40%, while the remaining clustered with the SHH-subgroup. Copy-number changes were notably infrequent (&amp;lt;20%). All tumors harboured hypermutation and microsatellite instability in contrast to non-RRD controls (p&amp;lt;0.0001). Pathogenic variants were frequent in POLE/POLD1 (80%), TP53 (48%) and SHH-pathway genes (PTCH1, SUFU, SMO: 56%). Interestingly, some tumors additionally had glioma-driver alterations (ATRX, NF1: ~50%), similar to RRD cerebellar glioblastoma, but distinct from both non-RRD medulloblastoma (n=791) and hemispheric glioblastoma (n=733) controls. Moreover, although bulk-transcriptome matched non-RRD SHH medulloblastoma, single-nuclei analyses suggested an earlier cell-of-origin potentially explaining the heterogenous phenotypes resulting from the driver mutations. Uniquely, immune analyses (gene expression and immunohistochemistry) demonstrated high intra-tumoral CD8 T-cell infiltration. Three-year progression-free survival was 60%. Outcome was worse for RRD-medulloblastoma harbouring TP53-mutation (p=0.04) and failing subgroup-classification (p=0.004). Nestin/Cre MSH2-/- POLE;p.S459F mice developing medulloblastoma recapitulated human disease and demonstrated response to anti-PD1 monotherapy. Recurrent/progressive human tumors including those harbouring TP53-mutations exhibited radiological responses to anti-PD1 monotherapy, which was associated with prolonged ongoing survival in comparison to those not treated with checkpoint-inhibitors (p=0.02). CONCLUSIONS Hypermutant RRD-medulloblastoma reveal heterogenous phenotypes due to unique spectrum of driver mutations and an early cell of origin. Their immune-hot microenvironment allows successful salvage treatment with checkpoint-inhibitors for those failing chemo-radiation, including the high-risk SHH/TP53-mutant and unclassified subgroups.

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.