Defects In Mismatch Repair Genes Research Articles

Molecular Biomarkers in Colorectal Cancer in the Era of Precision Medicine: A Review

Colorectal cancer (CRC) is a primary cause of death among cancer patients. This heterogeneous disease is characterized by alterations in many molecular pathways during its development. Mutations in RAS, combined with the mismatch repair gene defect are currently widely studied in clinics. Such biomarkers provide information for patient risk classification and for the selecting of the appropriate therapy alternatives. Nevertheless, likely, and robust prognostic markers that can identify “high-risk” CRC patients, who can benefit from adjuvant chemotherapy, in early stages, are currently absent. To solve this gap, genomic information has lately gained interest as a potential technique for estimating the likelihood of recurrence. However, due to several limitations of gene-based signatures, these have not yet been realistically used. In this review, we summarise the multiple molecular markers in clinical use for CRC, highlight potential indicators that might become indispensable over the next years, discuss recently discovered gene expression-based assays and emphasise the difficulties in biomarker development.

Linking the Defect of Gamma-Aminobutyric Acid Type A Receptor Subunit Delta Gene to the Microsatellite Instability in Colorectal Cancer

Abstract Introduction: There is primitive data suggest the role of gamma-aminobutyric acid type A receptor subunit delta (GABRD) in the pathogenesis of colorectal cancer. This research aimed to investigate the role of GABRD in patients with microsatellite instability (MSI)-associated colorectal cancer. Materials and Methods: In total, 58 patients diagnosed with MSI-associated adenocarcinoma of colorectal cancer participated in this study. The level of GABRD expression in both cancerous and noncancerous colonic and rectal tissue was done first by immunohistochemistry and then confirmed by quantitative real-time polymerase chain reaction (PCR). In addition, GABRD expression level was correlated with other pathologic parameters of patients. Results: Overall, 72.41% of cases expressed a high level of GABRD at the genetic level, and 67.24% expressed GABRD at the protein level. Moreover, the adjacent normal tissue did not show specific expression of GABRD protein by immunohistochemical analysis. Also, this study demonstrates a significant correlation between GABRD expression to the advanced stages of MSI-associated adenocarcinoma of colorectal cancer. Conclusion: GABRD can be a new and important diagnostic marker in patients with MSI and can be targeted for the treatment of patients with colorectal cancer having MSI. Further studies with larger sample sizes and in vitro studies will be of great value to uncover the pathway by which this gene controls the defect in mismatch repair (MMR) genes.

MLH1 Promotor Hypermethylation in Colorectal and Endometrial Carcinomas from Patients with Lynch Syndrome

Screening for Lynch syndrome (LS) in colorectal cancer (CRC) and endometrial cancer patients generally involves immunohistochemical staining of the mismatch repair (MMR) proteins. In case of MLH1 protein loss, MLH1 promotor hypermethylation (MLH1-PM) testing is performed to indirectly distinguish the constitutional MLH1 variants from somatic epimutations. Recently, multiple studies have reported that MLH1-PM and pathogenic constitutional MMR variants are not mutually exclusive. This study describes 6 new and 86 previously reported MLH1-PM CRCs or endometrial cancers in LS patients. Of these, methylation of the MLH1 gene promotor C region was reported in 30 MLH1, 6 MSH2, 6 MSH6, and 3 PMS2 variant carriers at a median age at diagnosis of 48.5 years [interquartile range (IQR), 39-56.75 years], 39 years (IQR, 29-51 years), 58 years (IQR, 53.5-67 years), and 68 years (IQR, 65.6-68.5 years), respectively. For 31 MLH1-PM CRCs in LS patients from the literature, only the B region of the MLH1 gene promotor was tested, whereas for 13 cases in the literature the tested region was not specified. Collectively, these data indicate that a diagnosis of LS should not be excluded when MLH1-PM is detected. Clinicians should carefully consider whether follow-up genetic MMR gene testing should be offered, with age <60 to 70 years and/or a positive family history among other factors being suggestive for a potential constitutional MMR gene defect.

DNA Damage Response and Mismatch Repair Gene Defects in Advanced and Metastatic Prostate Cancer.

Alterations in DNA damage response (DDR) and related genes are present in up to 25% of advanced prostate cancers (PCa). Most frequently altered genes are involved in the homologous recombination repair, the Fanconi anemia, and the mismatch repair pathways, and their deficiencies lead to a highly heterogeneous spectrum of DDR-deficient phenotypes. More than half of these alterations concern non- BRCA DDR genes. From a therapeutic perspective, poly-ADP-ribose polymerase inhibitors have demonstrated robust clinical efficacy in tumors with BRCA2 and BRCA1 alterations. Mismatch repair-deficient PCa, and a subset of CDK12-deficient PCa, are vulnerable to immune checkpoint inhibitors. Emerging data point to the efficacy of ATR inhibitors in PCa with ATM deficiencies. Still, therapeutic implications are insufficiently clarified for most of the non- BRCA DDR alterations, and no successful targeted treatment options have been established.

PATH-07. GENOMIC AND IMMUNE ANALYSIS OF PRIMARY REPLICATION-REPAIR DEFICIENT (RRD) GLIOMAS REVEALS THREE SUBGROUPS WITH DISTINCT DRIVERS AND RESPONSE TO IMMUNOTHERAPY: AN IRRDC REPORT.

Abstract BACKGROUND Replication-repair deficiency (RRD) caused by germline/somatic defects in mismatch repair (MMRD) and/or polymerase-proofreading genes (PPD) drives 5-10% of high-grade gliomas in children, adolescents, and young adults (CAYA). Despite harbouring high mutation-burden (TMB), the basis of their heterogenous responses to immune-checkpoint inhibitors (ICI) is unknown. METHODS We performed multi-omic and immune analyses on human RRD-glioma specimens and developed murine models to investigate their differential responses to ICI. RESULTS RRD-gliomas in 202 CAYA-patients uniformly harbored high genomic microsatellite-instability and hypermutation (median TMB: 302-mutations/megabase), with specific MMRD mutational signatures contributing to frequent mutations in TP53 (88%), ATRX (83%), RAS/MAPK (77%) and IDH1/2 (17%). Strikingly, common pediatric mutations (K27M, G34R/V and BRAF;p.V600E) unrelated to MMRD signatures were absent, and a unique methylation profile distinct from non-RRD gliomas was observed. Molecularly, RRD-gliomas segregated into three genomic subgroups: RRD1 (MMRD+PPD; 56%), RRD2 (MMRD-only; 27%), and RRD3 (MMRD+IDH1/2; 17%). RRD1 included glioblastomas that developed early in germline CMMRD patients across the cerebral hemispheres and posterior-fossa, harbored extreme TMB (median: 420-mutations/megabase), balanced copy-number profiles, and immunogenic PPD signatures. RRD3 predominated in Lynch syndrome, presented in older children, were located in the forebrain, harbored lower TMB (median: 31-mutations/megabase) and demonstrated specific chromosomal gains/losses. Enrichment for immune-resistant JAK-STAT signalling in the RRD2/RRD3 transcriptome, and presence of high CD8+ T-cells in RRD1-glioma microenvironment, correlated with significantly improved 24-month post-ICI survival of 75% in RRD1 versus 43% and 33% for RRD2/3, respectively (p &amp;lt; 0.0001). Genetically engineered mouse models recapitulated the immuno-genomic phenotype and ICI-responses of the human RRD-subgroups, allowing successful preclinical testing of combinatorial checkpoint-inhibition strategies. Significant temporal and spatial heterogeneity linked to genomic instability and post-therapy immune-escape were observed. CONCLUSION Our data emphasizes an immediate need to distinguish RRD-glioma subgroups in clinical practice and translate our preclinical discoveries to subgroup-specific ICI-based combinatorial clinical trials.

Mutational spectrum of DNA damage and mismatch repair genes in prostate cancer.

Over the past few years, a number of studies have revealed that a significant number of men with prostate cancer had genetic defects in the DNA damage repair gene response and mismatch repair genes. Certain of these modifications, notably gene alterations known as homologous recombination (HRR) genes; PALB2, CHEK2 BRCA1, BRCA2, ATM, and genes for DNA mismatch repair (MMR); MLH1, MSH2, MSH6, and PMS2 are connected to a higher risk of prostate cancer and more severe types of the disease. The DNA damage repair (DDR) is essential for constructing and diversifying the antigen receptor genes required for T and B cell development. But this DDR imbalance results in stress on DNA replication and transcription, accumulation of mutations, and even cell death, which compromises tissue homeostasis. Due to these impacts of DDR anomalies, tumor immunity may be impacted, which may encourage the growth of tumors, the release of inflammatory cytokines, and aberrant immune reactions. In a similar vein, people who have altered MMR gene may benefit greatly from immunotherapy. Therefore, for these treatments, mutational genetic testing is indicated. Mismatch repair gene (MMR) defects are also more prevalent than previously thought, especially in patients with metastatic disease, high Gleason scores, and diverse histologies. This review summarizes the current information on the mutation spectrum and clinical significance of DDR mechanisms, such as HRR and MMR abnormalities in prostate cancer, and explains how patient management is evolving as a result of this understanding.

A unique case of rectal cancer with coexistence of multiple pathways of carcinogenesis

BackgroundColorectal cancer with a global incidence of 10% has multiple pathways implicated in its carcinogenesis. WNT signaling is the principal underlying pathway via APC gene, while defective mismatch repair genes and epigenetic changes also are known to contribute.Case presentationHere, we present an unusual case of rectal adenocarcinoma in a woman, with germline MSH6 and PMS1 mutations, and simultaneous somatic APC and TP53 mutations treated with surgery and adjuvant capecitabine.ConclusionsThe case is unique suggesting a possible interaction between the two pathways and contributing to carcinogenesis in this patient. This also suggests need for a thorough germline and somatic mutation evaluation in select colorectal cancer patients to direct a tailored therapy.

BG13 A rare case of Muir–Torre syndrome

Abstract Muir–Torre syndrome is a rare variant of hereditary nonpolyposis colorectal cancer, also known as Lynch syndrome. It is caused by a defect in mismatch repair genes, which are most commonly inherited in an autosomal dominant fashion. It is characterized clinically by sebaceous neoplasms of the skin associated with at least one visceral malignancy, with colon cancer being the most frequent. The most common genetic defects responsible are in MLH1, MSH2 and MSH6, which results in an increased frequency of unrepaired base mismatching during DNA replication and subsequent microsatellite instability, predisposing to cancer. We present a case of a 59-year-old man with a history of colon cancer who was referred with a rapidly growing irregular nodule on his lower back for the past 2 months, concerning for an amelanocytic melanoma. An excision biopsy was arranged, and subsequent histology demonstrated a sebaceoma. Immunohistochemistry for mismatch repair genes showed loss of expression for MSH2 and MSH6. Upon further questioning, it was found that he had a strong family history of colon cancer (father, brother and two sisters), and he developed colon cancer at 36 years of age, which was treated with a pancolectomy. Genetic testing revealed a mutation in MSH2. Given this history, annual follow-up was arranged for skin surveillance.

HGG-26. GENOMIC AND IMMUNE ANALYSIS OF PRIMARY REPLICATION-REPAIR DEFICIENT (RRD) GLIOMAS REVEALS THREE SUBGROUPS WITH DISTINCT DRIVERS AND RESPONSE TO IMMUNOTHERAPY: AN IRRDC REPORT

Abstract BACKGROUND Replication-repair deficiency (RRD) caused by germline/somatic defects in mismatch repair (MMRD) and/or polymerase-proofreading genes (PPD) drives 5-10% of gliomas in children, adolescents, and young adults (CAYA). Although RRD-gliomas harbour high mutation-burden (TMB), the basis of their heterogenous biology, clinical behavior, and response to immune-checkpoint inhibitors (ICI) is unknown. METHODS We analyzed the genome (whole exome, low-coverage genome), methylome, transcriptome (bulk, single-nuclei), and the immune-microenvironment of RRD-gliomas in a large cohort of IRRDC patients and correlated these with clinical outcomes and response to ICI. RESULTS Gliomas in 202 CAYA-patients uniformly harbored hypermutation and genomic microsatellite-instability. Median TMB was 297-mutations/megabase, with frequent mutations in TP53 (90%), ATRX (85%), RAS/MAPK (80%) and IDH1/2 (15%). MMRD (but not PPD) mutational signatures contributed to the enrichment of driver mutations in POLE, IDH1, and TP53 while common pediatric mutations (K27M, G34R/V and BRAF;p.V600E) not driven by MMRD signatures were absent. Paired analyses suggested acquisition of novel variants driven by the mutational signatures that also impacted the immune microenvironment. Multi-omic analyses classified RRD-gliomas into three subgroups: RRD1 (MMRD+PPD; 60%), RRD2 (MMRD-only; 23%), and RRD3 (MMRD+IDH1/2; 16%). All RRD1-gliomas were glioblastomas with earlier age of onset, enrichment in CMMRD, arose at diverse locations including the posterior-fossa, classified in proximity to methylation-RTK1-subclass, exhibited balanced copy number profiles, harbored the highest TMB (median: 409-mutations/megabase), and immunogenic PPD signatures. Conversely, RRD3-gliomas were frequent in Lynch syndrome, presented at an older age, with predominant localization in the forebrain, more complex genomic instability, clustered in proximity to IDH1-gliomas, and harbored lower TMB (median: 33-mutations/megabase). RRD1-gliomas revealed the highest immune infiltrates with significantly improved median post-ICI survival of 52-months versus &amp;lt;12-months for RRD2/3 (p&amp;lt;0.0001). CONCLUSIONS The distinct genomic subgroups of RRD-gliomas can explain their diverse clinical outcomes, highlighting the need for developing subgroup-specific, immune-directed treatment approaches for these patients.

MDB-12. 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 RRD-medulloblastoma (n=40) were enriched for anaplasia (55%) and localised disease (85%). 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.

Abstract 2737: A gene therapy with a novel PD-L1 inhibitory peptide secretory gene inhibits the growth of colon carcinoma in mice

Abstract In the United States, colorectal cancer (CRC) is the second leading cause of cancer-related death in both sexes. Immune checkpoint blockade therapy (ICBT) has emerged as a powerful new tool for cancer therapy. However, only CRCs associated with microsatellite instability (MSI) or DNA mismatch repair gene defects (dMMR, ~15% of all CRCs) are sensitive to this therapy. This poor sensitivity to ICBT is either due to poor tumor infiltration of functional T cells or T cell exhaustion. To overcome this, we investigated a combination treatment with an oncolytic virus and an immune checkpoint inhibitor (ICI). Because tumor cell oncolysis generates neoantigens and increases tumor immunogenicity, T cell infiltration into the tumor tissue will be increased and the efficacy of ICBT will be enhanced. Accordingly, local immune checkpoint inhibition coupled with oncolysis may be an ideal ICBT. In the present study, a novel peptide that interferes with the PD-1/PD-L1 immune checkpoint pathway, termed PD-L1 inhibitory peptide 3 (PD-L1ip3), was computationally designed, experimentally validated for its specific binding to PD-L1, and evaluated for its antitumor effects in cell culture and in a mouse colon carcinoma syngeneic murine model. Fourteen candidate peptide sequences were generated using the PinaColada algorithm and the x-ray structure of the PD-1:PD-L1 complex and screened in molecular dynamics simulations on the microsecond timescale. Two were chosen for experimental testing, wherein the peptide denoted PD-L1ip3 showed a binding affinity for PD-L1 in the micromolar range. In cell culture studies, treatment with PD-L1ip3, but not a similar peptide with a scrambled sequence, substantially increased death of CT26 colon carcinoma cells when co-cultured with murine CD8+ T cells primed by antigens from CT26 cells. In immunocompetent mice, growth of CT26 tumor cells transduced with the PD-L1ip3 gene by an adenovirus vector was significantly slower than that of un-transduced CT26 cells. This tumor growth attenuation was further enhanced by cotreatment with the peptide form of PD-L1ip3. The present study suggests that this peptide can stimulate host antitumor immunity via a blockade of the PD-1/PD-L1 pathway, thereby increasing CD8+ T cell-induced death of colon carcinoma cells. The tumor site-specific inhibition of PD-L1 by an adenovirus carrying the PD-L1ip3 gene, together with direct peptide treatment, may be used as a local immune checkpoint blockade therapy to inhibit colon carcinoma growth. This research was supported by Kansas State University Johnson Cancer Research center (MT), K-INBRE Scholar Award (MB), National Cancer Institute (MT, JC) and the National Science Foundation (JC). Citation Format: Susumu Ishiguro, Deepa Upreti, Molly Bassette, E. R. Azhagiya Singam, Ravindra Thakkar, Mayme Loyd, Makoto Inui, Jeffrey Comer, Masaaki Tamura. A gene therapy with a novel PD-L1 inhibitory peptide secretory gene inhibits the growth of colon carcinoma in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2737.

Concurrent inactivating mutations and expression losses of RGS2, HNF1A, and CAPN12 candidate tumor suppressor genes in colon cancers.

Microsatellite instability-high (MSI-H) colorectal cancer (CRC) is different from microsatellite stable (MSS) CRC concerning biological, and clinical features. In MSI-H CRCs, defects of mismatch repair genes produce increased mutation accumulation in repetitive DNA sequences. To see whether candidate tumor suppressor genes (TSGs) are altered in MSI-H CRC, we studied frameshift mutation and protein expression of candidate TSGs of RGS2, HNF1A, HNF1B, CAPN12, RCBTB2, ATE1, PKNOX1, and USP19. We found frameshift mutations of RGS2 in 5 (5%), HNF1A in 6 (6%), HNF1B in 2 (2%), CAPN12 in 3 (3%), RCBTB2 in 4 (4%), ATE1 in 2 (2%), PKNOX1 in 2 (2%), and USP19 in 2 (2%) MSI-H CRCs. However, we found no such mutations in MSS CRCs. RCBTB2, CAPN12, HNF1A, and HNF1B frameshift mutations revealed the regional difference in the same tumors. In addition, we identified loss of RGS2, HNF1A, and CAPN12 protein expression irrespective of MSI phenotype in 13-29% of CRCs. The results indicate that many TSGs harbor concurrent inactivating mutations and protein loss in MSI-H CRCs with intratumoral mutational heterogeneity, and that MSS CRCs are altered by protein losses. These alterations could contribute to CRC development and underlying mechanisms and consequences of the TSG alterations remain to be clarified.

Pathogenic Germline Variants in Multiple Myeloma

BACKGROUND: There is growing evidence supporting inherited predisposition to multiple myeloma (MM). Epidemiologic studies have shown that 1st-degree relatives of MM patients (pts) have a 2-4 fold increase in risk of developing MGUS or MM. Genome-wide association studies (GWAS) have identified common SNPs as well as rare high-penetrance variants that collectively explain ~16% of the estimated heritability of multiple myeloma (PMID 30213928). Pathogenic/likely-pathogenic germline variants (PGV) in hereditary cancer genes (HCG) are common in adult cancer patients (~8%, PMID 29625052), but prevalence in MM is not known. The aim of our study is to investigate the occurrence of PGV in newly-diagnosed MM (NDMM), and to describe clinical characteristics & outcomes of carriers.METHODS: We analyzed MMRF CoMMpass data (version IA16) and identified 895 NDMM pts for whom whole-exome sequencing of germline DNA was available. We used the clinical annotation pipeline from Sema4, a CLIA/CAP certified genetic testing laboratory, to identify pts with PGV according to ACMG variant classification guidelines. We compared clinical characteristics & disease phenotypes of PGV carriers vs non-carriers. We used Chi-Square and Fisher's Exact tests to assess statistical significance, which we defined as a two-sided p value < 0.05. Logistic regression models were used for multivariate analyses. Kaplan-Meier method and Cox proportional-hazards models were used for uni- and multivariate survival analysis, respectively. Bonferroni method was used to account for multiple testing.RESULTS: We identified 83 PGV in 31 distinct HCG in 79 (8.8%) of 895 NDMM pts (83% European ancestry) [Figure 1A]. Most PGV involved DNA damage repair (DDR) genes (78%), and homologous recombination (HR) genes were the most commonly mutated (34%). PGV in CHEK2 were the most common (n=10, 1.1% of all MM pts). 2 pts carried PGV in TP53 and reported extensive family history of Li-Fraumeni-associated cancers (breast, sarcoma, gastric & melanoma). 6 pts had germline mismatch repair (MMR) gene defects (1:149, considerably higher than the estimated prevalence of Lynch syndrome in Western populations). 4 pts carried PGV in BRCA2 (previously identified in a family study as a potential MM predisposition gene, PMID 11904319). MM pts with a family history of hematologic malignancy (leukemia, lymphoma or MM) in a 1st or 2nd-degree relative were significantly more likely to carry PGV (22 vs 7.6%, OR=3.3, p<0.001), an association that remained significant in MVA (OR=4.1, p<0.001). CHEK2 variants emerged as leading drivers of this correlation (OR 18.2, 95% CI 4.1-75, adjusted p<0.01), & especially protein-truncating founder variant c.1100delC. Likelihood of being diagnosed w/ MM before age 40 was significantly higher in PGV carriers (6.3 vs 1.8%, OR=3.7, p=0.025). 25% of those younger than 40 y/o carried PGV, but none of these were in DDR-HR genes, a notable difference with other age groups (0 vs 41%, p=0.02). 2/6 MMR PGV were detected in pts diagnosed before age 40. In univariate survival analysis, DDR-PGV carriers had a significant PFS1 advantage over non-carriers (median 52 vs 35 months, p=0.008), as well as a non-significant OS advantage (p=0.08). PFS1 difference remained significant in MVA after adjusting for age, ISS stage, high-risk cytogenetics, treatment type & transplant status (OR 0.65, 95% CI 0.44-0.97, p=0.03) [Figure 1B].CONCLUSIONS: PGV in HCG were common (8.8%) in this large cohort of NDMM pts of predominantly European ancestry, especially in those with a family history of hematologic malignancy (1:4, with high prevalence of CHEK2 variants & particularly protein-truncating founder variant c.1100delC), and in those diagnosed before age 40 (1:4). Routine screening in high-prevalence subgroups might be warranted, as carriers may benefit from counseling and enrollment in early cancer detection programs. We observed a clinically and statistically significant PFS1 advantage in carriers of PGV in DDR genes, possibly due to increased sensitivity to MM therapies, a well-described phenomenon in other cancer types (PMID 33158305). Prospective validation of these findings is needed to better understand prognostic & therapeutic implications of PGV in MM. [Display omitted] DisclosuresChari: Karyopharm: Consultancy; Takeda Pharmaceutical Company: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; Pharmacyclics: Research Funding; Amgen: Consultancy, Research Funding; Novartis Pharmaceuticals: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Janssen Pharmaceuticals: Consultancy, Research Funding; Sanofi Genzyme: Consultancy; Oncopeptides: Consultancy; Antegene: Consultancy; Glaxosmithkline: Consultancy; Secura Bio: Consultancy. Richard: Karyopharm, Janssen: Honoraria. Richter: Sanofi: Consultancy; Antengene: Consultancy; Karyopharm: Consultancy; BMS: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Adaptive Biotechnologies: Speakers Bureau; Celgene: Speakers Bureau; Janssen: Speakers Bureau; X4 Pharmaceuticals: Consultancy; Oncopeptides: Consultancy; Adaptive Biotechnologies: Consultancy; Secura Bio: Consultancy; Astra Zeneca: Consultancy. Parekh: Foundation Medicine Inc: Consultancy; Amgen: Research Funding; PFIZER: Research Funding; CELGENE: Research Funding; Karyopharm Inv: Research Funding.

Low prevalence of biliary tract cancer with defective mismatch repair genes in a Japanese hospital-based population.

Recent studies have reported that immune checkpoint inhibitors are effective against various defective mismatch repair (dMMR)/microsatellite instability-high (MSI-H) cancers. A limited number of reports are available on the frequency of dMMR/MSI-H carcinoma in biliary tract cancer (BTC), describing its clinicopathological characteristics and prognosis. The latter carcinoma is also associated with Lynch syndrome (LS). The present study was performed to investigate the frequency of patients with dMMR/MSI-H in BTC and the clinical characteristics of BTC with dMMR/MSI-H in a single institution in Japan. A total of 116 patients with BTC who underwent curative surgical resection at Kagawa University Hospital between January 2008 and December 2017 were included. The protein expression levels of the mismatch repair (MMR) genes [mutL homolog 1 (MLH1), mismatch repair endonuclease PMS2 (PMS2), MutS homolog (MSH)2 and MSH6] were assessed by immunohistochemistry (IHC) using formalin-fixed paraffin-embedded tissue specimens. Subsequently, MSI testing was performed on patients who exhibited loss of MMR protein expression. Loss of expression of one or more proteins was detected in five cases (4.3%). Loss of MLH1/PMS2 expression was observed in one case of intrahepatic cholangiocarcinoma, whereas loss of PMS2 expression was noted in one case of perihilar cholangiocarcinoma. Loss of MSH2/MSH6 and MSH6 expression was noted in two cases of distal cholangiocarcinoma and loss of PMS2 expression in one case of ampullary carcinoma. Out of the five patients, two demonstrated MSI-H. Microsatellite stability was observed in two cases and for one case, no data were available. Two MSI-H cases were patients with loss of expression of MLH1/PMS2 and MSH2/MSH6. None of the five patients exhibited a past medical history or family history of suspected LS. The frequency of dMMR in BTC was ~5%, which was similar to that reported by similar studies performed in other countries. In the present study, IHC appeared to be more useful than MSI testing for detecting MMR abnormalities with regards to the detection rate. Furthermore, there may only be a limited number of patients with BTCs who are likely to benefit from the therapeutic effects of treatment with immune checkpoint inhibitors.

P-063: Pathogenic germline variants in hereditary cancer genes in patients with Multiple Myeloma

<h3>Background</h3> GWAS have identified common SNPs & rare high-penetrance variants that explain ~16% of the estimated heritability of multiple myeloma (MM)(PMID 30213928). Pathogenic/likely-pathogenic germline variants (PGV) in hereditary cancer genes (HCG) are common in adult cancer pts (~8%), but prevalence in MM is not known. The aim of our study is to investigate the occurrence of PGV in newly-diagnosed MM (NDMM) and describe clinical characteristics & outcomes of carriers. <h3>Methods</h3> We analyzed MMRF CoMMpass data & identified 895 NDMM pts with whole-exome sequencing of germline DNA. We used the clinical annotation pipeline from Sema4, a CLIA/CAP certified genetic testing laboratory, to identify pts with PGV according to ACMG variant classification guidelines. We compared clinical characteristics & disease phenotypes of PGV carriers vs non-carriers. We used Chi-Square and Fisher's Exact tests to assess statistical significance. <h3>Results</h3> We identified 83 PGV in 31 distinct HCG in 79 (8.8%) of 895 NDMM pts (83% European ancestry). Most PGV involved DNA damage repair (DDR) genes (78%), & homologous recombination (HR) genes were the most commonly mutated (34%). PGV in CHEK2 were most common (n=10,1.1% of all MM pts). 2 pts carried PGV in TP53, & 6 pts had mismatch repair (MMR) gene defects (1:149). MM pts with family history (FH) of hematologic malignancy (HM) in a 1st or 2nd-degree relative were more likely to carry PGV (22 vs 7.6%,OR=3.3,p<0.001), an association that remained significant in multivariate analysis (MVA) (OR=4.1,p<0.001). CHEK2 variants were leading drivers of this correlation (OR 18,adjusted p<0.01), & especially protein-truncating founder variant c.1100delC. Likelihood of MM dx before age 40 was significantly higher in PGV carriers (6.3 vs 1.8%,OR=3.7,p=0.025). 25% of those <40y/o carried PGV, but none of these were in DDR-HR genes, a notable difference with other age groups (0 vs 41%,p=0.02). 2/6 MMR PGV were detected in pts <40y/o. In univariate analysis, DDR-PGV carriers had a significant PFS1 advantage (median 52 vs 35 months,p=0.008) & a non-significant OS advantage (p=0.08). PFS1 difference remained significant in MVA adjusting for age, ISS stage, high-risk cytogenetics, treatment type & transplant status (OR 0.65,95% CI 0.44-0.97,p=0.03). <h3>Conclusions</h3> PGV in HCG were common (8.8%) in this large cohort of NDMM pts of predominantly European ancestry, especially in those with FH of HM (1:4, with high prevalence of CHEK2 variants), and in those <40y/o (1:4). Routine screening in high-prevalence subgroups may be warranted, as carriers can benefit from counseling & enrollment in early cancer detection programs. We observed a clinically & statistically significant PFS1 advantage in PGV carriers, possibly due to increased sensitivity to MM therapies, a well-described phenomenon in other cancers (PMID 33158305). Prospective validation of these findings is needed to better understand prognostic & therapeutic implications of PGV in MM.

Evaluating morphological features for predicting microsatellite instability status in colorectal cancer

BackgroundColorectal cancer (CRC) is one of the commonest cancers worldwide, with incidence rates in India being around 4%. It is a heterogeneous disease with multiple established prognostic factors. Ten to fifteen percent originate from microsatellite instability (MSI) pathway, characterized by defect in mismatch repair (MMR) gene. Identification of MMR defective protein is relevant for diagnosis, prognosis, and prediction. Certain clinical and histological features are known to be associated with defective MMR genes. The objectives of this study are to find the prevalence of MSI in CRC to identify features associated with MSI and assess the value of histopathology in predicting MSI. MethodsWe evaluated various clinical and histological parameters for identifying prognostically favorable colon cancers in a tertiary hospital. One hundred fifty colon cancers were evaluated, and MSI status was correlated with clinicopathologic variables. ResultsThe prevalence of MSI in CRC was found to be 11.3%. The factors associated with MSI were tumor differentiation, stage, tumor site, tumor size, tumor-infiltrating lymphocytes, Crohn’s-like lymphoid reaction, and dirty necrosis. We have defined a “P” score for prediction of MSI using the clinicohistological parameters, which could be used to select patients who are to be tested for MSI. ConclusionAssessment of clinical and histopathological features will help in patient stratification and selection of patients for MSI testing. The evaluation is economical, reproducible, and easy to apply.

Abstract 868: Heritable cancer mutations in multiple myeloma

Abstract Background: Epidemiologic studies have shown familial aggregations of multiple myeloma (MM) and other hematologic malignancies, but little is known about heritable genetic susceptibilities in these patients. The purpose of this study is to investigate the prevalence of known germline cancer-predisposing mutations (CPM) in patients with multiple myeloma (MM). Methods: We analyzed a set of 895 newly diagnosed MM patients from the Multiple Myeloma Research Foundation (MMRF) CoMMpass registry for whom whole-exome sequencing of germline DNA data was available. We used the clinical annotation pipeline from Sema4, a CLIA- and CAP-certified genetic testing laboratory, in a research context, to identify patients with pathogenic or likely pathogenic CPM according to ACMG variant classification guidelines, and compared their characteristics against those without CPM. Statistical significance was assessed using the Chi-Square and Fisher's Exact tests and defined as a two-sided p value &amp;lt;0.05. Results: We identified 83 germline CPM in 31 distinct genes in 79 (8.8%) of 895 patients. Homologous recombination (HR) pathway gene mutations were the most common (n = 38, 46%), with CHEK2 being the most frequently mutated gene (n = 10, 12%). Notably, the number of mismatch repair (MMR) gene defects was considerably higher (n = 6, 1:149) than the estimated prevalence in the general population. CPM carriers were significantly more likely to be diagnosed with MM before age 40 (6.3% vs 1.8%, p = 0.025) and to have a family history (FH) of MM or leukemia (16.4% vs 6.3%, p = 0.01) than those without a CPM. Likelihood of having a FH of any malignancy was also significantly higher in the CPM group (70.9% vs 54.5%, p = 0.028). Interestingly, common CHEK2 founder mutations were observed at a much higher rate in patients with a FH of MM or leukemia (8.7% vs 0.68%, p = 0.0013). Conclusions: Our results suggest that a significant proportion of patients with MM carry germline cancer-predisposing mutations, especially those diagnosed at a very young age (&amp;lt;40 years old), and those with a family history of MM or leukemia. The observation that 6 out of 895 patients with MM had pathogenic MMR variants suggests that MM may be a previously unsuspected component of Lynch syndrome. Common CHEK2 founder mutations were strikingly frequent in patients with a FH of MM, suggesting that FH modifies the penetrance of these variants in MM predisposition, as has been observed for other cancers. Citation Format: Santiago Thibaud, Aaron Etra, Ryan Subaran, Zachry Soens, Scott Newman, Rong Chen, Ajai Chari, Hearn J. Cho, Sundar Jagannath, Deepu Madduri, David T. Melnekoff, Shambavi Richard, Joshua Richter, Larysa Sanchez, Alessandro Lagana, Samir Parekh, Kenan Onel. Heritable cancer mutations in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 868.

Abstract PS18-18: Microsatellite instability high (MSI-H) detection utilizing targeted plasma based genotyping in metastatic breast cancer

Abstract Background: Microsatellite instability (MSI) occurs in some tumors from defects in mismatch repair genes. Immune checkpoint inhibition is approved for treatment of MSI-high (MSI-H) tumors. Plasma-based genotyping assays are being used more commonly in breast cancer to identify targetable mutations. The objective of this study was to evaluate MSI-H detection by plasma-based genotyping in metastatic breast cancer (MBC) and understand the co-existing genomic landscape. In selected cases, correlations of MSI-H with clinical characteristics were determined. Methods:Patients who had MSI-H detected by cell-free DNA (cfDNA) analysis via Guardant360™ testing (next-generation sequencing (NGS), up to 74 gene panel) between 9/27/2018 and 3/12/2020 with a diagnosis of breast cancer reported on the test requisition form were identified from a de-identified database. MSI detection is based on plasma NGS of 90 microsatellite sites and combining observed read sequences and molecular barcoding information in a probabilistic model (Willis, 2019). A retrospective review was conducted to identify demographic and genomic characteristics of patients with MSI-H findings. For 11 patients, clinical data was provided by the treating physician for evaluation of demographics and response to immunotherapy. Results:Of 7824 patients with breast cancer with alterations in plasma NGS, 40 (0.5%) were MSI-H. Of these 40 patients, 40 (100%) were female. The median age was 61 (range 39-92). The median number of genomic alterations per sample was 13 (range 4-69), and the median maximum allelic fraction (MAF) was 15.9% (range 0.92-54.2), compared to 4 and 2.7%, respectively, in all breast cancer samples reported in this time period. Table 1 depicts the most common co-existing non-synonymous mutations, which included DNA damage repair genes (ATM and BRCA2). Clinical data was available for 11/40 MSI-H patients with MBC. Of these 11 patients, 4 (36%) had triple-negative MBC (mTNBC), and 7 (64%) had hormone receptor positive (HR+)/HER2- MBC. None had known Lynch syndrome. Seven patients received treatment with an immune checkpoint inhibitor (2 atezolizumab/nab-paclitaxel, 3 pembrolizumab, 1 pembrolizumab/capecitabine followed by pembrolizumab/eribulin, and 1 nivolumab). Treatment duration was available for 5 patients, and the median duration of treatment was 108 days (range 65-273 days). Two patients had durable benefit (1 with stable disease for 10 cycles, and another on treatment for &amp;gt; 152 days), both of whom had mTNBC and were treated in the first-line setting with atezolizumab/nab-paclitaxel. Conclusions:Plasma-based genotyping assays can identify the presence of MSI-H in breast cancer, including in patients with mTNBC and HR+/HER2- MBC. MSI-H breast cancers had a higher number of somatic alterations and MAF, suggesting higher tumor burden and genomic instability. The co-existing genomic landscape is heterogeneous, and mutations in TP53, PI3KCA, ESR1, RB1, NOTCH1, and ARID1A, and DNA damage repair genes (ATM and BRCA2) may be present. Since plasma based genotyping is increasingly being utilized to identify actionable mutations including PI3KCA, the ability to detect additional genomic alterations such as MSI-H extends the potential clinical application. However, the clinical utility of MSI detection by cfDNA needs to be determined and further prospective research is needed to validate the use of immunotherapy in cfDNA detected MSI-H MBC. Table 1.GeneNumber (%) of MSI-H patients with ≥ 1 mutationTP5331 (78)PI3KCA25 (63)ESR120 (50)RB116 (40)NOTCH115 (38)ARID1A15 (38)ATM14 (35)BRAF14 (35)EGFR13 (33)PDGFRA11 (28)PTEN11 (28)APC11 (28)MET10 (25)KIT10 (25)FGFR210 (25)BRCA210 (25) Citation Format: Neelima Vidula, Katherine Hesler, Kristin Price, Andrew Lipman, Georges Azzi, Ahmed Elkhanany, Tarek Sabagh, Dejan Juric, Estelamari Rodriguez, Shumei Kato, Joyce O'Shaughnessy, Aditya Bardia. Microsatellite instability high (MSI-H) detection utilizing targeted plasma based genotyping in metastatic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-18.

Abstract PR008: Identifying mechanisms of immune evasion in microsatellite instable endometrial cancer mouse models

Abstract Introduction: Microsatellite instability (MSI), caused by defects in DNA mismatch repair genes, including MLH1 and MSH2, occur in 30% of endometrial cancers (EC). High mutational loads associated with MSI have been associated with greater immunogenicity, and an increase in immune inhibitory factors such as PD-1 and PD-L1. While immunotherapy with anti-PD-L1 has shown promising results in MSI ECs, up to 40% did not respond, suggesting there are alternate immunosuppressive mechanisms. To improve understanding of response/resistance to immunotherapy, we evaluated immune activation and evasion using a novel immunocompetent mouse model of MSI EC. Methods: Endometrial lesions were evaluated in mice with MSH2 loss targeted to the endometrium (PRCre+MSH2flox/flox). When aged to 12-16 months, 22% develop hyperplasia and 18% develop spontaneous EC. MSI status was confirmed by PCR. Immune infiltration and function were assessed by immunohistochemistry (IHC) and Nanostring digital spatial profiling. Tumors were evaluated for tumor infiltrating lymphocytes (TIL) based on CD8a expression and found to separate into TILHIGH and TILLOW groups and further characterized to determine immune activation/resistance mechanisms using transcriptome analysis with Affymetrix Clariom D assay and validation by RT-PCR. A gene signature score approach was used to quantify immune-related expression changes for tumors from PRCre+MSH2flox/flox mice and MSI EC patient data from TCGA. Cell lines were generated from tumors and used to evaluate a syngeneic orthotopic mouse model for immunotherapy studies, where immune infiltration was characterized. Results: All endometrial tumors were MSI and included endometrioid, serous, and mixed histologies. Tumors showed varying degrees of CD8+ T cell infiltration, irrespective of histology. TILHIGH tumors had increased infiltration of dendritic cells and Type I interferon response, including genes IFI203 (fold-change, FC=18.5), IFI204 (FC=11.1) and CXCL9 (FC=5.7). There was no significant difference in type II interferon response. TILLOW tumors had low expression of IFN-associated genes. TILHIGH tumors also exhibited increased immune exhaustion markers such as TIM3 (FC=2.4) and LAG3 (FC=3.2). Tumoral PD-L1 expression was negative (&amp;lt;1% of positive cells). Similar to our mouse model, TILHIGH MSI uterine cancer patients from TCGA also showed activation of type I interferon, increased dendritic cell infiltration, and increased T cell exhaustion markers. Primary cell lines used in an orthotopic mouse model retained the immune profile of the original tumor. Conclusions: Elucidating immune activation and evasion mechanisms is critical for improving immunotherapies. The PRCre+MSH2flox/flox mouse model reflects the spectrum of immunogenicity observed in patients. A panel of primary cell lines generated from this model are an important tool that enable studies of immunotherapy efficacy and resistance mechanisms, in both immunogenic and non-immunogenic tumors. Citation Format: Brenda Melendez, Emily Hinchcliff, Nisha Gokul, Xiaoping Su, Daniel McGrail, Elizabeth Whitley, Russell Broaddus, Rosemarie Schmandt, Karen Lu, Melinda Yates. Identifying mechanisms of immune evasion in microsatellite instable endometrial cancer mouse models [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PR008.

Progress Report: New insights into the prevention of CRC by colonoscopic surveillance in Lynch syndrome.

Lynch syndrome is the most frequent hereditary colorectal cancer (CRC) syndrome, affecting approximately 1 in 300 in the Western population. It is caused by pathogenic variants in the mismatch repair (MMR) genes including MLH1, MSH2 (EPCAM), MSH6 and PMS2, and is associated with high risks of CRC, endometrial cancer and other cancers. In view of these risks, carriers of such variants are encouraged to participate in colonoscopic surveillance programs that are known to substantially improve their prognosis. In the last decade several important studies have been published that provide detailed cancer risk estimates and prognoses based on large numbers of patients. These studies also provided new insights regarding the pathways of carcinogenesis in CRC, which appear to differ depending on the specific MMR gene defect. In this report, we will discuss the implications of these new findings for the development of new surveillance protocols.