Microsatellite Unstable Cancers Research Articles

Effector Function Characteristics of Exhausted CD8+ T-cell in Microsatellite Stable and Unstable Gastric Cancer.

Gastric cancer exhibits molecular heterogeneity, with the microsatellite instability high (MSI-H) subtype drawing attention for its distinct features. Despite a higher survival rate, MSI-H gastric cancer lack significant benefits from conventional chemotherapy. The immune checkpoint inhibitors (ICIs), presents a potential avenue, but a deeper understanding of the tumor immune microenvironment of MSI-H gastric cancer is essential. We explored the molecular characteristics of CD8+ T cell subtypes in three MSI-H and three microsatellite stable (MSS) gastric cancer samples using single-cell RNA sequencing and spatial transcriptome analysis. In MSI-H gastric cancer, significantly higher proportions of effector memory T cell (Tem), exhausted T cell (Tex), proliferative exhausted T cell (pTex), and proliferative T cell were observed, while MSS gastric cancer exhibited significantly higher proportions of mucosal-associated invariant T (MAIT) cell and NKT cell. In MSI-H gastric cancer, Tex and pTex exhibited a significant upregulation of the exhaustion marker LAG3, as well as elevated expression of effector function markers such as IFNG, GZMB, GZMH, and GZMK, compared to those in MSS gastric cancer. The IFN-γ signaling pathway of Tex and pTex was retained compared to those of MSS gastric cancer. The spatial transcriptome analysis demonstrates the IFN-γ signaling pathway between neighboring Tex and malignant cell, showcasing a significantly elevated interaction in MSI-H gastric cancer. Our study reveals novel finding indicating that IFN-γ signaling pathway is retained in Tex and pTex of MSI-H gastric cancer, offering a comprehensive perspective for future investigations into immunotherapy for gastric cancer.

Novel WRN Helicase Inhibitors Selectively Target Microsatellite Unstable Cancer Cells.

Microsatellite-unstable (MSI) cancers require WRN helicase to resolve replication stress due to expanded DNA (TA)n-dinucleotide repeats. WRN is a promising synthetic lethal target for MSI tumours, and WRN inhibitors are in development. Here, we used CRISPR-Cas9 base editing to map WRN residues critical for MSI cells, validating the helicase domain as the primary drug target. Fragment-based screening led to the development of potent and highly selective WRN helicase covalent inhibitors. These compounds selectively suppressed MSI model growth In vitro and In vivo by mimicking WRN loss, inducing DNA double-strand breaks at expanded TA-repeats and DNA damage. Assessment of biomarkers in preclinical models linked TA-repeat expansions and mismatch repair (MMR) alterations to compound activity. Efficacy was confirmed in immunotherapy-resistant organoids and patient-derived xenograft (PDX) models. The discovery of potent, selective covalent WRN inhibitors provides proof of concept for synthetic-lethal targeting of WRN in MSI cancer and tools to dissect WRN biology.

Clinical, pathological, and adjuvant chemotherapy use differences among microsatellite unstable and microsatellite stable colon cancers

BackgroundColon cancers are categorized into mismatch repair deficient/microsatellite unstable (MSI-H) and mismatch repair proficient/microsatellite stable (MSS) cancers. This study aims to compare the disease characteristics and trends in the utilization of cancer therapies across different age groups and stages in these two groups. MethodsMSI-H and MSS colon adenocarcinomas from 2010 to 2016 were identified using the National Cancer Database. We compared patient and disease characteristics between the two groups and evaluated the use of adjuvant chemotherapy across age groups and cancer stages. Within MSI-H and MSS groups, we conducted a landmark analysis after propensity score matching for adjuvant chemotherapy versus no chemotherapy to determine its effect on survival. ResultsOf the 542,368 patients that met inclusion criteria, 120,751 (22%) had mismatch repair results available—out of these 96,928 (80%) had MSS colon cancers while 23,823 (19.7%) had MSI-H cancers. MSI-H disease had a bimodal age distribution (<40 years = 22%; ≥75 years = 26%) and was frequent among females (22%) and non-Hispanic Whites (20%). Among those < 65 years, 15% of low-risk stage 2 MSI-H patients and 40% of high-risk stage 2 MSI-H patients received adjuvant chemotherapy. More than two-thirds of stage 3 patients <65 years received adjuvant chemotherapy in both groups. After conducting propensity-score matching for age, gender, and co-morbidities, we found that adjuvant chemotherapy use had a trend towards lower overall survival (OS) in low-risk stage 2 MSI-H (HR = 1.8 [95% CI, 0.8–4.02]) and high-risk stage 2 MSI-H (HR = 1.42 [95% CI, 0.96–2.12]) groups. Adjuvant chemotherapy significantly improved OS in stage 3 colon cancer patients irrespective of microsatellite status or risk category of disease. ConclusionsMSI-H colon cancer had bimodal age distribution. Among stage 2 MSI-H patients <65 years, a notable proportion received adjuvant chemotherapy. Among MSI-H stage 2 patients, adjuvant chemotherapy use was associated with lower survival while it significantly improved survival for stage 3 patients, irrespective of MSI status.

Abstract 6590: Novel WRN helicase inhibitors selectively target microsatellite unstable cancer cells

Abstract Microsatellite-unstable (MSI) cancers depend on the WRN helicase to resolve replication stress from expanded (TA)-dinucleotide repeats. WRN is a promising synthetic lethal target for MSI tumours and WRN inhibitors are being developed. Here, we used CRISPR-Cas9 base editing to map WRN residues critical for MSI synthetic lethality, validating the helicase domain as the primary target and guiding inhibitor discovery. Fragment-based screening led to the discovery of potent and highly selective WRN helicase covalent inhibitors from the same chemical series. These compounds strikingly and selectively suppressed MSI model growth in vitro and in vivo by mimicking genetic WRN loss, inducing DNA double-strand breaks at expanded TA repeats and DNA damage response. We characterized WRN inhibition across cancer cell lines and patient-derived tumour organoids, enabling biomarker assessment of TA-repeat expansions and MMR status to refine patient selection, with compound efficacy correlating with TA-repeat expansions in MSI cells. Efficacy was also confirmed in patient-derived organoids and PDX models refractory to immunotherapy. The discovery of potent and selective covalent WRN inhibitors provides proof-of-concept for targeting MSI cancers and provides tools to unlock the molecular dissection of the WRN biology and MSI synthetic lethality. Citation Format: Gabriele Picco, Yanhua Rao, Angham Al Saedi, Sara F. Vieira, Yang Lee, Ray Shenje, Shriram Bhosle, Samantha Walker, Kieron May, Carmen Herranz-Ors, Cansu Dicer, Freddy Gibson, Ruby Banerjee, Thilo Werner, Josh Cottom, Yang Peng, Nanhua Deng, Phil Landis, Thomas Angel, Jacob Bush, Matthew G. Rees, Melissa M. Ronan, Jennifer A. Roth, Howard Lightfoot, Francesca Zappacosta, Jonathan Pettinger, Syd Barthorpe, Chris Eberl, Brian Jones, Jess Schneck, Dennis Murphy, Josh Taygerly, Mike DeMartino, Matthew Coelho, Jon Houseley, Benjamin Schwartz, Mathew J. Garnett. Novel WRN helicase inhibitors selectively target microsatellite unstable cancer cells [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 6590.

Comprehensive mapping of cell fates in microsatellite unstable cancer cells supports dual targeting of WRN and ATR.

Addiction to the WRN helicase is a unique vulnerability of human cancers with high levels of microsatellite instability (MSI-H). However, while prolonged loss of WRN ultimately leads to cell death, little is known about how MSI-H cancers initially respond to acute loss of WRN-knowledge that would be helpful for informing clinical development of WRN targeting therapy, predicting possible resistance mechanisms, and identifying useful biomarkers of successful WRN inhibition. Here, we report the construction of an inducible ligand-mediated degradation system in which the stability of endogenous WRN protein can be rapidly and specifically tuned, enabling us to track the complete sequence of cellular events elicited by acute loss of WRN function. We found that WRN degradation leads to immediate accrual of DNA damage in a replication-dependent manner that curiously did not robustly engage checkpoint mechanisms to halt DNA synthesis. As a result, WRN-degraded MSI-H cancer cells accumulate DNA damage across multiple replicative cycles and undergo successive rounds of increasingly aberrant mitoses, ultimately triggering cell death. Of potential therapeutic importance, we found no evidence of any generalized mechanism by which MSI-H cancers could adapt to near-complete loss of WRN. However, under conditions of partial WRN degradation, addition of low-dose ATR inhibitor significantly increased their combined efficacy to levels approaching full inactivation of WRN. Overall, our results provide the first comprehensive view of molecular events linking upstream inhibition of WRN to subsequent cell death and suggest that dual targeting of WRN and ATR might be a useful strategy for treating MSI-H cancers.

ALK, ROS1, RET and NTRK1-3 Gene Fusions in Colorectal and Non-Colorectal Microsatellite-Unstable Cancers.

This study aimed to conduct a comprehensive analysis of actionable gene rearrangements in tumors with microsatellite instability (MSI). The detection of translocations involved tests for 5'/3'-end expression imbalance, variant-specific PCR and RNA-based next generation sequencing (NGS). Gene fusions were detected in 58/471 (12.3%) colorectal carcinomas (CRCs), 4/69 (5.8%) gastric cancers (GCs) and 3/65 (4.6%) endometrial cancers (ECs) (ALK: 8; RET: 12; NTRK1: 24; NTRK2: 2; NTRK3: 19), while none of these alterations were observed in five cervical carcinomas (CCs), four pancreatic cancers (PanCs), three cholangiocarcinomas (ChCs) and two ovarian cancers (OCs). The highest frequency of gene rearrangements was seen in KRAS/NRAS/BRAF wild-type colorectal carcinomas (53/204 (26%)). Surprisingly, as many as 5/267 (1.9%) KRAS/NRAS/BRAF-mutated CRCs also carried tyrosine kinase fusions. Droplet digital PCR (ddPCR) analysis of the fraction of KRAS/NRAS/BRAF mutated gene copies in kinase-rearranged tumors indicated that there was simultaneous co-occurrence of two activating events in cancer cells, but not genetic mosaicism. CRC patients aged above 50 years had a strikingly higher frequency of translocations as compared to younger subjects (56/365 (15.3%) vs. 2/106 (1.9%), p = 0.002), and this difference was particularly pronounced for tumors with normal KRAS/NRAS/BRAF status (52/150 (34.7%) vs. 1/54 (1.9%), p = 0.001). There were no instances of MSI in 56 non-colorectal tumors carrying ALK, ROS1, RET or NTRK1 rearrangements. An analysis of tyrosine kinase gene translocations is particularly feasible in KRAS/NRAS/BRAF wild-type microsatellite-unstable CRCs, although other categories of tumors with MSI also demonstrate moderate occurrence of these events.

Cancer-specific functional profiling in microsatellite-unstable (MSI) colon and endometrial cancers using combined differentially expressed genes and biclustering analysis.

Microsatellite-unstable (MSI) cancers have distinct genetic and clinical features from microsatellite-stable cancers, but the molecular functional differences between MSI cancers originating from different tissues or organs have not been well studied because the application of usual differentially expressed gene (DEG) analysis is error-prone, producing too many noncancer-specific normally functioning genes. To maximize therapeutic efficacy, biomarkers reflecting cancer-specific differences between MSI cancers of different tissue origins should be identified. To identify functional differences between MSI colon and endometrial cancers, we combined DEG analysis and biclustering instead of DEG analysis alone and refined functionally relevant biclusters reflecting genuine functional differences between the 2 tumors. Specifically, using The Cancer Genome Atlas and genome-tissue expression as data sources, gene ontology (GO) enrichment tests were performed after routinely identifying DEGs between the 2 tumors with the exclusion of DEGs identified in their normal counterparts. Cancer-specific biclusters and associated enriched GO terms were obtained by biclustering with enrichment tests for the preferences for cancer type (either colon or endometrium) and GO enrichment tests for each cancer-specific bicluster, respectively. A novel childness score was developed to select functionally relevant biclusters among cancer-specific biclusters based on the extent to which the enriched GO terms of the biclusters tended to be child terms of the enriched GO terms in DEGs. The selected biclusters were tested using survival analysis to validate their clinical significance. We performed multiple sequential analyses to produce functionally relevant biclusters from the RNA sequencing data of MSI colon and endometrial cancer samples and their normal counterparts. We identified 3066 cancer-specific DEGs. Biclustering analysis revealed 153 biclusters and 41 cancer-specific biclusters were selected using Fisher exact test. A mean childness score over 0.6 was applied as the threshold and yielded 8 functionally relevant biclusters from cancer-specific biclusters. Functional differences appear to include gland cavitation and the TGF-β receptor, G protein, and cytokine pathways. In the survival analysis, 6 of the 8 functionally relevant biclusters were statistically significant. By attenuating noise and applying a synergistic contribution of DEG results, we refined candidate biomarkers to complement tissue-specific features of MSI tumors.

Abstract 6652: Overcoming immune resistance in DNA mismatch repair deficient tumors

Abstract Patients with Lynch Syndrome, an inherited mismatch repair deficiency, have an increased risk for developing microsatellite unstable (MSI-H) cancers. Our team recently identified shared immunogenic frameshift peptides in patient tumors that can be targets of T cell surveillance and preventative MSI-H cancer vaccines. We hypothesize that in Lynch Syndrome some premalignant lesions are capable of evading immune surveillance from cytotoxic T lymphocytes due to immune checkpoint expression and immunosuppression from tumor, myeloid and stromal cell populations. Immune checkpoint blockade (ICB) showed promising results in the treatment of MSI-H tumors and are being evaluated in the adjuvant setting of patients with Lynch syndrome post surgical resection. However, a significant percentage of advanced MSI-H tumors resist ICB suggesting evolving mechanisms of immune resistance. We will use a MSI-H in vivo mouse model and additionally develop 3D spheroids cocultured with immune cells to characterize the underlying immune resistance mechanisms. The MSI-H mouse model will first be applied to identify shared frameshifts in MSI-H tumors by WES to develop vaccination approaches preventing tumor growth. Then, we will quantify MSI-H tumor growth and immune infiltration in the MSI-H mouse model, with or without ICB. We will analyze the presence of immunosuppressive pathways and myeloid subsets in high growth resisting tumors by immunohistochemistry, scRNAseq, spatial transcriptomics and flow cytometry. In addition, we will perform short-term in vitro spheroid-splenic cell co-cultures to better characterize the early immune resistance mechanisms. We will also perform these spheroid-immune cell co-cultures using the immune cells taken from mice after vaccination or ICB to identify the early myeloid immune resistance mechanisms. Overall, the identification of shared immune frameshifts in MSI-H tumors will allow developing peptide vaccination approaches to prevent tumor growth. Further, the identification of myeloid resistance pathways will guide therapeutic strategies against tumor resisting to ICB and/or vaccination. Citation Format: Guillaume Mestrallet. Overcoming immune resistance in DNA mismatch repair deficient tumors [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 6652.

Landscape of KRAS, BRAF, and PIK3CA Mutations and Clinical Features of EBV-Associated and Microsatellite Unstable Gastric Cancer

Landscape of KRAS, BRAF, and PIK3CA Mutations and Clinical Features of EBV-Associated and Microsatellite Unstable Gastric Cancer

Landscape of KRAS, BRAF, and PIK3CA Mutations and Clinical Features of EBV-Associated and Microsatellite Unstable Gastric Cancer

Personalization of gastric cancer (GC) treatment is an urgent problem because of the clinical heterogeneity and aggressive course of the disease. Four GC subtypes were isolated based on molecular characteristics by The Cancer Genome Atlas researchers in 2014: Epstein-Barr virus positive (EBV^(+)), microsatellite unstable (MSI), chromosomally unstable (CIN), and genomically stable (GS). There is no unified method to detect the CIN and GS subtypes today, while MSI and EBV status assessments are used routinely and are of great clinical importance. A total of 159 GC samples were tested for MSI, EBV DNA, and somatic mutations in codons 12-13 (exon 2), 61 (exon 3), and 146 (exon 4) of the KRAS gene; codons 597-601 (exon 15) of the BRAF gene; and codons 542-546 (exon 9), 1047-1049 (exon 20) of the PIK3CA gene. EBV^(+) GC was detected in 8.2% of samples; and MSI, in 13.2%. MSI and EBV+ were found to be mutually exclusive. The mean ages at GC manifestation were 54.8 and 62.1 years in patients with EBV^(+) and MSI GCs, respectively. EBV^(+) GC affected men in 92.3% of cases, 76.2% of the patients were older than 50 years of age. Diffuse and intestinal adenocarcinomas were diagnosed in 6 (46.2%) and 5 (38.5%) EBV^(+) cases, respectively. MSI GC equally affected men (n = 10, 47.6%) and women (n = 11, 52.4%). The intestinal histological type was the most prevalent (71.4%); the lesser curvature was affected in 28.6% of the cases. The E545K variant of PIK3CA was observed in one EBV^(+) GC case. A combination of clinically significant variants of KRAS and PIK3CA was found in all MSI cases. The BRAF V600E mutation, which is specific to MSI colorectal cancer, was not detected. The EBV^(+) subtype was associated with better prognosis. The five-year survival rates were 100.0 and 54.7% for MSI and EBV^(+) GCs, respectively.

A simple approach for detecting HLA-A*02 alleles in archival formalin-fixed paraffin-embedded tissue samples and an application example for studying cancer immunoediting.

The HLA system represents a central component of the antigen presentation machinery. As every patient possesses a defined set of HLA molecules, only certain antigens can be presented on the cell surface. Thus, studying HLA type-dependent antigen presentation can improve the understanding of variation in susceptibility to various diseases, including infectious diseases and cancer. In archival formalin-fixed paraffin-embedded (FFPE) tissue, the HLA type is difficult to analyze because of fragmentation of DNA, hindering the application of commonly used assays that rely on long DNA stretches. Addressing these difficulties, we present a refined approach for characterizing presence or absence of HLA-A*02, the most common HLA-A allele in the Caucasian population, in archival samples. We validated our genotyping strategy in a cohort of 90 samples with HLA status obtained by an NGS-based method. 90% (n=81) of the samples could be analyzed with the approach. For all of them, the presence or absence of HLA-A*02 alleles was correctly determined with the method, demonstrating 100% sensitivity and specificity (95% CI: 91.40%-100% and 91.19%-100%). Furthermore, we provide an example of application in an independent cohort of 73 FFPE microsatellite-unstable (MSI) colorectal cancer samples. As MSI cancer cells encompass a high number of mutations in coding microsatellites, leading to the generation of highly immunogenic frameshift peptide antigens, they are ideally suited for studying relations between the mutational landscape of tumor cells and interindividual differences in the immune system, including the HLA genotype. Overall, our method can help to promote studying HLA type-dependency during the pathogenesis of a wide range of diseases, making archival and historic tissue samples accessible for identifying HLA-A*02 alleles.

S1-END-seq reveals DNA secondary structures in human cells

DNA becomes single stranded (ssDNA) during replication, transcription, and repair. Transiently formed ssDNA segments can adopt alternative conformations, including cruciforms, triplexes, and quadruplexes. To determine whether there are stable regions of ssDNA in the human genome, we utilized S1-END-seq to convert ssDNA regions to DNA double-strand breaks, which were then processed for high-throughput sequencing. This approach revealed two predominant non-B DNA structures: cruciform DNA formed by expanded (TA)n repeats that accumulate in microsatellite unstable human cancer cell lines and DNA triplexes (H-DNA) formed by homopurine/homopyrimidine mirror repeats common across a variety of cell lines. We show that H-DNA is enriched during replication, that its genomic location is highly conserved, and that H-DNA formed by (GAA)n repeats can be disrupted by treatment with a (GAA)n-binding polyamide. Finally, we show that triplex-forming repeats are hotspots for mutagenesis. Our results identify dynamic DNA secondary structures in vivo that contribute to elevated genome instability.

Critical aspects of microsatellite instability testing in endometrial cancer: a comparison study

The identification of mismatch repair deficient (dMMR) and microsatellite unstable (MSI) endometrial cancers (ECs) is important in screening, diagnosis, and therapeutic stratification of patients. We compared the diagnostic performance of 4 MSI molecular tests based on fragment length assay in capillary electrophoresis (OncoMate™ MSI assay, Promega) and in microcapillary electrophoresis (TapeStation 4200, Agilent); with high-resolution melting (HRM) analysis approaches (Idylla™ MSI Test, Biocartis; EasyPGX® ready MSI, Diatech Pharmacogenetics) on a series of 56ECs, which was well characterized for MMR status with immunohistochemical approach (IHC, nonmolecular reference test). The concordance of fluorescence capillary electrophoresis with IHC (AUC 0.98) was higher respect to the other molecular methodologies. Otherwise, HRM approaches and microcapillary electrophoresis platform failed to detect MSI-ECs showing minimal microsatellite shifts. In conclusion, in colorectal site, several technologies are eligible for MSI test, whereas in ECs, MSI test should be based on fluorescent capillary electrophoresis as it identifies a higher proportion of cases that could be misdiagnosed with other strategies.

Single-cell and bulk transcriptome sequencing identifies two epithelial tumor cell states and refines the consensus molecular classification of colorectal cancer

The consensus molecular subtype (CMS) classification of colorectal cancer is based on bulk transcriptomics. The underlying epithelial cell diversity remains unclear. We analyzed 373,058 single-cell transcriptomes from 63 patients, focusing on 49,155 epithelial cells. We identified a pervasive genetic and transcriptomic dichotomy of malignant cells, based on distinct gene expression, DNA copy number and gene regulatory network. We recapitulated these subtypes in bulk transcriptomes from 3,614 patients. The two intrinsic subtypes, iCMS2 and iCMS3, refine CMS. iCMS3 comprises microsatellite unstable (MSI-H) cancers and one-third of microsatellite-stable (MSS) tumors. iCMS3 MSS cancers are transcriptomically more similar to MSI-H cancers than to other MSS cancers. CMS4 cancers had either iCMS2 or iCMS3 epithelium; the latter had the worst prognosis. We defined the intrinsic epithelial axis of colorectal cancer and propose a refined ‘IMF’ classification with five subtypes, combining intrinsic epithelial subtype (I), microsatellite instability status (M) and fibrosis (F).

Lynch Syndrome and MSI-H Cancers: From Mechanisms to "Off-The-Shelf" Cancer Vaccines.

Defective DNA mismatch repair (dMMR) is associated with many cancer types including colon, gastric, endometrial, ovarian, hepatobiliary tract, urinary tract, brain and skin cancers. Lynch syndrome – a hereditary cause of dMMR – confers increased lifetime risk of malignancy in different organs and tissues. These Lynch syndrome pathogenic alleles are widely present in humans at a 1:320 population frequency of a single allele and associated with an up to 80% risk of developing microsatellite unstable cancer (microsatellite instability – high, or MSI-H). Advanced MSI-H tumors can be effectively treated with checkpoint inhibitors (CPI), however, that has led to response rates of only 30-60% despite their high tumor mutational burden and favorable immune gene signatures in the tumor microenvironment (TME). We and others have characterized a subset of MSI-H associated highly recurrent frameshift mutations that yield shared immunogenic neoantigens. These frameshifts might serve as targets for off-the-shelf cancer vaccine designs. In this review we discuss the current state of research around MSI-H cancer vaccine development, its application to MSI-H and Lynch syndrome cancer patients and the utility of MSI-H as a biomarker for CPI therapy. We also summarize the tumor intrinsic mechanisms underlying the high occurrence rates of certain frameshifts in MSI-H. Finally, we provide an overview of pivotal clinical trials investigating MSI-H as a biomarker for CPI therapy and MSI-H vaccines. Overall, this review aims to inform the development of novel research paradigms and therapeutics.

Adoption of universal next generation sequencing for Lynch syndrome screening in endometrial cancer

<h3>Objectives:</h3> To assess implementation of a novel microsatellite instability (MSI) screen using next generation sequencing (NGS) in endometrial cancer (EC) through comparison to prior Lynch syndrome screening strategies, as well as to compare characteristics of patients with microsatellite stable (MSS) and microsatellite unstable endometrial cancer. <h3>Methods:</h3> Retrospective review was performed of patients with newly diagnosed endometrial cancer at a single academic institution during three time periods and using different screening strategies: 1) 01/2010 - 01/2013 with provider-directed mismatch repair immunohistochemistry (MMR IHC) in women ≤60, 2) 01/2013 - 01/2016 with universal MMR IHC in all women ≤60, and 3) 10/2018 - 10/2019 with universal screening for MSI using NGS regardless of age. In cohorts 1 and 2, women with abnormal MMR IHC were referred to a genetic counselor who ordered MLH1 promoter methylation or germline genetic testing as indicated. In cohort 3, reflexive MLH1 promoter methylation was performed for patients with MSI and no MMR germline or somatic mutations, with referral to genetic counseling for suspected germline MMR mutation or unexplained MSI. Statistical analysis was performed using a t-test for continuous variables and Fisher's exact test for categorical variables. ≤60, 2) 01/2013 - 01/2016 with universal MMR IHC in all women ≤60, and 3) 10/2018 - 10/2019 with universal screening for MSI using NGS regardless of age. In cohorts 1 and 2, women with abnormal MMR IHC were referred to a genetic counselor who ordered MLH1 promoter methylation or germline genetic testing as indicated. In cohort 3, reflexive MLH1 promoter methylation was performed for patients with MSI and no MMR germline or somatic mutations, with referral to genetic counseling for suspected germline MMR mutation or unexplained MSI. Statistical analysis was performed using a t-test for continuous variables and Fisher's exact test for categorical variables. <h3>Results:</h3> There were 133 subjects in cohort 1, 135 subjects in cohort 2, and 140 subjects in cohort 3. The proportion of patients screened for Lynch syndrome increased over time (39%, 94%, and 100% of intended screening population). The proportion of positive screens was similar between the 3 cohorts (27%, 21%, and 27%). The proportion with positive screens who underwent confirmatory testing with germline genetic testing or MLH1 promoter methylation was 50% in cohort 1 and 59% in cohort 2. Following implementation of reflexive MLH1 promoter methylation testing when indicated, the proportion increased to 89% (p=0.001). Attendance at genetic counseling appointments following referral was 50%, 59%, and 61% in each cohort. Eleven patients were diagnosed with Lynch syndrome across the 3 cohorts (4%, 3%, and 1% respectively). In cohort 3, subjects with MSI EC had similar clinicopathologic characteristics compared with MSS cancers, and over half of MSI cancers were identified in subjects over the age of 60 (71%). Eight subjects had incidental somatic mutations identified through NGS, including 6 <i>POLE</i> mutations, 1 <i>BRIP1</i> mutation, and a subject with mutations in <i>TP53, AKT1,</i> and <i>FBXW7.</i> At our institution, MSI testing by NGS was billed at $905 versus $1200 for MMR IHC screening. <h3>Conclusions:</h3> Transition to universal screening using NGS with reflexive MLH1 promoter methylation testing increased the number of subjects completing genetic assessment for Lynch syndrome after a positive screen, likely as somatic causes of MSI were ruled out, allowing more focused genetics referrals. Similar characteristics between subjects with MSI and MSS EC supports universal screening regardless of clinical characteristics. In addition to identifying Lynch syndrome, detection of MSI through NGS can identify somatic alterations with therapeutic implications, and can be performed at a lower cost than MMR IHC.

Characterization of Clonal Evolution in Microsatellite Unstable Metastatic Cancers through Multiregional Tumor Sequencing.

Microsatellites are short, repetitive segments of DNA, which are dysregulated in mismatch repair-deficient (MMRd) tumors resulting in microsatellite instability (MSI). MSI has been identified in many human cancer types with varying incidence, and microsatellite instability-high (MSI-H) tumors often exhibit increased sensitivity to immune-enhancing therapies such as PD-1/PD-L1 inhibition. Next-generation sequencing (NGS) has permitted advancements in MSI detection, and recent computational advances have enabled characterization of tumor heterogeneity via NGS. However, the evolution and heterogeneity of microsatellite changes in MSI-positive tumors remains poorly described. We determined MSI status in 6 patients using our previously published algorithm, MANTIS, and inferred subclonal composition and phylogeny with Canopy and SuperFreq. We developed a simulated annealing-based method to characterize microsatellite length distributions in specific subclones and assessed the evolution of MSI in the context of tumor heterogeneity. We identified three to eight tumor subclones per patient, and each subclone exhibited MMRd-associated base substitution signatures. We noted that microsatellites tend to shorten over time, and that MMRd fosters heterogeneity by introducing novel mutations throughout the disease course. Some microsatellites are altered among all subclones in a patient, whereas other loci are only altered in particular subclones corresponding to subclonal phylogenetic relationships. Overall, our results indicate that MMRd is a substantial driver of heterogeneity, leading to both MSI and subclonal divergence. IMPLICATIONS: We leveraged subclonal inference to assess clonal evolution based on somatic mutations and microsatellites, which provides insight into MMRd as a dynamic mutagenic process in MSI-H malignancies.

Repeat expansions confer WRN dependence in microsatellite-unstable cancers.

The RecQ DNA helicase WRN is a synthetic lethal target for cancer cells with microsatellite instability (MSI), a form of genetic hypermutability that arises from impaired mismatch repair1-4. Depletion of WRN induces widespread DNA double-strand breaks in MSI cells, leading to cell cycle arrest and/or apoptosis. However, the mechanism by which WRN protects MSI-associated cancers from double-strand breaks remains unclear. Here we show that TA-dinucleotide repeats are highly unstable in MSI cells and undergo large-scale expansions, distinct from previously described insertion or deletion mutations of a few nucleotides5. Expanded TA repeats form non-B DNA secondary structures that stall replication forks, activate the ATR checkpoint kinase, and require unwinding by the WRN helicase. In the absence of WRN, the expanded TA-dinucleotide repeats are susceptible to cleavage by the MUS81 nuclease, leading to massive chromosome shattering. These findings identify a distinct biomarker that underlies the synthetic lethal dependence on WRN, and support the development of therapeutic agents that target WRN for MSI-associated cancers.

The shared frameshift mutation landscape of microsatellite-unstable cancers suggests immunoediting during tumor evolution

The immune system can recognize and attack cancer cells, especially those with a high load of mutation-induced neoantigens. Such neoantigens are abundant in DNA mismatch repair (MMR)-deficient, microsatellite-unstable (MSI) cancers. MMR deficiency leads to insertion/deletion (indel) mutations at coding microsatellites (cMS) and to neoantigen-inducing translational frameshifts. Here, we develop a tool to quantify frameshift mutations in MSI colorectal and endometrial cancer. Our results show that frameshift mutation frequency is negatively correlated to the predicted immunogenicity of the resulting peptides, suggesting counterselection of cell clones with highly immunogenic frameshift peptides. This correlation is absent in tumors with Beta-2-microglobulin mutations, and HLA-A*02:01 status is related to cMS mutation patterns. Importantly, certain outlier mutations are common in MSI cancers despite being related to frameshift peptides with functionally confirmed immunogenicity, suggesting a possible driver role during MSI tumor evolution. Neoantigens resulting from shared mutations represent promising vaccine candidates for prevention of MSI cancers.

Abstract 5587: Characterization of shared immunogenic frameshift mutations in microsatellite unstable cancers to guide the design of off-the-shelf immunotherapies

Abstract Microsatellite instability-high (MSI-H) cancers are an important model for evaluating neoantigen-based immunotherapies, given their high loads of tumor-specific antigens, evidence of T cell infiltration and exceptional response rates to checkpoint blockade. Importantly, MSI-H tumors are enriched in short nucleotide insertion/deletions in microsatellite regions (“hot spots”) within open reading frames, potentially leading to frameshift mutations shared across patients with MSI-H tumors and Lynch Syndrome, a hereditary cancer predisposition syndrome with MSI-H phenotype. We hypothesized that MSI-H tumors are enriched in recurrent frameshift mutations encoding immunogenic neoantigens and pre-selection of an optimal set of recurrent frameshift mutations, based on the multiplicity of encoded neoepitopes, diversity of interaction with common major histocompatibility complex (MHC) alleles and immunogenicity, will inform the design of off-the-shelf immune therapies for patients with MSI-H phenotype. Utilizing the whole-exome sequencing information available at The Cancer Genome Atlas, we evaluated the neoantigen profiles of patients with colorectal (n=461), stomach (n=443) and endometrial carcinoma (n=560) (COAD, STAD and UCEC, respectively) according to the aforementioned criteria and identified 37, 23 and 9 frameshift mutations encoding potentially immunogenic neoantigens, which were shared across the MSI-H population in COAD, STAD and UCEC, respectively. In an array of immunological assays utilizing peripheral blood mononuclear cells (PBMCs) from healthy donors, we characterized the immunogenicity of the neoantigens derived from the 9 frameshift peptides shared in MSI-H UCEC to assess the extent and specificity of the engendered T cell immunity. We found that each frameshift peptide could elicit antigen-specific CD8+ T cell responses in a subset of subjects tested, which suggests that MSI-H UCEC patients have an increased frequency of high-quality T cell epitopes derived from shared frameshift peptides, binding to a broad spectrum of MHC alleles, capable of inducing CD8+ T responses. Currently, utilizing blood and tissue specimens from patients with MSI-H COAD, STAD and UCEC, we are interrogating (1) the expression levels and frequencies of the selected shared neoantigens, (2) whether these neoantigens are presented in the context of MHC and (3) can elicit endogenous T cell responses. Findings from this research will guide the selection of shared neoantigens eliciting antitumor T cell immunity in patients with MSI-H tumors and will ultimately lay the foundation for common neoantigen-based, off-the-shelf cancer immunotherapy designs, which can be utilized in therapeutic and preventative settings in MSI-H cancers and Lynch Syndrome, respectively. Citation Format: Cansu Cimen Bozkus, Vladimir Roudko, Theofani Orfanelli, Stephanie Blank, Benjamin Greenbaum, Nina Bhardwaj. Characterization of shared immunogenic frameshift mutations in microsatellite unstable cancers to guide the design of off-the-shelf immunotherapies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5587.