MMR Genes Research Articles

Microsatellite instability in mismatch repair proficient colorectal cancer: clinical features and underlying molecular mechanisms

Both defects in mismatch repair (dMMR) and high microsatellite instability (MSI-H) have been recognised as crucial biomarkers that guide treatment strategies and disease management in colorectal cancer (CRC). As MMR and MSI tests are being widely conducted, an increasing number of MSI-H tumours have been identified in CRCs with mismatch repair proficiency (pMMR). The objective of this study was to assess the clinical features of patients with pMMR/MSI-H CRC and elucidate the underlying molecular mechanism in these cases. From January 2015 to December 2018, 1684 cases of pMMR and 401 dMMR CRCs were enrolled. Of those patients, 93 pMMR/MSI-H were identified. The clinical phenotypes and prognosis were analysed. Frozen and paraffin-embedded tissue were available in 35 patients with pMMR/MSI-H, for which comprehensive genomic and transcriptomic analyses were performed. In comparison to pMMR/MSS CRCs, pMMR/MSI-H CRCs exhibited significantly less tumour progression and better long-term prognosis. The pMMR/MSI-H cohorts displayed a higher presence of CD8+ T cells and NK cells when compared to the pMMR/MSS group. Mutational signature analysis revealed that nearly all samples exhibited deficiencies in MMR genes, and we also identified deleterious mutations in MSH3-K383fs. This study revealed pMMR/MSI-H CRC as a distinct subgroup within CRC, which manifests diverse clinicopathological features and long-term prognostic outcomes. Distinct features in the tumour immune-microenvironment were observed in pMMR/MSI-H CRCs. Pathogenic deleterious mutations in MSH3-K383fs were frequently detected, suggesting another potential biomarker for identifying MSI-H. This work was supported by the Science and Technology Commission of Shanghai Municipality (20DZ1100101).

Novel insights into tumorigenesis revealed by molecular analysis of Lynch syndrome cases with multiple colorectal tumors.

Lynch syndrome (LS) is an autosomal dominant multi-organ cancer syndrome with a high lifetime risk of cancer. The number of cumulative colorectal adenomas in LS does not generally exceed ten, and removal of adenomas via routine screening minimizes the cancer burden. However, abnormal phenotypes may mislead initial diagnosis and subsequently cause suboptimal treatment. Currently, there is no standard guide for the care of multiple colorectal adenomas in LS individuals. We aimed to shed insight into the molecular features and reasons for multiplicity of adenomas in LS patients. We applied whole exome sequencing on nine adenomas (ten samples) and three assumed primary carcinomas (five samples) of an LS patient developing the tumors during a 21-year follow-up period. We compared the findings to the tumor profiles of two additional LS cases ascertained through colorectal tumor multiplicity, as well as to ten adenomas and 15 carcinomas from 23 unrelated LS patients with no elevated adenoma burden from the same population. As LS associated cancers can arise via several molecular pathways, we also profiled the tumors for CpG Island Methylator Phenotype (CIMP), and LINE-1 methylation. All tumors were microsatellite unstable (MSI), and MSI was present in several samples derived from normal mucosa as well. Interestingly, frequent frameshift variants in RNF43 were shared among substantial number of the tumors of our primary case and the tumors of LS cases with multiple tumors but almost absent in our control LS cases. The RNF43 variants were completely absent in the normal tissue, indicating tumor-associated mutational hotspots. The RNF43 status correlated with the mutational signature SBS96. Contrary to LS tumors from the reference set with no elevated colorectal tumor burden, the somatic variants occurred significantly more frequently at C>T in the CpG context, irrespective of CIMP or LINE-1 status, potentially indicating other, yet unknown methylation-related mechanisms. There were no signs of somatic mosaicism affecting the MMR genes. Somatic variants in APC and CTNNB1 were unique to each tumor. Frequent somatic RNF43 hot spot variants combined with SBS96 signature and increased tendency to DNA methylation may contribute to tumor multiplicity in LS.

Incidence and molecular characteristics of deficient mismatch repair conditions across nine different tumors and identification of germline variants involved in Lynch-like syndrome

BackgroundBased on molecular characteristics, deficient DNA mismatch repair (dMMR) solid tumors are largely divided into three categories: somatically MLH1-hypermethylated tumors, Lynch syndrome (LS)-associated tumors, and Lynch-like syndrome (LLS)-associated tumors. The incidence of each of these conditions and the corresponding pathogenic genes related to LLS remain elusive.MethodsWe identified dMMR tumors in 3609 tumors from 9 different solid organs, including colorectal cancer, gastric cancer, small-bowel cancer, endometrial cancer, ovarian cancer, upper urinary tract cancer, urinary bladder cancer, prostate cancer, and sebaceous tumor, and comprehensively summarized the characterization of dMMR tumors. Characterization of dMMR tumors were performed as loss of at least one of MMR proteins (MLH1, MSH2, MSH6, and PMS2), by immunohistochemistry, followed by MLH1 promotor methylation analysis and genetic testing for MMR genes where appropriate. Somatic variant analysis of MMR genes and whole exome sequencing (WES) were performed in patients with LLS.ResultsIn total, the incidence of dMMR tumors was 5.9% (24/3609). The incidence of dMMR tumors and the proportion of the three categorized dMMR tumors varied considerably with different tumor types. One to three likely pathogenic/pathogenic somatic MMR gene variants were detected in 15 out of the 16 available LLS tumors. One patient each from 12 patients who gave consent to WES demonstrated non-MMR germline variants affect function (POLQ or BRCA1).ConclusionsOur data regarding the LS to LLS ratio would be useful for genetic counseling in patients who are suspected to have LS, though the genetic backgrounds for the pathogenesis of LLS need further investigation.

ERH Impacts Patient Prognosis and Tumor Immune Microenvironment: A Pan-Cancer Analysis.

The enhancer of rudimentary homolog (ERH) has been shown to play significant roles in tumorigenesis and progression. However, few systematic pan-cancer analyses about ERH have been described. From the tumor immune estimation resource web server2.0 (TIMER2.0), the Genotype-Tissue Expression database (GTEx) and the Gene Expression Profile Interactive Analysis version 2 (GEPIA2) databases, we explored the expression profiles and prognostic significance of ERH in 33 cancers. The Clinical Proteomic Tumor Analysis Consortium (CPTAC) and the Human Protein Atlas (HPA) databases were further used to examine the differential expression of ERH at the protein level. The genetic alteration profile was obtained from the cBioPortal. The correlation between ERH expression and the quantities of immune infiltrating cells was examined by the TIMER tool. Spearman's correlation test was conducted to analyze the association between ERH expression status and a number of prognostic indicators, including immune checkpoints, TMB, MSI, immune neoantigen, MMR genes, and DNA methyltransferases. ProteinProtein Interaction analyses were performed in the String and GeneMANIA databases, and enrichment analysis and predicted signaling pathways were identified through GO and KEGG. To make our results convincing, we validated them in six datasets in the Gene Expression Omnibus (GEO) database. In addition, we verified the expression of ERH between gastric cancer tissues and adjacent normal tissues by RT-qPCR. ERH expression was elevated in numerous tumors, and it was not associated with the patient's prognosis. Furthermore, the quantities of immune infiltrating cells and immune checkpoint genes were remarkably associated with ERH. TMB and MSI were related to ERH expression in 14 and 15 cancer types, respectively. Moreover, the expression of ERH was strongly associated with MMR defects in multiple cancer types, and almost all tumors showed coexpression of ERH and four DNA methyltransferases. The results of GO and KEGG analysis confirmed that ERH potentially impacts several important signaling pathways. Both the GEO datasets and the RT-qPCR experiment validated our previous analysis. Our pan-cancer analysis demonstrated the characterization of ERH in multiple tumors. ERH may be a valuable novel biological indicator for assessing immunotherapy efficacy and prognosis in various malignancies.

Tumor analysis of MMR genes in Lynch-like syndrome: Challenges associated with results interpretation.

Up to 70% of suspected Lynch syndrome patients harboring MMR deficient tumors lack identifiable germline pathogenic variants in MMR genes, being referred to as Lynch-like syndrome (LLS). Previous studies have reported biallelic somatic MMR inactivation in a variable range of LLS-associated tumors. Moreover, translating tumor testing results into patient management remains controversial. Our aim is to assess the challenges associated with the implementation of tumoral MMR gene testing in routine workflows. Here, we present the clinical characterization of 229 LLS patients. MMR gene testing was performed in 39 available tumors, and results were analyzed using two variant allele frequency (VAF) thresholds (≥5% and ≥10%). More biallelic somatic events were identified at VAF ≥ 5% than ≥10% (35.9% vs. 25.6%), although the rate of nonconcordant results regarding immunohistochemical pattern increased (30.8% vs. 20.5%). Interpretation difficulties question the current utility of the identification of MMR somatic hits in the diagnostic algorithm of suspected LS cases.

Circulating Biomarkers for Tumour Monitoring, Prognosis Predictions and Detection of Temozolomide Resistance Mutations in Glioma

INTRODUCTION: The potential benefits of circulating biomarkers have been well demonstrated in other cancers. Despite this, no circulating biomarker exists for glioma due to poor sensitivity and hetereogeneity of results. METHODS: Serum miRNA and plasma ctDNA was isolated in a cohort of 212 gliomas using next-generation sequencing (NGS) and digital droplet PCR (ddPCR). Differentially expressed miRNAs were discovered using a machine learning algorithm and compared to tumor volume on MRI. A Lasso-regression model identified miRNAs that were associated with both progression-free and overall survival. Tumor mutations and ctDNA was compared in 49 plasma samples from 10 patients with tumor tissue available from at least two operations using NGS. Identified mutations were validated using ddPCR in the whole cohort. RESULTS: A 9-gene miRNA signature was found that could distinguish between glioma and healthy controls with 99.8% accuracy, including two that demonstrated dynamic changes that correlated with tumor volume on MRI. We found a separate 4-gene miRNA signature that was predictive of survival outcomes and could be incorporated into a clinical nomogram. Common glioma mutations were identified within 93% of plasma samples, including 25% that were not found in the tumor tissue. Mutations in the mismatch repair genes (MMR) were frequently detected in the plasma following temozolomide treatment and observed prior to their appearance in tissue at progression. CONCLUSIONS: We have identified serum miRNA biomarker signatures that can monitor tumour burden and improve prognostic predictions. We also show that contrary to previous reports, ctDNA can be reliably detected in the plasma of gliomas. We found that ctDNA offers complementary information to tissue biopsy alone and provides early evidence of temozolomide resistance through mutations in MMR genes.

Abstract 6102: Clinicopathological and genetics features of endometrial cancer in Algerian women: The first nation-wide study

Abstract Background: Endometrial cancer represents the fourth common cancer in women in Algeria and the fifth cause of cancer mortality. In the present study, we aimed to determine, clinical, tumor and genetics characteristics associated with endometrial cancer in Algerian women. In addition, we screened for germline pathogenic variants in MMR genes in endometrial cancer patients with strong family history of Lynch syndrome (LS). Materials and Methods: Our study population included 273 patients diagnosed with endometrial cancer between 2010 and 2021. Data were collected from three public hospitals that covered 29 provinces among 48 in Algeria. Patient and tumor information included: age at diagnosis, menopausal status, histological type, histological grade, TNM stage, FIGO stage, tumor markers CA125, ACE, CA19-9, family history with cancer and age at menarche . MLH1 (exons 1, 9, 10, 13, 16), MSH2 (exons 5, 6, 7, 12), MSH6 (exons 4 and 8) and PMS 2(exons 6 and 10) were screened by PCR-Sanger direct sequencing in 14 LS families . Results: The mean age at diagnosis was 58.88 years. The mean age at menarche was 13.5 years. The proportion of endometrial cancer patients with premenopausal status was 21.62%. The commonest histological type was endometrioid adenocarcinoma (66.79%) followed by clear cell carcinoma (8.10%), carcinosarcomas (3.86%), serous carcinoma (3.47%) and mucinous carcinoma (2.7%). We found that the proportion of tumors with histological grade I (34.74%), grade II (27.41%) and grade IV (21.40%) was commonest in 83.55% of the patients. Our results showed that 73.74% of the patients were diagnosed at stage I (50.19%), stage II (12.74%) and stage III (10.81%), respectively. For the FIGO staging, our results showed that 74.12% of the patients were diagnosed at stage I (52.89%), stage III (10.81%) and stage I (10.42%), respectively. 16.21%, 3.08%, 6.94% of the patients were positive for CA125, ACE and CA19-9, respectively. We noticed that 56 patients (20.43%) had a positive family history with HBOC syndrome (17), Lynch syndrome (26) and Cowden syndrome (13), respectively. We identified two distinct germline pathogenic variants in MLH1 gene in two LS families. We detected the new germline pathogenic variant MLH1 c.53_63delinsT in young patient diagnosed with endometrial cancer and colorectal cancer (CRC) at age 43y and 44 y, respectively. The rare pathogenic germline variant MLH1 c.1546C>T has been detected in young woman diagnosed with CRC at age 41y and endometrial and ovarian cancers at age 52y, respectively. Conclusions : In this first study, we reported some clinical, biological, tumor and genetics features of endometrial cancer in Algerian women. Interestingly, we noticed that the median age of diagnosis was younger that the average age in Europe and America. Genetic counseling and testing is recommended for endometrial cancer patients diagnosed before age 50 with a family history of Lynch syndrome. Citation Format: Farid Cherbal, Chiraz Mehemmai, Mouchira Saidi, Yasmine Santoudji, Djamel-Eddine Seddik, Asma-Lamia Boumehdi, Fatiha Gouaref, Kamel Bentabak, Hassen Mahfouf, Mohammed Oukkal. Clinicopathological and genetics features of endometrial cancer in Algerian women: The first nation-wide study [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 6102.

Abstract 1445: Emergence of frameshift mutations during tumorigenesis in VCMsh2 mouse model of Lynch syndrome

Abstract Monoallelic germline mutations in MMR genes (e.g., MLH1, MSH2, MSH6, and PMS2) predispose individuals to Lynch syndrome (LS) with microsatellite instability (MSI) throughout the genome. Mutations at coding mononucleotide repeats (MNR) usually result in frameshift mutations (FSMs). Recurrent FSMs are thought to play a central role in the increased risk of different types of cancer. Neoantigens produced by FSMs have been shown to elicit immune responses, which is the basis for frameshift neoantigen-based vaccines. To develop a prophylactic vaccine and prevention strategy for this high-risk population, we assessed FSMs during tumorigenesis from histologically normal mucosa and fecal DNA of a LS mouse model using targeted sequencing and a panel of FSMs in MNR regions. Msh2LoxP/LoxP;Villin-Cre mice (VCMsh2) started developing detectable tumors at 7-8 months and median survival was 11.5 months with 100% penetrance. Interestingly, FSMs were detectable not only in tumors and mucosa at 7-8 months, but also in young mice (1 month), embryos and pups although FSMs detected and variant allele frequency (VAF) were very low, indicating that FSMs accumulated over time, and FSMs alone may be not sufficient for tumorigenesis since tumors emerge at older age. To determine whether Msh2 was absent in embryos and pups, immunohistochemical (IHC) analysis was performed. Msh2 was lost in almost all intestine epithelial cells in 2-month-old mice but still present in young pups and embryos with very few cells absent of Msh2, indicating that emerging FSMs in these young pups may be due to decreased Msh2 expression because of delayed Cre function. To determine whether low Msh2 expression could lead to the emergence of FSMs, intestine mucosa from 8-, 10-, and 12-month-old Msh2LoxP/+;Villin-Cre heterozygous mice was sequenced. FSMs were detectable with low VAF, although they didn’t develop tumors. IHC staining revealed that Msh2 was absent in only a few intestine epithelial cells in these heterozygous mice, suggesting that loss of heterozygosity was a late event and rare at 12 months. To determine whether MSI emerged in young VCMsh2 pups and heterozygous mice due to haploinsufficiency of Msh2, MSI was assessed using seven markers and fragment size analysis. One marker (mBat67) showed instability in the intestinal mucosa of heterozygous mice and three showed instability in fecal DNA of one month old VCMsh2 mice. Interestingly, mucosal and fecal samples from a time course study in VCMsh2 mice showed progressive increase in MSI with a good correlation between MSI and FSMs during the tumorigenesis process. In summary, FSMs emerged at an early stage of tumorigenesis in VCMsh2 mice, which correlated with MSI status. Our data indicates that FSMs and MSI status can be used to monitor the tumor development of LS colorectal cancer. Funded by the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261201500003I Citation Format: Yurong Song, Lei Wei, Shaneen S. Baxter, Brandon Somerville, Holli Loomans-Kropp, Chelsea Sanders, Ryan N. Baugher, Stephanie D. Mellott, Todd B. Young, Heidi E. Lawhorn, Teri M. Plona, Qiang Hu, Song Liu, Alan Hutson, Simone Difilippantonio, Ligia Pinto, Steven M. Lipkin, Matthias Kloor, Shizuko Sei, Robert H. Shoemaker. Emergence of frameshift mutations during tumorigenesis in VCMsh2 mouse model of Lynch syndrome [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 1445.

Abstract 1451: Replication repair deficient mouse models provide insights into gliomagenesis and response to immunotherapy

Abstract Background: Replication Repair Deficiency (RRD), caused by germline monoallelic (Lynch Syndrome) or biallelic (Constitutional Mismatch Repair Deficiency, CMMRD) mutations in MMR genes, is present in 5-10% of glioblastomas in children, adolescents, and young adults. RRD glioblastomas are chemoradiation-resistant, but respond favorably to immune checkpoint inhibition (ICI). Representative immunocompetent animal models are urgently needed for 3 recently identified subgroups based on specific somatically-acquired mutations, survival, and immunotherapy response (RRD1: MMRD with POLE mutations, RRD2: MMRD associated with TP53 mutations, and RRD3: MMRD harboring IDH1 mutations). Methods: Using germline mutations and brain-specific Cre-drivers, we genetically engineered mouse models that recapitulate each human RRD-subgroup. Results: All mouse models robustly developed brain tumors displaying phenotypic variation. RRD1 (Nestin- and Olig2-Cre+/Msh2LoxP/LoxP/PoleS459F/+ and LSL-PoleP286R/+): CMMRD-like Nestin-Cre-driven mice develop posterior-fossa glioma-like or embryonal (EB)-like tumors at ~2.7 months. Olig2-Cre-driven mice display hemispheric gliomas at ~10 months, suggesting distinct cell-of-origin. RRD2 (Nestin-Cre+/Trp53LoxP/LoxP and Msh2LoxP/LoxP or Mlh1−/−): tumors develop primarily in the hindbrain in germline Mlh1 mice, and majority arise in the forebrain of Nestin-Cre/Msh2 mice, highlighting the timeline of mutagenesis. Strikingly, germline Mlh1 tumors occur earlier than Nestin-Cre-driven RRD2 tumors, indicating early developmental mutation accumulation in CMMRD-patients. Lynch-like RRD1/2 mice succumb exclusively to gliomas >13 months (p<0.0001). RRD3 (Olig2-Cre+/Msh2LoxP/LoxP/Trp53LoxP/LoxP/LSL-Idh1R132H/+): mice succumb to brain tumors at >11 months and are hemispheric. Significance: These observations recapitulate human data, where CMMRD-patients develop glioblastoma/EB earlier than Lynch-patients (8.6 vs. 14-years; p<0.0001), and posterior-fossa glioblastoma/EB presents earlier than hemispheric gliomas (p=0.04). Additionally, tumor onset and location vary by subgroup (RRD1: 7.6-years, RRD2: 8.3-years, both hemispheric/posterior-fossa; RRD3: 12-years, hemispheric; p=0.005). In both mice and humans, RRD1 exhibits ultra-hypermutation, high immune infiltration, and response to ICI, whereas RRD2 harbors lower mutational burden, are immune-cold, and ICI-monotherapy resistant. Temporal dynamics of RRD tumor development is currently being tracked by serial MRI to define previously undetermined biologically relevant time points of tumor progression. Conclusion: Our models accurately mimic the human condition and provide unique insights into RRD tumorigenesis, allowing optimization of subgroup-tailored therapeutic approaches. Citation Format: Zoya Aamir, Melissa Galati, Emma Gattoni, Owen Crump, Nuno M. Nunes, Anirban Das, Nicholas Fernandez, Nicholas Fernandez, Angel K. Wong, Jerome Fortin, Lucie Stengs, Vanessa Bianchi, Melissa Edwards, Logine Negm, Cynthia Hawkins, David Malkin, Sean Egan, Uri Tabori. Replication repair deficient mouse models provide insights into gliomagenesis and response to immunotherapy [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 1451.

Clinical features and impact of p53 status on sporadic mismatch repair deficiency and Lynch syndrome in uterine cancer.

The clinical features of sporadic mismatch repair deficiency (MMRd) and Lynch syndrome (LS) in Japanese patients with endometrial cancer (EC) were examined by evaluating the prevalence and prognostic factors of LS and sporadic MMRd in patients with EC. Targeted sequencing of five LS susceptibility genes (MLH1, MSH2, MSH6, PMS2, and EPCAM) was carried out in 443 patients with EC who were pathologically diagnosed with EC at the National Cancer Center Hospital between 2011 and 2018. Pathogenic variants in these genes were detected in 16 patients (3.7%). Immunohistochemistry for MMR proteins was undertaken in 337 of the 433 (77.9%) EC patients, and 91 patients (27.0%) showed absent expression of at least one MMR protein. The 13 cases of LS with MMR protein loss (93.8%) showed a favorable prognosis with a 5-year overall survival (OS) rate of 100%, although there was no statistically significant difference between this group and the sporadic MMRd group (p = 0.27). In the MMRd without LS group, the 5-year OS rate was significantly worse in seven patients with an aberrant p53 expression pattern than in those with p53 WT (53.6% vs. 93.9%, log-rank test; p = 0.0016). These results suggest that p53 abnormalities and pathogenic germline variants in MMR genes could be potential biomarkers for the molecular classification of EC with MMRd.

Identification of people with Lynch syndrome from those presenting with colorectal cancer in England: baseline analysis of the diagnostic pathway

It is believed that >95% of people with Lynch syndrome (LS) remain undiagnosed. Within the National Health Service (NHS) in England, formal guidelines issued in 2017 state that all colorectal cancers (CRC) should be tested for DNA Mismatch Repair deficiency (dMMR). We used a comprehensive population-level national dataset to analyse implementation of the agreed diagnostic pathway at a baseline point 2 years post-publication of official guidelines. Using real-world data collected and curated by the National Cancer Registration and Analysis Service (NCRAS), we retrospectively followed up all people diagnosed with CRC in England in 2019. Nationwide laboratory diagnostic data incorporated somatic (tumour) testing for dMMR (via immunohistochemistry or microsatellite instability), somatic testing for MLH1 promoter methylation and BRAF status, and constitutional (germline) testing of MMR genes. Only 44% of CRCs were screened for dMMR; these figures varied over four-fold with respect to geography. Of those CRCs identified as dMMR, only 51% underwent subsequent diagnostic testing. Overall, only 1.3% of patients with colorectal cancer had a germline MMR genetic test performed; up to 37% of these tests occurred outside of NICE guidelines. The low rates of molecular diagnostic testing in CRC support the premise that Lynch syndrome is underdiagnosed, with significant attrition at all stages of the testing pathway. Applying our methodology to subsequent years’ data will allow ongoing monitoring and analysis of the impact of recent investment. If the diagnostic guidelines were fully implemented, we estimate that up to 700 additional people with LS could be identified each year.

Evolutionary origin of germline pathogenic variants in human DNA mismatch repair genes

BackgroundMismatch repair (MMR) system is evolutionarily conserved for genome stability maintenance. Germline pathogenic variants (PVs) in MMR genes that lead to MMR functional deficiency are associated with high cancer risk. Knowing the evolutionary origin of germline PVs in human MMR genes will facilitate understanding the biological base of MMR deficiency in cancer. However, systematic knowledge is lacking to address the issue. In this study, we performed a comprehensive analysis to know the evolutionary origin of human MMR PVs.MethodsWe retrieved MMR gene variants from the ClinVar database. The genomes of 100 vertebrates were collected from the UCSC genome browser and ancient human sequencing data were obtained through comprehensive data mining. Cross-species conservation analysis was performed based on the phylogenetic relationship among 100 vertebrates. Rescaled ancient sequencing data were used to perform variant calling for archeological analysis.ResultsUsing the phylogenetic approach, we traced the 3369 MMR PVs identified in modern humans in 99 non-human vertebrate genomes but found no evidence for cross-species conservation as the source for human MMR PVs. Using the archeological approach, we searched the human MMR PVs in over 5000 ancient human genomes dated from 45,045 to 100 years before present and identified a group of MMR PVs shared between modern and ancient humans mostly within 10,000 years with similar quantitative patterns.ConclusionOur study reveals that MMR PVs in modern humans were arisen within the recent human evolutionary history.

Survival of patients with microsatellite instability-high and Lynch syndrome-associated pancreatic ductal adenocarcinomas.

640 Background: Pancreatic ductal adenocarcinoma (PDAC) remains a challenging disease due to its aggressive nature, late-stage diagnosis, and limited treatment options. There is an increased interest in cancers which harbor microsatellite instability (MSI) due to their susceptibility to immune checkpoint inhibitors. MSI in PDAC is rare, and little is known about survival in this subtype of pancreas cancers. Our aim was to assess overall survival in surgically resected high microsatellite instability (MSI-H), mismatch repair deficient (MMRd), and Lynch Syndrome (LS) associated PDACs treated in our center. Methods: Patients with surgically resected PDACs from 1990 to 2023 were identified. A total of 936 cases were sequenced utilizing MSK-IMPACT, a targeted next generation sequencing platform. Patients with germline or sporadic pathogenic variants or likely pathogenic variants in one of the DNA MMR genes MLH1, MSH2, MSH6, PMS2, or 3’ end of EpCAM were identified. MSI-H, MMRd, and LS-associated cases were matched to patients with microsatellite stable or MMR proficient, non-LS-associated PDAC in a 1:2 ratio with a direct match on age (±5 years), gender, and year of surgery (±3 years). Kaplan-Meier curves were used to visualize the data. A generalized estimating equation (GEE) cox model with robust sandwich estimator was used to compare matched cohorts. Results: 17 patients with MSI-H, MMRd or LS-associated surgically resected PDACs were identified. Five patients were classified as MSI-H, 3 patients were MSI-indeterminate, and 9 patients were microsatellite stable. Eight patients were found to have pathogenic germline variants in LS-associated genes. The remaining nine patients had sporadic pathogenic DNA MMR gene variants ( MLH1 n=3, MSH6 n=6). Median age at surgery was 66 (interquartile range (IQR) 60-77), 41% of the patients were female, and median year of surgery was 2014. MSI-H, MMRd or LS-associated PDACs were matched to 34 control patients. The median survival time of the cases was 12 years vs. 1.9 years in the control group. Five-year survival rate was 81% (IQR 60-100%) and 18% (7.4%-43%), respectively. LS-associated, MSI-H or MMRd status in resectable PDAC was a strong predictor of good prognosis (hazard ratio 0.17, 95% CI 0.07-0.38, p value <0.001). Conclusions: MSI-H, MMRd or LS-associated PDACs displayed significantly better survival compared to their MSI-stable, MMR proficient, non-LS-associated counterparts, in the era before routine use of immunotherapy. It is expected that survival will be increased further with more frequent availability of immunotherapy.

Non-canonical antigens are the largest fraction of peptides presented by MHC class I in mismatch repair deficient murine colorectal cancer

BackgroundImmunotherapy based on checkpoint inhibitors is highly effective in mismatch repair deficient (MMRd) colorectal cancer (CRC). These tumors carry a high number of mutations, which are predicted to translate into a wide array of neoepitopes; however, a systematic classification of the neoantigen repertoire in MMRd CRC is lacking. Mass spectrometry peptidomics has demonstrated the existence of MHC class I associated peptides (MAPs) originating from non-coding DNA regions. Based on these premises we investigated DNA genomic regions responsible for generating MMRd-induced peptides.MethodsWe exploited mouse CRC models in which the MMR gene Mlh1 was genetically inactivated. Isogenic cell lines CT26 Mlh1+/+ and Mlh1-/- were inoculated in immunocompromised and immunocompetent mice. Whole genome and RNA sequencing data were generated from samples obtained before and after injection in murine hosts. First, peptide databases were built from transcriptomes of isogenic cell lines. We then compiled a database of peptides lost after tumor cells injection in immunocompetent mice, likely due to immune editing. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and matched next-generation sequencing databases were employed to identify the DNA regions from which the immune-targeted MAPs originated. Finally, we adopted in vitro T cell assays to verify whether MAP-specific T cells were part of the in vivo immune response against Mlh1-/- cells.ResultsWhole genome sequencing analyses revealed an unbalanced distribution of immune edited alterations across the genome in Mlh1-/- cells grown in immunocompetent mice. Specifically, untranslated (UTR) and coding regions exhibited the largest fraction of mutations leading to highly immunogenic peptides. Moreover, the integrated computational and LC-MS/MS analyses revealed that MAPs originate mainly from atypical translational events in both Mlh1+/+ and Mlh1-/- tumor cells. In addition, mutated MAPs—derived from UTRs and out-of-frame translation of coding regions—were highly enriched in Mlh1-/- cells. The MAPs trigger T-cell activation in mice primed with Mlh1-/- cells.ConclusionsOur results suggest that—in comparison to MMR proficient CRC—MMRd tumors generate a significantly higher number of non-canonical mutated peptides able to elicit T cell responses. These results reveal the importance of evaluating the diversity of neoepitope repertoire in MMRd tumors.

Abstract B042: Clinical and molecular characterization of Lynch syndrome-associated pancreas adenocarcinoma (PDAC)

Abstract Background: Lynch syndrome (LS) is a pan-cancer predisposition syndrome caused by pathogenic germline alterations (gPVs) in DNA mismatch repair genes (PMS2, MSH6, MLH1, MSH2, EPCAM). It is characterized by microsatellite instability (MSI)/deficient DNA mismatch repair (dMMR) of associated tumors, which predicts immune checkpoint blockade (ICB) response. Clinical and molecular features of LS-PDAC are not well defined. Herein we sought to describe clinical characteristics, MSI prevalence, and ICB response. Methods: Patients (pts) with LS-associated gPV and PDAC who had consented to IRB-approved protocol of matched tumor-normal sequencing (MSK-IMPACT from which MSIsensor status derivable) were included. MiMSI utilized to assess microsatellite stable (MSS) PDACs to account for low tumor content. MMR status defined with immunohistochemistry (IHC). Composite MSI adjudicated as either loss of MMR protein expression by IHC or MSI by MSISensor or MiMSI. FACETS algorithm assessed biallelic MMR gene alteration status. Mutational signature analysis performed on MSI tumors with deconstructSigs. Results: N=29 pts identified with LS-associated gPVs and PDAC: 10 MSH2 (34%), 9 MSH6 (31%), 7 PMS2 (24%), 2 MLH1 (7%), 1 EPCAM (3%). Majority white (24, 83%) and female (15, 52%). At diagnosis 62% pts early-stage disease (8 stage II, 10 stage III), 11 stage IV. Median age at diagnosis: 68 yrs (range 45-88). Eighteen (62%) had first-degree family history of LS spectrum cancers. Four pts had metachronous cancers (2 CRC, 1 uterine, 1 CRC and uterine). Orthogonal tumor testing feasible in 22 pts (76%). MiMSI (conducted for 16) reclassified 4 as MSI that were indeterminate by MSISensor; IHC (conducted for 15) classified 1/8 tumors as dMMR previously MSS on MSISensor. Whole cohort MSI status: 16 composite MSI (59%), 11 MSS (41%), and 2 MS unknown. gPVs in N= 16 with MSI tumors: 9 MSH2, 4 MSH6, 2 MLH1, and 1 PMS2; amongst N= 11 MSS tumors: 6 gMSH6, 4 gPMS2, and 1 EPCAM. Amongst 24 evaluable pts biallelic loss identified in 6 pts with MSI disease. The dominant mutational signature in 4/6 was Clock/aging and not MSI; 2 of these 4 harbored gMSH2, and 2/4 gMSH6. KRAS and TP53 mutations more common in sporadic MSS PDAC than LS-PDAC (p=0.001, p=0.005 respectively); KRAS G12R was enriched in LS PDAC vs sporadic PDAC (31.6% vs 15.3%, p=0.06). N= 13 (47%) received ICB, inclusive of 4 reclassified by MiMSI and 8 with metastatic disease. In evaluable pts with MSI disease, overall response rate 60% (2 CR, 4 PR, 4 SD). N= 1 MSS PDAC had POD as best response. Median ICB duration: 27.7 months (95% CI 9.0, NR). Conclusion: In this LS PDAC cohort we observed a higher proportion of MSH6, MSH2, PMS2 gPVs relative to other cohorts. LS-PDAC MSI arises frequently in association with gMSH2, gMLH1. MSI status determination with orthogonal testing is critical to appropriately select pts for ICB. Outcome to ICB in LS PDAC MSI comparable to other tumor types highlighting the importance of MSI as a predictive biomarker in PDAC. Citation Format: Emily Harrold, Catherine A. O'Connor, David Lin, Andrea Gazzo, Henry Walch, Megha Ranganathan, Amanda Catchings, Sarah Kane, Fergus Keane, Deborah M. Thurtle-Schmidt, Fiyinfolu Balogun, Anna Varghese, Kenneth Yu, David Kelsen, Alicia Latham, Britta Weigelt, Wungki Park, Zsofia Stadler, Eileen M. O'Reilly. Clinical and molecular characterization of Lynch syndrome-associated pancreas adenocarcinoma (PDAC) [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B042.

Molecular and Clinicopathologic Characterization of Mismatch Repair-Deficient Endometrial Carcinoma Not Related to MLH1 Promoter Hypermethylation

Universal tumor screening in endometrial carcinoma (EC) is increasingly adopted to identify individuals at risk of Lynch syndrome (LS). These cases involve mismatch repair-deficient (MMRd) EC without MLH1 promoter hypermethylation (PHM). LS is confirmed through the identification of germline MMR pathogenic variants (PV). In cases where these are not detected, emerging evidence highlights the significance of double-somatic MMR gene alterations as a sporadic cause of MMRd, alongside POLE/POLD1 exonuclease domain (EDM) PV leading to secondary MMR PV. Our understanding of the incidence of different MMRd EC origins not related to MLH1-PHM, their associations with clinicopathologic characteristics, and the prognostic implications remains limited.In a combined analysis of the PORTEC-1, -2, and -3 trials (n = 1254), 84 MMRd EC not related to MLH1-PHM were identified that successfully underwent paired tumor–normal tissue next-generation sequencing of the MMR and POLE/POLD1 genes. Among these, 37% were LS associated (LS-MMRd EC), 38% were due to double-somatic hits (DS-MMRd EC), and 25% remained unexplained. LS-MMRd EC exhibited higher rates of MSH6 (52% vs 19%) or PMS2 loss (29% vs 3%) than DS-MMRd EC, and exclusively showed MMR-deficient gland foci. DS-MMRd EC had higher rates of combined MSH2/MSH6 loss (47% vs 16%), loss of >2 MMR proteins (16% vs 3%), and somatic POLE-EDM PV (25% vs 3%) than LS-MMRd EC. Clinicopathologic characteristics, including age at tumor onset and prognosis, did not differ among the various groups.Our study validates the use of paired tumor–normal next-generation sequencing to identify definitive sporadic causes in MMRd EC unrelated to MLH1-PHM. MMR immunohistochemistry and POLE-EDM mutation status can aid in the differentiation between LS-MMRd EC and DS-MMRd EC. These findings emphasize the need for integrating tumor sequencing into LS diagnostics, along with clear interpretation guidelines, to improve clinical management. Although not impacting prognosis, confirmation of DS-MMRd EC may release patients and relatives from burdensome LS surveillance.

Analiza genetskih mutacija TP53, APC, KRAS i MMR kod kolorektalnog karcinoma - pregled literature

Introduction: Colorectal cancer (CRC) is one of the most common malignancies with significant global health and economic implications. Genetic mutations in genes such as TP53, APC, KRAS, and MMR play a crucial role in the development and progression of this cancer. This review paper analyzes current knowledge about the impact of these mutations on colorectal carcinogenesis, using available literature. Objective: To provide a comprehensive review of the role of genetic mutations in TP53, APC, KRAS, and MMR genes in the development of colorectal cancer and to consider their impact on diagnosis and treatment. Materials and Methods: This review examines peer-reviewed research articles and reports sourced from databases such as PubMed, Google Scholar, and other academic sources. The focus was on studies investigating genetic mutations, their prevalence, and their role in the pathogenesis of CRC. Results: Mutations in the TP53 gene, present in more than 50% of CRC cases, are critical for malignant cell transformations. KRAS mutations, found in about 50% of cases, lead to abnormal signaling contributing to unchecked proliferation. APC mutations are associated with hereditary predisposition to CRC, while MMR genes, such as MLH1 and MSH2, play a key role in DNA repair and are linked to hereditary nonpolyposis colorectal cancer. Conclusion: Genetic mutations in TP53, APC, KRAS, and MMR genes play a significant role in the development of colorectal cancer. A deeper understanding of these mutations may significantly enhance diagnostic and therapeutic strategies, guiding future research in this rapidly evolving field.

SOMATIC DEFICIENT MISMATCH REPAIR ASSESSED BY IMMUNOHISTOCHEMISTRY AND CLINICAL FEATURES IN BRAZILIAN GLIOBLASTOMA PATIENTS.

Glioblastoma (GBM) is the most frequent primary malignant CNS tumor. Deficient mismatch repair (dMMR) is associated with better prognosis and is a biomarker for immunotherapy. Evaluation of MMR by immunohistochemistry (IHC) is accessible, cost effective, sensitive, and specific. Our objective was to investigate MMR proteins in adult GBM patients. We retrospectively analyzed 68 GBM samples to evaluate the proficiency of MMR genes expression assessed by IHC. Clinicopathologic and molecular features were compared in proficient (pMMR) or dMMR. 10 (14.7%) samples showed dMMR, and the most frequent was MSH6 (100%) followed by MSH2, PMS2, and MLH1. We observed heterogeneous expression of dMMR in 5 GBMs. The median overall survival did not differ between pMMR (19.8 months; 0.2-30) and dMMR (16.9 months; 6.4-27.5) (p = 0.31). We observed a significantly higher overall survival associated with gross total resection compared to subtotal resection or biopsy (30.7 vs. 13.6 months, p = 0.02) and MGMT methylated status (29.6 vs. 19.8 months, p = 0.049). At the analysis time, 10 patients were still alive, all in the pMMR group. Our data demonstrated dMMR phenotype assessed by IHC in an expressive portion of GBM patients, however without significant impact on overall survival.

Treatment Options in Late-Line Colorectal Cancer: Lessons Learned from Recent Randomized Studies.

Systemic treatment of metastatic colorectal cancer (mCRC) has improved considerably over the past 20 years. First- and second-line combinations of 5FU, oxaliplatin, and irinotecan, with or without anti-angiogenic and/or anti-EGFR antibodies, were approved shortly after the turn of the millennium. Further triumphs were not seen for almost 10 years, until the approval of initially regorafenib and shortly after trifluridine/tipiracil. A growing understanding of tumor biology through molecular profiling has led to further treatment options. Here, we review the most recent clinical data for late-line treatment options in mCRC, focusing on randomized trials if available. We include recommendations for options in unselected patients and therapies that should only be offered in patients with distinct tumor profiles (e.g., BRAF mutations, KRAS G12C mutations, HER2 amplification, deficient MMR, or NTRK gene fusions).

Abstract A001: Reshaping the immune microenvironment after temozolomide priming in metastatic colorectal cancer patients in ARETHUSA clinical trial

Abstract Mismatch repair (MMR) proficient (MMRp) tumors, known for their weak immune response, often exhibit limited effectiveness in immunotherapy. In contrast, responsive mismatch repair deficient (MMRd) tumors, characterized by heightened immune activity, show a stronger response to immune checkpoint blockade (ICB). In the ARETHUSA clinical trial (NCT03519412), we revealed that treatment with temozolomide (TMZ) can pharmacologically deactivate the MMR machinery, as indicated by mutational signature analysis and the emergence of MMR gene mutations. We focused our investigation on a subset of initially MMRp CRC patients who experienced sustained disease stabilization with ICB after receiving TMZ priming treatment. This treatment resulted in the emergence of an inactivating MSH6 mutation and the TMZ mutational signature. Additionally, TMZ treatment induced diverse genomic changes, leading to the identification of three distinct subtypes through analysis of blood and tissue samples. Notably, we observed a dose-dependent accumulation of mutations with a specific molecular signature visible at the clonal level (subtype B2) in 10% of patients, at the subclonal level (subtype B1) in 71% of patients, and the absence of these mutations (subtype A) in 19% of patients within our cohort of 21 patients. In conjunction with previous genetic findings, in this new study we performed an analysis of T-cell receptors (TCRs) in the tumor microenvironment following TMZ treatment. Our results revealed expanded clonotypes in tumors that exhibited a clonal increase in mutations following priming treatment. Furthermore, the diversity of TCRs within the immune infiltrate confirmed the categorization of tumors into three distinct classes based on Tumor Mutational Burden (TMB) and mutational signatures. We also established a significant linear correlation (p-value 0.0048) between the diversity of T-cells in the microenvironment and the number of mutations induced by TMZ. Based on these clinically-oriented preliminary findings, we propose that increasing the number of TMZ-induced mutations could enhance the likelihood of TMZ-driven neoantigens triggering clonal expansion of specific T-cell clonotypes within the tumor. Interestingly, the subset of TMZ-treated tumors displaying an acquired MSH6 mutation, TMZ mutational signature, and increased TMB not only achieved temporary disease stabilization with ICB but also exhibited clonal expansion of T-cells in the tumor microenvironment. These clinically relevant findings suggest that the inactivation of MMR achieved through TMZ priming has the potential to reshape the immune microenvironment modulating the immune response in metastatic colorectal cancer. Citation Format: Giovanni Crisafulli, Andrea Sartore-Bianchi, Luca Lazzari, Filippo Pietrantonio, Paolo Battuello, Alice Bartolini, Federica Di Nicolantonio, Silvia Marsoni, Salvatore Siena, Alberto Bardelli. Reshaping the immune microenvironment after temozolomide priming in metastatic colorectal cancer patients in ARETHUSA clinical trial [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A001.