DNA Mismatch Repair Deficiency Research Articles

MSI-XGNN: an explainable GNN computational framework integrating transcription- and methylation-level biomarkers for microsatellite instability detection.

Microsatellite instability (MSI) is a hypermutator phenotype caused by DNA mismatch repair deficiency. MSI has been reported in various human cancers, particularly colorectal, gastric and endometrial cancers. MSI is a promising biomarker for cancer prognosis and immune checkpoint blockade immunotherapy. Several computational methods have been developed for MSI detection using DNA- or RNA-based approaches based on next-generation sequencing. Epigenetic mechanisms, such as DNA methylation, regulate gene expression and play critical roles in the development and progression of cancer. We here developed MSI-XGNN, a new computational framework for predicting MSI status using bulk RNA-sequencing and DNA methylation data. MSI-XGNN is an explainable deep learning model that combines a graph neural network (GNN) model to extract features from the gene-methylation probe network with a CatBoost model to classify MSI status. MSI-XGNN, which requires tumor-only samples, exhibited comparable performance with two well-known methods that require tumor-normal paired sequencing data, MSIsensor and MANTIS and better performance than several other tools. MSI-XGNN also showed good generalizability on independent validation datasets. MSI-XGNN identified six MSI markers consisting of four methylation probes (EPM2AIP1|MLH1:cg14598950, EPM2AIP1|MLH1:cg27331401, LNP1:cg05428436 and TSC22D2:cg15048832) and two genes (RPL22L1 and MSH4) constituting the optimal feature subset. All six markers were significantly associated with beneficial tumor microenvironment characteristics for immunotherapy, such as tumor mutation burden, neoantigens and immune checkpoint molecules such as programmed cell death-1 and cytotoxic T-lymphocyte antigen-4. Overall, our study provides a powerful and explainable deep learning model for predicting MSI status and identifying MSI markers that can potentially be used for clinical MSI evaluation.

Mismatch repair deficiency is not sufficient to elicit tumor immunogenicity.

DNA mismatch repair deficiency (MMRd) is associated with a high tumor mutational burden (TMB) and sensitivity to immune checkpoint blockade (ICB) therapy. Nevertheless, most MMRd tumors do not durably respond to ICB and critical questions remain about immunosurveillance and TMB in these tumors. In the present study, we developed autochthonous mouse models of MMRd lung and colon cancer. Surprisingly, these models did not display increased T cell infiltration or ICB response, which we showed to be the result of substantial intratumor heterogeneity of mutations. Furthermore, we found that immunosurveillance shapes the clonal architecture but not the overall burden of neoantigens, and T cell responses against subclonal neoantigens are blunted. Finally, we showed that clonal, but not subclonal, neoantigen burden predicts ICB response in clinical trials of MMRd gastric and colorectal cancer. These results provide important context for understanding immune evasion in cancers with a high TMB and have major implications for therapies aimed at increasing TMB.

Japanese Society of Medical Oncology/Japan Society of Clinical Oncology/Japanese Society of Pediatric Hematology/Oncology-led clinical recommendations on the diagnosis and use of immunotherapy in patients with DNA mismatch repair deficient (dMMR) tumors, third edition

BackgroundClinical trials have reported the efficacy of immune checkpoint inhibitors in the treatment of mismatch repair-deficient (dMMR) advanced solid tumors. The accumulated evidence of tumor agnostic agent has been made since PD-1 inhibitor was approved and used in clinical practice. Therefore, we have revised the guideline “Japan Society of Clinical Oncology provisional clinical opinion for the diagnosis and use of immunotherapy in patients with deficient DNA mismatch repair tumors, cooperated by Japanese Society of Medical Oncology, First Edition”.MethodsClinical questions regarding medical care were formulated for patients with dMMR advanced solid tumors. Relevant publications were searched by PubMed and Cochrane Database. Critical publications and conference reports were added manually. Systematic reviews were performed for each clinical question for the purpose of developing clinical recommendations. The committee members identified by Japan Society of Clinical Oncology (JSCO), Japanese Society of Medical Oncology (JSMO), and Japanese society of pediatric hematology/oncology (JSPHO) voted to determine the level of each recommendation considering the strength of evidence, expected risks and benefits to patients, and other related factors. Thereafter, a peer review by experts nominated from JSCO, JSMO, and JSPHO and the public comments among all societies’ members were done.ResultsThe current guideline describes two clinical questions and eight recommendations for whom, when, and how MMR status should be tested.ConclusionIn this guideline, the committee proposed eight recommendations for performing MMR testing properly to select patients who are likely to benefit from immunotherapy.

Depicting the landscape of gut microbial-metabolic interaction and microbial-host immune heterogeneity in deficient and proficient DNA mismatch repair colorectal cancers

BackgroundAccumulating evidence has indicated the role of gut microbiota in remodeling host immune signatures, but various interplays underlying colorectal cancers (CRC) with deficient DNA mismatch repair (dMMR) and proficient DNA...

Aligning germline and somatic mutations in prostate cancer. Are genetics changing practice?

To review the current status of germline and somatic (tumour) genetic testing for prostate cancer, and its relevance for clinical practice. A narrative synthesis of various molecular profiles related to their clinical context was carried out. Current guidelines for genetic testing and its feasibility in clinical practice were analysed. We reported the main identified genetic sequencing results or functional genomic scores for prostate cancer published in the literature or obtained from the French PROGENE study. The molecular alterations observed in prostate cancer are mostly linked to disruption of the androgen receptor (AR) pathway or DNA repair deficiency. The main known germline mutations affect the BRCA2 and HOXB13 genes, whereas AR and TP53 are the genes with most frequent somatic alterations in tumours from men with metastatic prostate cancer. Molecular tests are now available for detecting some of these germline or somatic alterations and sometimes recommended by guidelines, but their utilisation must combine rationality and feasibility. They can guide specific therapies, notably for the management of metastatic disease. Indeed, following androgen deprivation, targeted therapies for prostate cancer currently include PARP inhibitors, immune checkpoint inhibitors and PSMA guided radiotherapy. The genetic tests currently approved for targeted therapies remain limited to the detection of BRCA1 and BRCA2 mutation and DNA mismatch repair deficiency, while large panels are recommended for germline analyses, not only for inherited cancer predisposing syndrome, but also for metastatic prostate cancer. Further consensus aligning germline with somatic molecular analysis in metastatic prostate cancer is required, including genomics scars, emergent immunohistochemistry or functional pre-screen imaging. With rapid advances in knowledge and technology in the field, continuous updating of guidelines to help the clinical management of these individuals, and well conducted studies to evaluate the benefits of genetic testing are needed.

Molecular typing and mutational characterization of rectal neuroendocrine neoplasms.

Rectal neuroendocrine neoplasms (NENs) are rare neoplasms with limited understanding of its genomic alterations and molecular typing. The paraffin-embedded tissue specimens of 38 patients with rectal NENs after surgery were subjected to whole gene sequencing (WGS), and mutation profilings were drawn to identify high-frequency mutation genes, copy-number variations (CNVs), tumor mutation burden (TMB), signal pathways, mutation signatures, DNA damage repair (DDR) genes, and molecular types. The differences of mutated genes and signaling pathways in different pathological grades and metastatic/non-metastatic groups were compared. It helped to search for potential targets. C > T and T > C transitions are the most common base substitutions in rectal NENs. DNA mismatch repair deficiency, DNA base modifications, smoking and exposure to ultraviolet light might play a role in the occurrence of rectal NENs. DAXX, KMT2C, BCL2L1, LTK, MERTK, SPEN, PKN1, FAT3, and LRP2 mutations were found in only low-grade rectal NETs, whereas APC, TP53, NF1, SOX9, and BRCA1 mutations were common in high-grade rectal NECs/MiNENs. These genes helped in distinguishing poorly-differentiated or well-differentiated rectal NENs. Alterations in P53, Wnt and TGFβ signaling pathways were more pronounced in rectal NECs and MiNENs. Alterations in Wnt, MAPK and PI3K/AKT signaling pathways promoted metastases. Rectal NENs were classified into two molecular subtypes by cluster analysis based on the mutant genes and signaling pathways combined with clinicopathological features. Patients with mutations in the LRP2, DAXX, and PKN1 gene showed a trend of well-differentiated and early-stage tumors with less metastasis (p = 0.000). This study evaluated risk factors for regional lymphatic and/or distant metastases, identified high-frequency mutated genes, mutation signatures, altered signaling pathways through NGS. Rectal NENs were divided into two molecular types. This helps to evaluate the likelihood of metastasis, formulate follow-up strategies for patients and provide a target for future research on precision treatment of rectal NENs. PARP inhibitors, MEK inhibitors, mTOR/AKT/PI3K and Wnt signaling pathway inhibitors may be effective drugs for the treatment of metastatic rectal NENs.

Evolution of systemic therapy for disseminated endometrial cancer: literature review

The TC combination regimen (paclitaxel + carboplatin) is the “gold standard” first-line therapy for disseminated endometrial cancer (EC). The use of hormone therapy (HT) in the first-line setting is limited. Until recently, patients with disseminated EC had unfavorable outcomes despite the standard-of-care treatment (chemotherapy (CHT) and HT). None of the available cytostatics could improve disease control and survival in patients who have received standard platinum-based therapy. Evidently, the poor treatment outcomes of disseminated EC suggested that therapeutic approaches should be changed, and more effective treatment regimens should be developed. The treatment of disseminated EC has been revolutionized with deeper understanding of carcinogenesis, a new molecular classification of EC, and stratification of treatment approaches according to the biological potential of the tumor. The most significant advances included understanding the role of microsatellite instability (MSI) and DNA mismatch repair (MMR) deficiencies as a predictor of high efficacy of immunotherapy, a novel class of systemic therapies for disseminated EC. This review article focuses on the evolution of systemic therapy for disseminated EC. Here we discuss in detail the results of key international trials of HT, first and second lines of chemotherapy, targeted therapy, immunotherapy, and immunotherapeutic/ targeted agents for disseminated EC. Biological markers, such as MSI and PD-L1, their correlation with the response rate, and the mechanism of synergy between pembrolizumab and lenvatinib are discussed in detail.

HGG-23. DECIPHERING THE RELATIONSHIP OF MICROSATELLITE-INSTABILITY AND MUTATIONAL BURDEN IN HIGH-GRADE GLIOMA PATIENTS WITH CONSTITUTIONALLY IMPAIRED DNA DAMAGE RESPONSE

Abstract Multiple large-scale genomic studies across pediatric tumors have reported an overall frequency of ~6-10% of patients with constitutional pathogenic variants, but associations of cancer predisposition with distinct tumor types vary between zero and 100%. We analyzed sequencing and DNA methylation data from 363 patients to explore the landscape of cancer predisposition in pediatric high-grade gliomas (pedHGG). Almost all pedHGG with constitutional alterations resulting in DNA mismatch repair deficiency (MMRD) were classified to IDH-mutant or pedRTK1a DNA methylation classes. Conversely, 70% of pedRTK1a and nearly 60% of IDH-mutant pedHGG (<18 years) arose in patients with either Lynch or cMMRD syndromes. Biallelic MMR inactivation (cMMRD) was found almost exclusively to affect the exchangeable part of the mismatch repair machinery complex (MSH6/PMS2; 75%), while heterozygous loss affected non-exchangeable core members MLH1 or MSH2 (93%). IDH-mutant MMRD tumors showed substantially lower overall tumor mutational burden (TMB < 40), less microsatellite instability, and frequent MSH6 mutation (60%), while pedRTK1-MMRD tumors often harbored additional alterations in POLE, leading to an ultra-mutator phenotype (TMB < 100), had higher microsatellite instability burden, and more often linked to PMS2 or MSH2 loss. Both IDH-mutant- and pedRTK1-MMRD tumors were associated with poor outcome, with MMRD-associated IDH-mutant pedHGG resulting in significantly shorter survival compared with sporadic IDH-mutant tumors (p=0.0015). We observed a nonlinear correlation between increasing numbers of point mutations, InDels and microsatellite instability. Exemplary, patients with pathogenic constitutional variants in MSH6 showed a significantly lower number of InDels compared with overall TMB, which might be due to repair ability of the replacement protein MSH3. As newer studies point towards an important role of InDels in antigen presentation and therefore as a potential biomarker for immune checkpoint inhibition, further exploration of these detailed molecular associations in conjunction with clinical outcome and response data is warranted.

Results of phase I-II bridging study for Nous-209, a neoantigen cancer immunotherapy, in combination with pembrolizumab as first line treatment in patients with advanced dMMR/MSI-h colorectal cancer.

e14665 Background: Microsatellite instability (MSI) is a molecular hallmark in which systemic deficiency of DNA mismatch-repair (dMMR) generates tumor-specific frameshift peptides (FSPs). Nous-209 is an off-the-shelf cancer vaccine targeting 209 FSPs selected from dMMR/MSI-H tumors (Leoni, G. et al., 2020). Treatment options for unresectable dMMR/MSI-H tumors have improved, but further efforts to increase long-term progression-free survival are needed. NOUS-209-01 Phase I demonstrated that Nous-209 and pembrolizumab is safe, well tolerated, and provides clinical benefit in advanced gastric and CRC dMMR/MSI-H tumors (Fakih et al., 2022). Moreover, Nous-209 induces durable specific anti-tumoral immune response, capable of successfully infiltrating the tumor (D’Alise et al., 2022). Here, we present data from NOUS-209-01 study Phase I-II expansion cohort at the recommended phase 2 dose (RP2D) of Nous-209 and pembrolizumab in adults with dMMR/MSI-H locally advanced unresectable or metastatic colorectal cancer (mCRC) eligible for 1st line immune checkpoint inhibition. Methods: NOUS-209-01 ( NCT04041310 ) Phase I-II bridge expansion cohort receives one GAd20-209-FSP prime intramuscularly at the 1st infusion of pembrolizumab, followed by 3 MVA-209-FSP boosts with the 2nd, 3rd and 4th pembrolizumab cycles. Patients can continue treatment with pembrolizumab for 18 months. Response assessment is evaluated every 9 (+/-1) weeks by CT imaging, according to RECIST v1.1 criteria. ctDNA assessments are at baseline, 11, 28, 49, 79 weeks and at objective progression on study. Results: Phase I-II bridge enrolled 8 patients with dMMR/MSI-H treatment-naïve mCRC. One patient had clinical progression before 1st scan and was excluded as non-evaluable and 7 were evaluable for tumor response. 5/7 (71%, CI: 95% 29.0, 96.3) patients achieved objective response: 1 (14%) complete response (CR) and 4 (57%) partial responses (PR). 2 patients (29%) had disease progression (PD) at the 1st scan. ctDNA assessments were available for 6 out of 7 patients (4 PR; 2 PD); ctDNA levels reduced >50% at week 11 in the 4 PR patients, in contrast to a 28% increase in 1 PD. Interestingly 1 PD patient had ctDNA > 50% reduction. The median follow-up at data cut-off was 7.5 months (range: 3.3-8.4), median progression free survival and median duration of response have not been reached. 6/7 (85.7%) patients had treatment emergent adverse events (TEAE), all mild or moderate grade; no TEAEs were serious or led to treatment discontinuation. Conclusions: The combination of Nous-209 and pembrolizumab is safe, well tolerated and shows encouraging clinical efficacy in patients with treatment-naive dMMR/MSI-H mCRC eligible for anti-PD-1 therapy. The study is ongoing and expanding to Phase II randomization with a new accelerated Nous-209 vaccination schedule. Clinical trial information: NCT04041310 .

DNA Mismatch Repair Deficiency in Colorectal Adenocarcinoma by a Two-Antibody Immunohistochemical Approach and its Association with Clinicopathological Features among Bangladeshi Patients

Background: Defective DNA Mismatch Repair (dMMR) genes cause dMMR/Microsatellite Instability (MSI)-related Colorectal Cancers (CRC) in humans which are different from Microsatellite Stable (MSS) tumors in terms of biological behavior, therapeutic response, and prognosis. We aimed to determine the frequency of dMMR CRCs by a two-antibody (PMS2 and MSH6) immunohistochemical approach and to evaluate their association with clinicopathological parameters to document the ever first report of such cases in Bangladesh. Methods: Fifty histopathologically confirmed resected bowel specimens of CRC were studied over a period of two years at a tertiary-level hospital in Bangladesh. Histopathological parameters like morphologic variants, histologic subtypes, grade, stage, Lymphovascular Invasion (LVI), intratumoral lymphocytic infiltrate, and Crohn-like peritumoral reaction were assessed. Immunohistochemistry using PMS2 and MSH6 was performed on representative paraffin blocks by DAKO EnVision method. Expression of both of the markers was evaluated in classified groups. Results: Mean age of study population was 48.60±14.6 years with a male to female ratio of 1.8:1. dMMR was recorded in 32% of cases. Expression of PMS2 and MSH6 were lost in 20% and 12% of cases, respectively. dMMR status was significantly associated with mucinous histology (p=0.014), lower pN staging (p=0.042), low LVI (p=0.002), exhibited intra-tumoral lymphocytosis (p=0.001), and Crohn like peritumoral reaction (p=0.001). No significant association with gender, age, right-sided location, histologic type, pT stage or grade was observed. Conclusion: Frequency of dMMR CRCs was comparatively higher in the Bangladeshi population than in other races. Identification of dMMR tumors by their protein expression pattern using at least two, preferably four antibodies is proposed for routine screening of CRC cases.

Pathobionts in the tumour microbiota predict survival following resection for colorectal cancer

Background and aimsThe gut microbiota is implicated in the pathogenesis of colorectal cancer (CRC). We aimed to map the CRC mucosal microbiota and metabolome and define the influence of the tumoral microbiota on oncological outcomes.MethodsA multicentre, prospective observational study was conducted of CRC patients undergoing primary surgical resection in the UK (n = 74) and Czech Republic (n = 61). Analysis was performed using metataxonomics, ultra-performance liquid chromatography-mass spectrometry (UPLC-MS), targeted bacterial qPCR and tumour exome sequencing. Hierarchical clustering accounting for clinical and oncological covariates was performed to identify clusters of bacteria and metabolites linked to CRC. Cox proportional hazards regression was used to ascertain clusters associated with disease-free survival over median follow-up of 50 months.ResultsThirteen mucosal microbiota clusters were identified, of which five were significantly different between tumour and paired normal mucosa. Cluster 7, containing the pathobionts Fusobacterium nucleatum and Granulicatella adiacens, was strongly associated with CRC (PFDR = 0.0002). Additionally, tumoral dominance of cluster 7 independently predicted favourable disease-free survival (adjusted p = 0.031). Cluster 1, containing Faecalibacterium prausnitzii and Ruminococcus gnavus, was negatively associated with cancer (PFDR = 0.0009), and abundance was independently predictive of worse disease-free survival (adjusted p = 0.0009). UPLC-MS analysis revealed two major metabolic (Met) clusters. Met 1, composed of medium chain (MCFA), long-chain (LCFA) and very long-chain (VLCFA) fatty acid species, ceramides and lysophospholipids, was negatively associated with CRC (PFDR = 2.61 × 10−11); Met 2, composed of phosphatidylcholine species, nucleosides and amino acids, was strongly associated with CRC (PFDR = 1.30 × 10−12), but metabolite clusters were not associated with disease-free survival (p = 0.358). An association was identified between Met 1 and DNA mismatch-repair deficiency (p = 0.005). FBXW7 mutations were only found in cancers predominant in microbiota cluster 7.ConclusionsNetworks of pathobionts in the tumour mucosal niche are associated with tumour mutation and metabolic subtypes and predict favourable outcome following CRC resection.E9vQ1bxk-JdT4E6GMYGhRXVideo

The role of immunotherapy in microsatellites stable metastatic colorectal cancer: state of the art and future perspectives.

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide, despite several advances has been achieved in last decades. Few prognostic and predictive biomarkers guide therapeutic choice in metastatic CRC (mCRC), among which DNA mismatch repair deficiency and/or microsatellite instability (dMMR/MSI) holds a crucial role. Tumors characterized by dMMR/MSI benefit from immune checkpoint inhibitors. However, most of the mCRC patients (around 95%) are microsatellite stable (MSS), thereby intrinsically resistant to immunotherapy. This represents a clear unmet need for more effective treatments in this population of patients. In this review, we aim to analyze immune-resistance mechanisms and therapeutic strategies to overcome them, such as combinations of immunotherapy and chemotherapy, radiotherapy or target therapies specifically in MSS mCRC. We also explored both available and potential biomarkers that may better select MSS mCRC patients for immunotherapy. Lastly, we provide a brief overview on future perspectives in this field, such as the gut microbiome and its potential role as immunomodulator.

ATM Inhibition-Induced ISG15/IFI27/OASL Is Correlated with Immunotherapy Response and Inflamed Immunophenotype.

Immune checkpoint blockade (ICB) therapy can improve the survival of cancer patients with a high tumor mutation burden (TMB-H) or deficiency in DNA mismatch repair (dMMR) in their tumors. However, most cancer patients without TMB-H and dMMR do not benefit from ICB therapy. The inhibition of ATM can increase DNA damage and activate the interferon response, thus modulating the tumor immune microenvironment (TIME) and the efficacy of ICB therapy. In this study, we showed that ATM inhibition activated interferon signaling and induced interferon-stimulated genes (ISGs) in cisplatin-resistant and parent cancer cells. The ISGs induced by ATM inhibition were correlated with survival in cancer patients who received ICB therapy. In oral cancer, high expressions of ISG15, IFI27, and OASL were associated with low expressions of ATM, the activation of inflamed immune pathways, and increased tumor-infiltrating scores of CD8+ T, natural killer, and dendritic cells. The high expressions of ISG15, IFI27, and OASL were also correlated with complete remission in patients with cervical cancer treated with cisplatin. These results suggest that ATM inhibition can induce the interferon response and inflamed TIME, which may benefit ICB therapy.

Microsatellite instability in endometrial cancer: the role of diagnostic methods and their clinical application. Expert consensus

Deficient DNA mismatch repair (dMMR) is a rare molecular disorder found in 20-30 % of endometrial tumors. Laboratory identification of dMMR/microsatellite instability (MSI) has a high diagnostic value, since these impairments are considered as biomarkers of endometrial adenocarcinoma. They help to identify patients at high risk of Lynch syndrome, evaluate the disease prognosis, and estimate the efficacy of immune checkpoint inhibitors and their combinations. This review details current concepts of MSI diagnostics and discusses its predictive value in patients with endometrial cancer. It also describes a new diagnostic algorithm for the detection of dMMR and MSI.

A tumor focused approach to resolving the etiology of DNA mismatch repair deficient tumors classified as suspected Lynch syndrome

Routine screening of tumors for DNA mismatch repair (MMR) deficiency (dMMR) in colorectal (CRC), endometrial (EC) and sebaceous skin (SST) tumors leads to a significant proportion of unresolved cases classified as suspected Lynch syndrome (SLS). SLS cases (n = 135) were recruited from Family Cancer Clinics across Australia and New Zealand. Targeted panel sequencing was performed on tumor (n = 137; 80×CRCs, 33×ECs and 24xSSTs) and matched blood-derived DNA to assess for microsatellite instability status, tumor mutation burden, COSMIC tumor mutational signatures and to identify germline and somatic MMR gene variants. MMR immunohistochemistry (IHC) and MLH1 promoter methylation were repeated. In total, 86.9% of the 137 SLS tumors could be resolved into established subtypes. For 22.6% of these resolved SLS cases, primary MLH1 epimutations (2.2%) as well as previously undetected germline MMR pathogenic variants (1.5%), tumor MLH1 methylation (13.1%) or false positive dMMR IHC (5.8%) results were identified. Double somatic MMR gene mutations were the major cause of dMMR identified across each tumor type (73.9% of resolved cases, 64.2% overall, 70% of CRC, 45.5% of ECs and 70.8% of SSTs). The unresolved SLS tumors (13.1%) comprised tumors with only a single somatic (7.3%) or no somatic (5.8%) MMR gene mutations. A tumor-focused testing approach reclassified 86.9% of SLS into Lynch syndrome, sporadic dMMR or MMR-proficient cases. These findings support the incorporation of tumor sequencing and alternate MLH1 methylation assays into clinical diagnostics to reduce the number of SLS patients and provide more appropriate surveillance and screening recommendations.

Abstract 4327: MSI cancer associated DNA (TA)n-dinucleotide repeat expansions and implications for Werner synthetic lethality

Abstract Microsatellite instability (MSI) is caused by deficient DNA mismatch repair (MMR) and is a ubiquitous feature of cancer. Werner syndrome (WRN) helicase is involved in genome stability and DNA repair. We identified WRN as a synthetic-lethal target in dMMR/MSI cancers and highlighted WRN inhibition as a therapeutic option for dMMR/MSI cancers refractory to available therapies. A previously unappreciated genetic feature of dMMR/MSI cancer cells, DNA (TA)n-dinucleotide repeat expansions, were recently reported to cause vulnerability to WRN depletion. Our mechanistic understanding of TA-dinucleotide repeat expansion biology is limited, and their potential therapeutic implications are unclear. To investigate the landscape of these alterations in cancer, we inferred (TA)-dinucleotide repeat expansions by performing coverage analysis in a collection of hundreds of preclinical cancer models and human tumors (PCAWG) profiled by whole genome sequencing (WGS). We validated our findings in cancer cell lines and organoid cultures by performing long-read WGS. Furthermore, we investigated TA-expansions in single-cell-derived clones from human MSI tumors and cancer organoids. Finally, we inferred TA repeats in laser capture microdissection (LCMB)-derived samples obtained from patients affected by familial cancer predisposition syndromes. Our analysis unveils the landscape of TA-repeats alterations in a large collection of tumors and preclinical models, informing on the level of inter-tumor heterogeneity and their association with variable levels of WRN dependency. In addition, we investigated intra-patient tumor heterogeneity of TA-repeats length both within clonal organoids expanded from normal and neoplastic colorectal stem cells, and within different subclones derived from MSI cancer organoids. Furthermore, analysis of non-neoplastic and neoplastic tissues from patients affected by familial cancer predisposition syndromes revealed the pattern of TA-repeats expansions associated with various DNA-repair pathway alterations. Finally, we will discuss the clinical implications of our findings, as TA-repeats heterogeneity may affect sensitivity and resistance to the future generation of WRN inhibitors. Our data provide fresh insights into the inter and intra-tumoral heterogeneity of TA-dinucleotide repeat expansions in human cancers. These data contribute to understanding the role of MMR in cancer and exploiting Werner as a therapeutic target in cancer. Citation Format: Gabriele Picco, Shriram Bhosle, Maria Kalyva, Elena Grassi, Freddy Gibson, Angham Al Saedi, Sara Vieira, Mathijs Sanders, Livio Trusolino, Andrea Bertotti, Isidro Cortes-Ciriano, Mathew Garnett. MSI cancer associated DNA (TA)n-dinucleotide repeat expansions and implications for Werner synthetic lethality. [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 4327.

Abstract 6648: Prediction of anti-PD-1 efficacy based on immune marker densities and their spatial distribution in colorectal cancer

Abstract Background: Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of patients with deficient DNA mismatch repair (d-MMR) metastatic colorectal cancer (CRC), yet less than half of patients receive clinical benefit. We hypothesized that immune contexture including spatial distribution of cells expressing immune molecules in the tumor microenvironment may predict immunotherapy outcome. Methods: Primary CRC tissues from consecutive patients with d-MMR metastatic CRC (N=33) were treated with anti-PD-1 antibodies at Mayo Clinic Comprehensive Cancer Center (2015-2018). Tumors were stained for PD-L1, PD-1, CD8, CD3, CD68, LAG3, TGFβR2, MHC-I, CD14, B2M, DAPI and pan-cytokeratin in 5 compartments (overall tumor, tumor epithelia, tumor stroma, peritumor inside vs outside) by multiplex immunofluorescence with digital image analysis. Features computed within each image analysis region included positive cell density, fraction of positive cell types, intensity of positive cells, and spatial distribution between distinct cell types (distances measured using image analysis software). Prior to model fitting, feature selection was performed using regularized Cox regression with LASSO. Regularization parameter was chosen based on 5-fold cross validation. A Cox proportional hazards model was fitted to predict patient progression-free survival (PFS). Results: Among patients with d-MMR CRCs, 16/33 (48.4%) were female, 10 received first-line ICI therapy, and 23 had received > 1 prior chemotherapy regimen. Median age was 61.6 years (IQR of 49.4, 73.7). Eight patients (24.2%) harbored BRAFV600E, 9 (27.2%) had mutant KRAS, and median PFS was 22.2 months (95% CI: 11.8, NR) with 20 events. PD-L1 was expressed in tumor cells, CD68+ macrophages, and CD3+ T lymphocytes. PD-1 expression on CD8+ T lymphocytes was also observed. By univariate analysis, only cell-cell distance readouts achieved statistical significance. Multivariable feature selection identified the mean number of PD-1+ cells within 10 microns of a PD-L1+ cell in the overall tumor as the strongest predictor of anti-PD-1 efficacy, and was significantly associated with PFS (HR=0.87, 95% CI: 0.79-0.95, p< 0.001). In contrast, the ratio of PD-1+/PD-L1+ cells was not predictive of treatment efficacy (p= 0.47), thereby underscoring the importance of spatial distribution for PFS prediction. Conclusion: The mean number of PD-1+ cells in proximity to PD-L1+ cells in tumors was a predictive biomarker of anti-PD-1 efficacy. Confirmatory studies are warranted. Citation Format: Bahar Saberzadeh-Ardestani, Rondell P. Graham, Qian Shi, Eze Ahanonu, Sara McMahon, Crystal Williams, Antony Hubbard, Wenjun Zhang, Andrea Muranyi, Dongyao Yan, Kandavel Shanmugam, Frank A. Sinicrope. Prediction of anti-PD-1 efficacy based on immune marker densities and their spatial distribution in colorectal cancer [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 6648.

Abstract 6463: Persistence of smoking mutational signatures in the non-small cell lung cancer genome

Abstract Mutational signatures offer insights into cancer etiology. Smoking tobacco is associated with three signatures involving single base substitutions (SBS4), double base substitutions (DBS2) and insertions/deletions (ID3). Combining mutational signatures with clinical data allows examination of temporal and quantitative aspects of carcinogenic exposures. We analyzed 132 non-small cell lung carcinoma (NSCLC) whole genomes, sequenced through the 100,000 Genomes Project (Genomics England), with paired clinical data available for 130/132 (98.5%). Clinical data included smoking status, duration, pack-years (py), and duration of smoking cessation. The majority were “ever-smokers” (ex-smokers: 90/130, 69.2%; current smokers: 32/130, 24.6%) and 120/122 (98.4%) had at least one smoking signature. DBS2 was the most sensitive signature for ever-smokers (118/122, 96.7%), although SBS4 was the optimal signature for diagnosing smoking-related NSCLCs (SBS4 positive likelihood ratio 7.15). An absence of ID3 best identified true never smoker NSCLCs (ID3 negative likelihood ratio 0.08). Ever-smokers had a higher tumor mutational burden (TMB) than never smokers (median 8.25 mutations/Mb vs 1.18 mutations/Mb, p = <0.0001), while TMB positively correlated with smoking pack-years (p = 0.044). Following smoking cessation, the median length of discontinuation was 11 years before a smoking-related NSCLC was diagnosed and, in some cases, smoking signatures persisted for up to 50 years prior to NSCLC diagnosis. To complement existing epidemiological data, 40 pack years was estimated as a critical cut-off for minimizing ongoing NSCLC risk. Prolonged smoking cessation did not reduce TMB (p = 0.055) or SBS4 mutations (p = 0.11), although this did produce lower DBS2 counts (p = 0.0067). Further signatures observed included SBS92 (previously seen in the bladder urothelium of ever-smokers) and a novel signature hypothesized to be linked to DNA mismatch repair deficiencies. NSCLCs from two patients with prior platinum chemo-radiotherapy harbored signatures of both chemotherapy- and radiotherapy-induced damage in their recurrent biopsy sample. These results highlight the importance of paired sequencing and clinical data, revealing that smoking-induced DNA damage can have long-lived effects, and strengthening health guidance of abstinence from smoking at the earliest possibility. Citation Format: Colin R. Lindsay, Pantelis Nicola, Andreas Gruber, Kate Brown, Mathew Carter, Helen Adderley, Shereen Rafee, Anna Moss, Andrew Wallace, George Burghel, Helene Schlecht, Katie Baker, Sharzad Moghadam, Jane Rogan, Jamie Weaver, Angeliki Malliri, William Newman, Fiona Blackhall, David Wedge, Genomics England. Persistence of smoking mutational signatures in the non-small cell lung cancer genome. [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 6463.

A noninvasive nomogram model based on CT features to predict DNA mismatch repair deficiency in gastric cancer.

DNA mismatch repair deficiency (dMMR) status has served as a positive predictive biomarker for immunotherapy and long-term prognosis in gastric cancer (GC). The aim of the present study was to develop a computed tomography (CT)-based nomogram for preoperatively predicting mismatch repair (MMR) status in GC. Data from a total of 159 GC patients between January 2020 and July 2021 with dMMR GC (n=53) and MMR-proficient (pMMR) GC (n=106) confirmed by postoperative immunohistochemistry (IHC) staining were retrospectively analyzed. All patients underwent abdominal contrast-enhanced CT. Significant clinical and CT imaging features associated with dMMR GC were extracted through univariate and multivariate analyses. Receiver operating characteristic (ROC) curve analysis, decision curve analysis (DCA) and internal validation of the cohort data were performed. The nomogram contained four potential predictors of dMMR GC, including gender (odds ratio [OR] 9.83, 95% confidence interval [CI] 3.78-28.20, P < 0.001), age (OR 3.32, 95% CI 1.36-8.50, P = 0.010), tumor size (OR 5.66, 95% CI 2.12-16.27, P < 0.001) and normalized tumor enhancement ratio (NTER) (OR 0.15, 95% CI 0.06-0.38, P < 0.001). Using an optimal cutoff value of 6.6 points, the nomogram provided an area under the curve (AUC) of 0.895 and an accuracy of 82.39% in predicting dMMR GC. The calibration curve demonstrated a strong consistency between the predicted risk and observed dMMR GC. The DCA justified the relatively good performance of the nomogram model. The CT-based nomogram holds promise as a noninvasive, concise and accurate tool to predict MMR status in GC patients, which can assist in clinical decision-making.

Are High Levels of Microsatellite Instability and Microsatellite Stability Identical in DNA Mismatch Repair-Deficient Colorectal Cancer Patients?

The purpose of the current study was to determine whether there is a difference between high levels of microsatellite instability (MSI-H) and microsatellite stability (MSS) in DNA mismatch repair-deficient (DMMR) colorectal cancer (CRC) patients. A total of 452 CRC patients with DMMR from December, 2014, to April, 2021, in our hospital were selected retrospectively. However, only 105 patients underwent Sanger or next-generation-sequencing (NGS) to confirm their microsatellite status. Ultimately, 55 MSI-H patients and 20 MSS patients with intact medical record information were included in this study. The MSS group was associated with a higher mutation rate in the KRAS gene (P=0.011). Meanwhile, MSI-H was related to colon cancer (P < 0.01). However, no significant differences in other clinical characteristics were observed between the two groups of patients. There was no significant difference between the MSI-H and MSS groups in terms of overall survival (OS) (P=0.398) and disease-free survival (DFS) (P=0.307). The MSI-H status was associated with colon cancer and a lower mutation rate of the KRAS gene in DMMR patients. In CRC-DMMR patients, the MSS group exhibited better OS and DFS than the MSI-H group, although these differences were not statistically significant. Accordingly, in clinical practice, we should not confuse these two types of patients.