ABSTRACT Immune checkpoint inhibitors (ICIs) can re-active the immune response and induce a complete response in mismatch repair-deficient and microsatellite instability-high (dMMR/MSI-H) colorectal cancer (CRC). However, most CRCs exhibit proficient mismatch repair and microsatellite stable (pMMR/MSS) phenotypes with limited immunotherapy response because of sparse intratumoral CD8+ T-lymphocyte infiltration. Cellular senescence has been reported to involve immune cell infiltration through a senescence-associated secretory phenotype (SASP). However, the relationship between CRC cellular senescence and CD8+ T-lymphocyte infiltration remains unclear. Through integrated analysis of clinical cohorts and transcriptomic data across mismatch repair (MMR) subtypes, we identified cellular senescence as a hallmark of dMMR tumors, accompanied by elevated expression of KDM4A (lysine demethylase 4A). Clinically, KDM4Ahigh CDKN2A/p16high expression correlated with improved CRC patient prognosis. Mechanistically, KDM4A upregulated AGT (angiotensinogen) expression through H3K9me3 demethylation and promoted CRC cellular senescence. Meanwhile, KDM4A-driven senescence suppressed tumor growth and enhanced intratumoral CD8+ T-lymphocyte infiltration via enhancing SASP-associated secretion. Furthermore, AGT disrupted PHB1 (prohibitin 1)-mediated basal mitophagy, triggering cytoplasmic mitochondrial DNA (mtDNA) accumulation that activated CGAS-STING1 signaling and enhanced SASP secretion. Crucially, KDM4A overexpression potentiated anti-PDCD1/PD1 efficacy in MSI-H CRC and reversed therapy resistance in MSS CRC. Conclusively, we established a KDM4A-AGT-PHB1 (KAP) grade system that robustly predicts immunotherapy responsiveness in pMMR CRC patients. Abbreviation: AGT: angiotensinogen; BafA: bafilomycin A1; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CRC: colorectal cancer; CDKN1A/p21: cyclin dependent kinase inhibitor 1A; CDKN2A/p16: cyclin dependent kinase inhibitor 2A; CHX: cycloheximide; Co-IP: co-immunoprecipitation; dMMR: deficient mismatch repair; EdU: 5-ethynyl-2’-deoxyuridine; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IL6: interleukin 6; IL8: interleukin 8; IHC: immunohistochemical; KDM4A: lysine demethylase 4A; mtDNA: mitochondrial DNA; MS: mass spectrometry; NFKB/NF-κB: nuclear factor kappa B; PHB1: prohibitin 1; PHB2: prohibitin 2; PINK1: PTEN induced kinase 1; pMMR: proficient mismatch repair; PRKN/parkin: parkin RBR E3 ubiquitin protein ligase; SASP: senescence-associated secretory phenotype; SA-GLB1/β-gal: senescence-associated galactosidase beta 1; TIMM23: translocase of inner mitochondrial membrane 23; TOMM20: translocase of outer mitochondrial membrane 20; TRIM21: tripartite motif containing 21; TUBB/beta-tubulin: tubulin beta class I.

Accounting for 15–30% of colorectal cancer cases, the serrated pathway remains poorly characterized compared to the adenoma–carcinoma sequence. It involves sessile serrated lesions as precursors and is characterized by BRAF mutations (BRAFV600E), CpG island hypermethylation, and microsatellite instability (MSI). Using label-free proteomics, we compared normal tissue margins from patients with diverticular disease, sessile serrated lesions, low-grade adenomas, and high-grade adenomas. We identified S100A14 as significantly overexpressed in sessile serrated lesions compared to low-grade adenomas, high-grade adenomas, and normal tissues. This overexpression was confirmed by immunohistochemical scoring in an independent cohort. Gene expression analyses of public datasets showed higher S100A14 expression in BRAFV600E-mutated and MSI-H colorectal cancers compared to microsatellite stable BRAFwt tumors. This finding was confirmed by immunohistochemical scoring in an independent colorectal cancer cohort. Furthermore, single-cell RNA sequencing analysis from the Human Colon Cancer Atlas revealed that S100A14 expression in tumor cells positively correlated with the abundance of tumoral CD8+ cytotoxic T cells, particularly the CD8+ CXCL13+ subset, known for its association with a favorable response to immunotherapy. Collectively, our results demonstrate for the first time that S100A14 is a potential biomarker of serrated neoplasia and further suggests its potential role in predicting immunotherapy responses in colorectal cancer.

Patients with metastatic colorectal cancer (CRC) presenting defective mismatch repair (dMMR) and/or high index of microsatellite instability (MSI-H) are suitable candidates for immunotherapy with immune checkpoint inhibitors (ICIs), such as pembrolizumab and nivolumab. However, the use of these ICIs as neoadjuvant treatment for non-metastatic and metastatic CRCs with these specific molecular alterations is still controversial. Thus, this systematic review (PROSPERO CRD42021258676) evaluated the benefits, toxicity profile, and clinical outcomes of pembrolizumab and nivolumab as neoadjuvant therapy for CRC with dMMR and/or MSI-H. For this, MEDLINE, Scopus, Cochrane Library, and Science Direct databases were used. Twenty-five case reports and four randomized clinical trials were included, and the main outcomes analyzed were overall survival, progression-free survival, pathological response through RECIST 1.1, and the Eastern Cooperative Oncology Group (ECOG) performance status, respectively. In general, the evaluated studies were well conducted according to the assessment tools, and the evidence suggests that combinations of nivolumab plus ipilimumab were more effective to treat metastatic CRC with dMMR and/or MSI-H than nivolumab alone. Regarding pembrolizumab, clinical evidence demonstrated its safety and efficiency for patients with this subtype of CRC in metastatic and locally advanced stages. This review indicates therapeutic promising potential of ICIs for patients with dMMR and/or MSI-H CRCs. Additionally, alterations in PI3K and JAK pathways are important mechanisms of resistance and represent an opportunity for the development of new drugs. Therefore, molecular investigations should be performed to uncover causes of therapeutic failure.

3573 Background: Activation of the complement cascade pathway is associated with pro-oncogenic inflammation and immune-suppressing, myeloid-derived M2 macrophages for many solid tumors. Most colorectal cancers (CRC) are microsatellite stable that do not respond to immunotherapy. The role of complement activation (CA) in the “immune cold” CRC phenotype remains poorly detailed. We sought to identify molecular annotations of CRC subpopulations enriched for CA to guide future therapeutic strategies. Methods: CRC tumors from 207 patients with stages II-IV CRC at MDACC underwent bulk RNA sequencing. Transcriptomes were analyzed per GSEA “Hallmark Complement” gene set to assign a normalized enrichment score (NES) for CA to each patient and considered “complement high” (“CH”; N = 103) or “complement low” (“CL”; N = 104) if the complement NES score was above or below the median. Associations between CA and clinical and pathologic characteristics - e.g., demographics, mutation status, and Consensus Molecular Subtype (CMS) - were evaluated by chi-squared analysis. Single cell RNA (scRNA) sequencing was performed on a separate cohort of CRC primary tumors (N = 85) and liver metastases (N = 60) to compare CA among different cell types using a Wilcoxon’s test. To assess for an association between CA and response to immunotherapy in a previously annotated clinical trial of patients with MSS, BRAF V600E metastatic CRC (NCT04017650), we evaluated pretreatment biopsies by bulk RNA sequencing and compared transcriptomic differences in CA between responders versus non-responders to encorafenib, cetuximab, and nivolumab (E+C+N). Results: CH CRC featured a higher prevalence for MSI-H CRC (21.3% vs 4.2%; p = .005), CMS1 (30.1% vs 6.7%; p < .001), and CMS4 (26.2% vs 7.7%; p < .001) relative to CL CRC. CMS2 was more common among CL CRC (54.8% vs 14.6%, p < .001). CH CRC was associated with BRAF V600E mutations (29.7% vs 9.2% for CL, p = .002) but not with KRAS/NRAS mutations or RAS/BRAF wild-type CRC (p = n.s. for both). On scRNA analysis, CA scores were highest in myeloid cells and lowest for B cells (p < .0001). Among patients with MSS, BRAF V600E CRC, CH signature was associated with non-response to E+C+N (fold-change 3.0 relative to responders, p = .046). Conclusions: Association of CH status with MSI-H CRC is a novel finding that warrants further study in understanding differential patterns of benefit to immune checkpoint blockade. CH CRC, associated uniquely with BRAF V600E CRC, was distributed bimodally across the immune-activated CMS1 and the immune-suppressing CMS4 CRC, similar to known transcriptomic heterogeneity of BRAF V600E CRC. Our data suggest high CA, linked to immune-suppressing myeloid cell subpopulations, as a negative predictive biomarker for response to immunotherapy in MSS BRAF V600E CRC and support broader study of complement-targeting agents to improve treatment for selected patients with CRC.

e14561 Background: Immune checkpoint inhibitors (ICI) are more effective than chemotherapy (CT) in treating MSI-H CRC in the metastatic and neoadjuvant settings. Currently, the impact of CT on ICIs efficacy in CRC is not well characterized, especially in stage 3 CRC adjuvant therapy. Methods: Based on data from published randomized control trials in CRC, we developed a mathematical modeling of Kaplan-Meier curves for stage 3 CRC pts treated with 1) CT, 2) ICI or 3) combination CT + ICI. Our modeling generated a hypothesis on the potential interactions of CT and ICI in CRC therapy. We retrospectively analyzed 30,500 CRC pts for MSI-H and/or TMB-H (MSI/TMB-H) status from multi-institutional electronic health records on an IRB-approved protocol between January 2009 and December 2024 to obtain real-world therapeutic CT and ICI interactions. Microsatellite status and genomic testing were done in CLIA-certified, CAP-accredited labs. Pt characteristics, pathology, in-depth clinical treatment history and genomics were summarized with descriptive statistics. Treatment efficacy (overall response rate [ORR], relapse free survival [RFS], progression-free survival [PFS], and overall survival [OS]) to CT and ICIs were determined in MSI/TMB-H CRC pts. Results: The Kaplan-Meier estimate may be represented by the equation f(t) as a function of time: f(t) = ((1/(at 2 +1) + b + ((1-b) e -ct ))/2. Mathematical modeling of Kaplan-Meier curves for stage 3 MSI-H and/or TMB-H CRC pts demonstrated superior efficacy with adjuvant ICI compared to combination CT + ICI, and both regimens are better than CT. The hypothesis that CT inhibits ICI efficacy in MSI/TMB-H CRC patients is supported by our retrospective study. Stage 4 MSI/TMB-H CRC ICI-treated pts who received prior CT demonstrated significantly decreased ORR, PFS and OS. Stage 3 MSI/TMB-H CRC pts had higher RFS and OS when treated with ICI compared to CT. Conclusions: Exposure to CT decreased ICI efficacy in CRC. Mathematical modeling shows adjuvant ICI is superior to combination ICI + CT in stage 3 CRC. Clinical trial investigation with adjuvant ICI compared to CT + ICI is warranted in stage 3 CRC.

TPS3643 Background: Currently, pembrolizumab is one of the front-line therapies for patients with MSI-H CRC. However, approximately 40% of patients who received pembrolizumab experienced disease progression early in the course of disease (KEYNOTE 177). Therefore, there is still an unmet need to enhance the efficacy of checkpoint inhibitors in MSI-H CRC. MSI-H CRC has a higher level of expression of VEGF in blood compared to patients compared to its MSS counterpart (Hansen et al. Colorectal Dis. 2011). Consistently, exploratory analysis of CALBG-80405 and PARADIGM trial showed that patients with MSI-H CRC were more likely to benefit from anti-VEGF therapy than anti-EGFR therapy regardless of the side of the tumor. NSABP C-08 also suggested that anti-VEGF therapy may have biological activity even in as adjuvant therapy for patients with MSI-H colon cancer. Regorafenib is a potent VEGF and multikinase inhibitor involved in pathologic processes such as oncogenesis, tumor angiogenesis, metastasis and tumor immunity, with preclinical evidence showing its immune modulatory effect in the tumor microenvironment. In this trial, we hypothesize that adding low-dose regorafenib to pembrolizumab may induce synergistic activity beyond their independent clinical efficacy and create deep and durable responses for patients with MSI-H CRC. Methods: In the lead arm of this prospective randomized study, 22 patients will be enrolled through Hoosier Cancer Research Network (HCRN-GI23-643). In this first line clinical trial, patients will receive regorafenib 60 mg daily in combination with pembrolizumab 200mg IV in cycle 1, followed by regorafenib 90mg in subsequent cycles to optimize the treatment tolerance and compliance. The primary outcome that will be measured is ORR, defined as the percentage of partial or complete response to the treatment within 12 months. ORR will be measured using RECIST 1.1. criteria. A formal one-sided hypothesis test will be conducted for futility, assuming that we will reject the null hypothesis of a target ORR only if we have strong evidence. In this study, we assume a null hypothesis that ORR is 0.60, which would reflect significant clinical improvement over the current standard of ORR = 0.43 from KEYNOTE 177. The alternative hypothesis is that ORR is less than 0.60. For the lead-in phase of the study, the emphasis is on controlling Type I error to be small, to be 0.05 or lower. An exact binomial test will be conducted, based on the number of ORRs in the 22 patients. The study is currently accruing through Hoosier Cancer Research Network was activated in July 2024. Clinical trial information: NCT06006923 .

Abstract Immune checkpoint blockade (ICB) therapy demonstrated limited efficacy in colorectal cancer (CRC), and identification of intrinsic factors modulating immune suppression in CRC is an unmet need. Here, we revealed that Chromosome 8 open reading frame 76 (C8orf76) drives immunosuppression and is a molecular target to boost ICB therapy in CRC. C8orf76 is upregulated in primary CRCs compared to adjacent normal tissues in 2 independent CRC cohorts and its expression predicts poor patient survival. Intestine-specific C8orf76 knockin in mice exacerbated AOM/DSS-induced CRC, accompanied by increased intratumoral myeloid-derived suppressor cells (MDSCs) but reduced IFN-γ+ and granzyme B+ CD8+ T cells, inferring that C8orf76 promotes immunosuppression in CRC. Consistently, genetic depletion of C8orf76 augmented antitumour immunity in CT26 (MSS-CRC) and MC38 (MSI-H-CRC) allograft models. Integrated RNA-seq and ChIP-seq revealed that C8orf76 functions as a transcription factor to drive NDST1 expression, which activates the PI3K-Akt-NF-κB signaling cascade. Activated NF-κB in turn promotes the expression and secretion of CXCL1, a major chemoattractant for MDSCs. Consequently, C8orf76-induced CXCL1 mediates the recruitment of MDSCs via CXCR2 to antagonize functional CD8+ T cells in tumor immune microenvironment of CRC. Confirming this, targeting NDST1 or CXCR2 could reverse C8orf76 induced immunosuppression in vivo. Finally, we tested the translational value of C8orf76 using genetic ablation or vesicle-like nanoparticles (VNPs)-encapsulated C8orf76-siRNA. In line with our hypothesis, we demonstrated that targeting C8orf76 by genetic ablation or VNPs-encapsulated C8orf76-siRNA potentiated the anti-PD1 efficacy in both MSI-H and MSS CRC models. In summary, C8orf76 drives immunosuppression through a NDST1-CXCL1/CXCR2 axis, and targeting C8orf76 is a promising approach to boost ICB therapy efficacy in CRC. Citation Format: Hongyan Gou, Shang Guo, Xiaohong Wang, Xia Jiang, Chi Chun Wong, Huarong Chen, Chunxian Wei, Lingxue Shi, Zengren Zhao, Jun Yu. C8orf76 promotes colorectal tumorigenesis by promoting an immunosuppressive microenvironment and is a therapeutic target for boosting anti-PD-1 efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB132.

Abstract Colorectal cancer (CRC) is the second leading cause of cancer-related mortality worldwide. Recent advancements have established immunotherapy as a promising treatment option, opening new avenues to address the therapeutic challenges faced by CRC patients. In response, Brenus Pharma has developed a novel therapeutic cancer vaccine, leveraging Stimulated Tumor Cells (STC) technology. This innovative approach involves exposing tumor cells to physical (irradiation and heat shock) or chemical (chemotherapy) stress mimicking the CRC treatment, coupled with haptenation to enhance their immunogenicity. The anti-tumor activity of STC technology was initially validated using a murine surrogate vaccine (mSTC-1010) composed of mouse tumor cell lines. These studies demonstrated that using three distinct cell lines significantly enhanced antigenicity anti-tumoral activity compared to a single cell line. Subsequently, this approach was used for the development of the human vaccine candidate, STC-1010, which consists of six drug substances (DS) derived from three human CRC cell lines (HCT116, HT29, and Lovo). These cells were also subjected to physical or chemical stress and haptenation processes that collectively ensure the cells are no longer capable of proliferation. Transcriptomic analyses revealed the activation of stress-related pathways specific to each DS, which enabled their quantification in STC-1010 using a deconvolution strategy. The preclinical efficacy of STC-1010 was extensively evaluated. In ex-vivo studies, STC-1010 demonstrated efficacy by activating CD8+ T cells through STC-1010-treated dendritic cells, resulting in significant cancer cell apoptosis. Dendritic cells (DC) primed with STC-1010 showed an increased expression score for the antigen processing and presentation pathway compared to vehicle controls. Interestingly, CD8 + T cells activated by STC-1010-primed DC induced significant cancer cell apoptosis. In the chicken embryo Chorio-Allantoic Membrane (CAM) model, STC-1010 treatment, in combination with GM-CSF, significantly reduced tumour weight and upregulated the secretion of IL-2, IL-8, IL-12, and IFN-gamma. Furthermore, this immune-reactive model exhibited increased tumor necrosis (p = 0.0267), a 49% regression in metastases, and enhanced infiltration of CD4+ and CD8+ T cells compared to controls confirming the high potential of the STC-1010 to induce immune response in vivo. Additionally, the efficacy of mSTC-1010 was evaluated in combination with an anti-PD-1 antibody in a subcutaneous CT26 in vivo mouse model. The results showed a significant improvement in overall survival among treated mice. Based on these preclinical findings, the approval process for a clinical trial of STC-1010 was in advanced stages as of early January 2025, with regulatory authority requests nearing completion for the first-in-human study. A phase I/II trial is planned for non resectable metastatic and advanced CRC patients, comprising a dose-escalation phase I and cohort-extension phase IIa. This trial aims to evaluate the safety and preliminary effectiveness of STC-1010 in combination with immunostimulants and mFOLFOX6, with or without bevacizumab, in MSS and MSI-H CRC patients. Citation Format: George Alzeeb, Iseulys Richert, Corentin Richart, Paul Marteau, Lionel Chalus, Marion Brun, Yan Wan, Corinne Tortorelli, Benoit Pinteur, Paul Bravetti, Céline Gongora, Antoine Italiano, François Ghirringhelli. Preclinical efficacy of an innovative therapeutic cancer vaccine: A new era for immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB383.

Abstract Colorectal cancer (CRC) is the third most common and lethal cancer type worldwide. Although CRC incidence in the older population has decreased in many countries, early-onset CRC cases (< 50 years age, EOCRC) have been rapidly increasing globally. The fecal microbiome has been reproducibly associated with CRC. However, microbiome differences according to age of onset, stage, anatomic location, and genomic instability remain only minimally explored. Here, we integrated data generated from the ONCOBIOME consortium, Cancer Grand Challenges PROSPECT project and the ARETHUSA study, providing seven new cohorts of gut microbiome samples from healthy, adenoma- and CRC individuals (n total = 1, 668) combined with 12 public studies, for a total of 3, 784 metagenomic samples, including 190 EOCRC and 270 age-matched control samples. Information on tumor stage, anatomic location, microsatellite stability/instability-high (MSS/MSI-H) has been curated and harmonized across datasets. Metagenomes were analyzed using the bioBakery suite for community-, species-, strain-level, and gene-level profiling, and downstream integrative analysis was performed via meta-analysis and leave-one-dataset-out learning tasks. We obtained increased microbiome-based CRC prediction capabilities (average leave-one-dataset-out AUC = 0.84) and accurate species- and strain-level characterization of CRC stages (e.g., Slackia exigua and Ruminococcus bicirculans, respectively, between stage 0-II and III/IV), while the microbiome in adenoma samples did not differ substantially from controls (average leave-one-dataset-out AUC < 0.6). The fecal microbiome of right-sided CRC cases presented lower species and higher oral commensals richness than left-sided cases. The gut microbiome composition of MSI-H CRC was significantly different than MSS, as reflected by machine learning discrimination performances (average cross-validation AUC = 0.71), and characterized by reduced alpha-diversity and fewer biomarkers than in MSS cases, such as Ligilactobacillus salivarius and Limosilactobacillus fermentum. We confirmed that EOCRC microbiome was significantly different from age-matched controls (average leave-one-dataset-out AUC = 0.84), harboring typical CRC-associated bacteria, such as F. nucleatum and D. pneumosintes. Overall, we defined cross-cohort reproducible microbiome associations across tumor stages, primary location, genetic instability, and EOCRC, providing improved resolution within the CRC microbiome and more refined targets for diagnosis and therapeutic stratification. Citation Format: Gianmarco Piccinno, Kelsey N. Thompson, Paolo Manghi, Luca Lazzari, Chiara Pozzi, Andrew R. Ghazi, Andrew M. Thomas, Aitor Blanco-Miguez, Francesco Asnicar, Katarina Mladenovic, Federica Pinto, Federica Armanini, Giulio Ferrero, Sonia Tarallo, Long H. Nguyen, Yan Yan, Mingyang Song, Veronika Vymetalkova, Mario Trompetto, Vaclav Liska, Tomas Hucl, Pavel Vodicka, Beatrix Bencsiková, Martina Čarnogurská, Vlad Popovici, Salvatore Siena, Andrea Sartore-Bianchi, Michela Lizier, Carmen Correale, Alessandra Raimondi, Filippo Pietrantonio, Federica Marmorino, Chiara Cremolini, Barbara Pardini, Francesca Cordero, Lisa Derosa, Silvia Marsoni, Alberto Bardelli, Maria Rescigno, Yin Cao, Andrew T. Chan, Laurence Zitvogel, Curtis Huttenhower, Alessio Naccarati, Eva Budinska, Nicola Segata. Microbiome alterations of colorectal cancer according to age of onset, stage, anatomic location, and mismatch repair deficiency. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2370.

Dipeptidase-1 (DPEP1) is highly upregulated in colorectal cancer (CRC), with its enzymatic function linked to invasion and metastasis. More recently, DPEP1 was found to serve as a receptor for neutrophils when expressed by activated endothelial cells. It is unknown whether neutrophils bind to DPEP1-expressing CRC cells and whether this impacts features of CRC. Neutrophils have been shown to be tumor promoting in cancers including CRC, where they act to exclude CD8+ T cells. Herein, we show that neutrophils bind DPEP1-expressing CRC cells. In addition, DPEP1 is preferentially expressed in microsatellite-stable (MSS) CRCs, in which there are a paucity of CD8+ T cells, whereas DPEP1 is negatively correlated with microsatellite-unstable (MSI-H) CRCs, which are T cell rich and are more responsive to immunotherapy. Remarkably, carcinogen-treated Dpep1-null mice develop multiple, large, plaque-like, locally invasive adenocarcinomas and squamous cell cancers in the distal colon. These adenocarcinomas exhibit a marked reduction in neutrophils and an influx CD8+ T cells, along with reduced expression of mismatch repair proteins, consistent with features of MSI-H CRC. These results establish DPEP1’s importance in maintaining MSS CRC and its ability to shape the tumor microenvironment.

Abstract INTRODUCTION: Immunotherapy outcomes have been underwhelming in most gastrointestinal malignancies. Microsatellite instable (MSI-H) colorectal cancers (CRC) are an exception. Immune checkpoint blockade (ICB) is standard of care for MSI-H CRC. However, a portion of MSI-H CRCs do not respond well to ICB, insufficiently explained by tumor mutational burden or clinical factors. We sought to understand transcriptional pathways of ICB resistance in MSI-H CRC through whole transcriptome analysis of archival formalin-fixed, paraffin-embedded (FFPE) samples. METHODS: Cases of MSI-H CRC were identified from the Duke Pathology lab and Molecular Registry of Tumors from 2017 to 2022. 12 durable responders (MSI-H-R) and 9 non-responders (MSI-H-N) were selected for analysis. 15 locally advanced MSI-H CRC (MSI-H-loc) samples, with a near 100% response rate to ICB in other settings, and 10 locally advanced MSS CRC samples were used as controls. 46 total samples (12 MSI-H-R, 9 MSI-H-N, 15 MSI-H-loc, and 10 MSS) were selected for FFPE-based whole transcriptome analysis using MACE-Seq (GenXPro), a 3’mRNA-sequencing approach. RESULTS: 44 samples (96%) were suitable for analysis with an average of 18,378 genes counted. The tumor suppressor GPRC5A was upregulated in the MSI-H-R group compared to MSI-H-N. MSI-H-N samples exhibited higher expression rates of genes coding for the extracellular matrix and mesenchymal stem cells (KLF2, Desmin, MUC1), hypoxia-induced genes including those related to metabolic processes (LDHD, PDK4) and the beta-globin subunit of hemoglobin (HBB), and terminal T cell exhaustion (TOX). Hypoxic states and LDHD have been shown to correlate inversely with immune cell infiltration and ICB response. HBB overexpression has been shown to suppress reactive oxygen species-mediated apoptosis. Gene set enrichment analysis using Gene Ontogeny (GO) Pathways demonstrated upregulation in neutrophil pathways and developmental signaling, such as bone morphogenesis (BMP), in the MSI-H-N group. We did not observe differences in T-cell markers between MSI-H groups, raising the possibility that non-T-cell mediated mechanisms may dictate response to ICB. MSI-H-R expression profiles resembled the MSI-H-loc group, apart from differences in PLA2G2A, ASS1, AGR2, and SPINK1, which may be markers of metastasis propensity. Gene expression profiling revealed substantive differences between MSI-H-N and MSS groups, indicating their resistance mechanisms to ICB may differ and supporting their fundamental differences. CONCLUSION: Given the rarity of MSI-H CRC and limited available data to predict response to ICB, additional studies are imperative to improve understanding of the therapeutic landscape for MSI-H CRC. MACE-seq is feasible on FFPE specimens and well suited for transcriptional comparisons between populations. Our study revealed a cohort of hypoxia-related genes to correlate with non-response to ICB in metastatic MSI-H CRC. Additional data collection is ongoing, and future studies will expand tumor types and implement mouse models to test genes of interest. Citation Format: Cameron Oswalt, Ashley Moyer, Y-Van Nguyen, Brent Hanks, John Strickler, David Hsu, William Jeck, Nicholas DeVito. Analyzing immunotherapy resistance in microsatellite instable colorectal FFPE samples using RNA sequencing [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A097.

TPS313 Background: Currently, pembrolizumab is one of the front-line therapies for patients with MSI-H CRC. However, approximately 40% of patients who received pembrolizumab experienced disease progression early in the course of disease (KEYNOTE 177). Therefore, there is still an unmet need to enhance the efficacy of checkpoint inhibitors in MSI-H CRC. MSI-H CRC has a higher level of expression of VEGF in blood compared to patients compared to its MSS counterpart (Hansen et al. Colorectal Dis. 2011). Consistently, exploratory analysis of CALBG-80405 and PARADIGM trial showed that patients with MSI-H CRC were more likely to benefit from anti-VEGF therapy than anti-EGFR therapy regardless of the side of the tumor. NSABP C-08 also suggested that anti-VEGF therapy may have biological activity even in as adjuvant therapy for patients with MSI-H colon cancer. Regorafenib is a potent VEGF inhibitor, with preclinical evidence showing its immune modulatory effect in the tumor microenvironment. In this trial, we hypothesize that adding low-dose regorafenib to pembrolizumab may induce synergistic activity beyond their independent clinical efficacy and create deep and durable responses for patients with MSI-H CRC. Methods: In the lead arm of this prospective randomized study, 22 patients will be enrolled through Hoosier Cancer Research Network (HCRN-GI23-643). Patients with treatment naïve MSI-H CRC will be enrolled in this front-line trial. One cycle of pembrolizumab and up to 3 cycles of chemotherapy prior to determination of MMR-D/MSI-H is allowed. Patients will receive regorafenib 60 mg daily in combination with pembrolizumab 200mg IV in cycle 1, followed by regorafenib 90mg in subsequent cycles to improve treatment tolerance. The primary outcome that will be measured is ORR, defined as the percentage of partial or complete response to the treatment within 12 months. ORR will be measured using RECIST 1.1. criteria. A formal one-sided hypothesis test will be conducted for futility, assuming that we will reject the null hypothesis of a target ORR only if we have strong evidence. In this study, we assume a null hypothesis that ORR is 0.60, which would reflect significant clinical improvement over the current standard of ORR = 0.43 from KEYNOTE 177. The alternative hypothesis is that ORR is less than 0.60. For the lead-in phase of the study, the emphasis is on controlling Type I error to be small, approximately 0.05. The test statistic will be the number of ORRs in the 22 patients, which we assume to follow a binomial distribution. Clinical trial information: NCT06006923 .

PurposeMicrosatellite high instability/deficient mismatch repair (MSI-H/dMMR) colorectal cancer (CRC) has an active tumor microenvironment, rendering it more sensitive to immune checkpoint inhibitors. Given that studies involving patients with MSI-H colorectal cancer with RAS mutations are scarce, we explored the effect of RAS mutations on the TME in patients with MSI-H/dMMR cancer and identified potential prognostic factors.MethodsSeventy-five patients diagnosed with MSI-H/dMMR colorectal cancer were retrospectively enrolled and divided into RAS-mutant and -wild-type groups. The expression levels of CD11c+ dendritic cells, CD4+ T cells, CD8+ T cells, and regulatory T cell (Treg) markers were detected, and prognostic factors were analyzed.ResultsRAS-mutant MSI-H colorectal patients were more likely to have: (1) higher platelet values; (2) shorter disease-free survival (DFS); (3) lower infiltrated numbers of CD11c+ dendritic cells, CD4+ T lymphocytes, and CD8+ T lymphocytes, and higher infiltrated numbers of Foxp3+ Treg cells. In MSI-H/dMMR CRC patients: (1) the high CD11c + , CD4 +, and CD8 + cells infiltration group had longer DFS than the low-infiltration group, and Foxp3 + cells infiltration was not significantly correlated with DFS; (2) the RAS mutation status, number of CD11c+ cells infiltrated, and carbohydrate antigen 19–9 (CA19–9) level were the potential prognostic factors.ConclusionRAS mutations in patients with MSI-H/dMMR CRC may reduce the infiltration of CD11c+ dendritic cells, CD4+ T cells, and CD8+ T cells, and increase the infiltration of Foxp3+ Treg cells to affect the tumor microenvironment of patients. RAS gene status, CD11c + cells infiltration, and CA19–9 level were potential prognostic factors for MSI-H/dMMR CRC.

179 Background: MMRd is a tumour agnostic biomarker predictive of response to immune checkpoint inhibitors (ICIs). Given the poor response to 5-FU based chemotherapy, ICIs are now being explored in early MSI-H CRC. The outcomes on this strategy are limited. Methods: We retrospectively evaluated early outcomes for patients receiving neoadjuvant ICIs for unresectable locally advanced/oligometastatic MMRd colorectal cancer (CRC), where intention of treatment was downstaging to permit curative resection. Following discussion at regional MDT, suitable patients were administered a PD-1 inhibitor 6-weekly until disease progression or improvement rendering suitability for surgical resection (to max of 2 years). Results: From October 2022-September 2024, ten patients with MMRd CRC were commenced on neoadjuvant ICIs. These comprised six right, one left sided colonic and three rectal tumours. Median age at diagnosis was 59 (IQR54–68). Four patients had family history of CRC. One patient had clinical stage II, seven stage III and two patients oligometastatic stage IV disease. All were patients with initially unresectable disease, with potential for curative resection pending response to ICI. Histologically, two tumours were poorly differentiated, two mucinous phenotype and six moderately differentiated adenocarcinomas. BRAF mutation was identified in three patients. None had KRAS mutation. Three rectal cancers received radiotherapy in addition to immunotherapy. Five required a defunctioning stoma either prior to or on ICI. Median follow-up was 12.5 months (IQR7-17). Objective response rate by RECIST 1.1 was 100%, with significant downstaging in 9 of 10 patients. The remaining patient is early in treatment course with no reimaging. To date, complete clinical response (cCR) has been observed in three patients (33%). All 10 patients are alive. Grade 3-4 treatment-related adverse events occurred in two patients (20%) who developed treatment-related strictures (one also had tumour perforation). This was successfully managed with defunctioning ileostomy and antibiotics, with subsequent cCR. Conclusions: We report impressive early outcomes of immunotherapy for locally advanced/metastatic MMRd CRC, with excellent downstaging and high predicted cCR. This highlights the importance of screening all CRC for MSI-H/MMRd. In the setting of initially unresectable CRC, an ICI-first approach can render patients eligible for surgery, however deep responses can result in local effects including strictures and/or perforation.

187 Background: NCCN guidelines recommend immunotherapy for MSI-H colon cancer (CC) with isolated peritoneal metastases (iPM); however, limited data support its benefit. This study aimed to evaluate the impact of immunotherapy in this population. Methods: Patients diagnosed with MSI-H CC with iPM from 2016-2019 were selected from the NCDB. Patients who received immunotherapy were compared to those who did not. Overall survival was assessed using Kaplan-Meier analysis and multivariate Cox proportional hazard regression. The trend of immunotherapy utilization from 2016-2020 was analyzed using the Cochran-Armitage test. Results: We analyzed a cohort of 444 patients, with 161 (36%) receiving immunotherapy. Patients in the immunotherapy group were, on average, younger (63 vs. 71 years; P<0.001), with balanced gender distribution between groups. Among those who received immunotherapy, 70% were treated as a first-line agent, and 30% after chemotherapy. Immunotherapy was more commonly administered in the adjuvant setting (66%) compared to neoadjuvant (7%) or without surgery (27%). Immunotherapy was associated with significantly longer median survival (33.6 vs. 14.9 months; P<0.001). In multivariate analysis, immunotherapy was associated with improved overall survival, whether given as a first-line agent (HR 0.54, 95% CI 0.39-0.74; P<0.001) or second-line after chemotherapy (HR 0.59, 95% CI 0.37-0.94; P=0.03). Additionally, primary tumor resection (HR 0.44, 95% CI 0.32-0.60; P<0.001), but not metastatectomy (HR 0.96, 95% CI 0.71-1.29; P=0.8), was associated with prolonged survival. Patients who received both immunotherapy and primary tumor resection (n=118) had a median survival of 43.8 months. From 2016 to 2020, the utilization of immunotherapy in this cohort increased from 29% to 37% (χ^2=0.23, P=0.63). Conclusions: Utilization of immunotherapy remains low in MSI-H CRC with iPM and did not change significantly during the study period. Immunotherapy is associated with improved overall survival and should be considered for appropriate candidates. The combination of primary tumor resection and immunotherapy yielded the best overall survival.

BackgroundMicrosatellite instability-high (MSI-H) metastatic colorectal cancer (CRC) patients are the dominant population in immune checkpoint blockade treatments, while more than half of them could not benefit from single-agent immunotherapy. We tried to identify the biomarker of MSI-H CRC and explore its role and mechanism in anti-PD-1 treatments. Tumor-specific MHC-II was linked to a better response to anti-PD-1 in MSI-H CRC and CD74 promoted assembly and transport of HLA-DR dimers.MethodsThe characteristic gene was screened by data analysis of single-cell and bulk transcriptome sequencing from public datasets. MSI-H CRC cells co-cultured with peripheral blood mononuclear cells and syngeneic model in C57BL/6 mice were performed to detect the sensitivity to anti-PD-1 treatments respectively.ResultsANXA10 was identified as a characteristic gene of MSI-H CRC and its expression was obviously greater in MSI-H than MSS CRC. ANXA10 significantly sensitized MSI-H CRC to anti-PD-1 treatments in vitro and in vivo. Specifically, ANXA10 promoted HLA-DR dimers in and on the surface of MSI-H CRC by increasing CD74 expression. Besides, this work demonstrated that ANXA10 contributed to better clinical benefits with anti-PD-1 therapy in MSI-H CRC patients.ConclusionsOur results provided a novel molecular marker ANXA10 to identify benefit population of MSI-H CRC for improving efficacy of anti-PD-1 and contributed to selection of treatment strategies.

Background: Colorectal cancer (CRC) is one of the leading causes of cancer-related morbidity and mortality worldwide. Microsatellite instability (MSI) is a crucial biomarker with prognostic and predictive value, influencing treatment decisions in CRC. Methods: This retrospective study investigated the impact of the MSI status on clinical outcomes in 184 CRC patients treated at the Oncology Institute of Vojvodina between 2018 and 2023. The MSI status was determined using the immunohistochemical analysis of mismatch repair proteins. Results: Among the cohort, 75% of tumors were microsatellite-stable (MSS), 4.3% exhibited low MSI (MSI-L), and 20.6% displayed high MSI (MSI-H). MSI-H tumors were significantly associated with right-sided CRC, mucinous differentiation, and female gender (p<0.05). The survival analyses revealed that the MSI-H patients with stage II disease had significantly better disease-free survival (DFS) and overall survival (OS) than the MSS counterparts (p=0.024 and p=0.006, respectively). Conversely, the MSI-H status in stage II patients receiving adjuvant therapy was linked to shorter DFS (p=0.000), highlighting the limited benefit from 5-fluorouracil-based regimens. In stage III CRC, the MSI status did not significantly affect DFS or OS. Conclusion: These findings underscore the dual role of MSI as a favorable prognostic marker in early-stage CRC and a predictor of the reduced benefit from adjuvant chemotherapy in stage II disease. This study emphasizes the need for individualized treatment strategies based on the MSI status and supports the potential integration of immunotherapy in the adjuvant setting for MSI-H CRC.

Growth differentiation factor 15 (GDF15) is a member of the transforming growth factor beta (TGF-β) superfamily and is related to metabolism, injury, and aging. GDF15 has both tumor-promoting and tumor-suppressing effects. However, its role in colorectal cancer (CRC) with high microsatellite instability (MSI-H) must be further clarified. In our study, we found that GDF15 is generally elevated in pancarcinoma, particularly in colorectal cancer, and serves as an early indicator of the development of colorectal cancer. IHC and WB confirmed that GDF15 was elevated in MSI-H CRC clinical tissues and MSI-H CRC cell lines (HCT-116 and LoVo). Suppressing GDF15 by siRNA resulted in a substantial decrease in cell viability and proliferation. Furthermore, suppressing GDF15 can increase the sensitivity of MSI-H CRC cells to 5-fluorouracil (5-FU), which decreases cell viability and increases the apoptosis rate. In vivo experiments also demonstrated that mouse xenografts with suppressed GDF15 expression were more susceptible to 5-FU chemotherapy. We examined alterations in mitochondria via electron microscopy and changes in the mitochondrial membrane potential, ferroptosis-related signals (MDA, Fe2+), and SLC7A11/GSH/GPX4 protein pathway. Our research indicates that inhibiting GDF15 affects ferroptosis-related pathways, leading to ferroptosis and improving the MSI-H CRC response to 5-FU therapy. As a result, GDF15 could be a promising target for diagnosing and treating MSI-H CRC, potentially enhancing the overall effectiveness of therapy for patients with MSI-H CRC.

BackgroundMicrosatellite instability-high (MSI-H) tumors, with elevated tumor mutational burden and expression of neoantigens, represent a distinct immune-activated subpopulation in colorectal cancer (CRC), characterized by strong lymph node reaction, locally advanced tumor and higher total lymph nodes harvested (TLN), but less metastatic lymph nodes and fewer incidence of III-IV stage. Host immune response to tumor and lymph nodes may be an important prognostic factor. However, N stage and LNR (Lymph-Node Ratio) have limitations in predicting the prognosis of MSI-H patients. Negative lymph node count (NLC) provided a more precise representation of immune activation status and extent of tumor metastasis. The study aims to detect prognostic significance of NLC in MSI-H CRC patients, and compare it with N stage, TLN and LNR.MethodsRetrospective data of 190 consecutive MSI-H CRC patients who received curative resection were collected. Survival analyses were performed using the Kaplan–Meier method. Clinicopathological variables including NLC, N stage, TLN and LNR were studied in univariate and multivariate COX regression analyses. ROC (receiver operating characteristic curve) and concordance index were employed to compare the differences in predictive efficacy between NLC, N stage, TLN and LNR.ResultsPatients with increased NLC experienced a significantly improved 5-years DFS and OS in Kaplan–Meier analysis, univariate analysis, and multivariate analysis, independent of potential confounders examined. Increased NLC corresponded to elevated 5-years DFS rate and 5-years OS rate. AUC (area under curve) and concordance index of NLC in DFS and OS predicting were both significantly higher than N stage, TLN and LNR.ConclusionsNegative lymph node is an important independent prognostic factor for MSI-H patients. Reduced NLC is associated with tumor recurrence and poor survival, which is a stronger prognostic factor than N stage, TLN and LNR.

e15580 Background: Colorectal cancer (CRC) is the third most common cancer in the US, with a dismal 5-year OS of 13% in mCRC. Concomitant metformin with chemotherapy or immune checkpoint inhibitors (ICIs) revealed improved outcomes in various cancers. Metformin's anti-cancer effects stem its effects on cancer metabolism, cell cycle modulation, cancer stem cells, gut microbiota, immunomodulatory properties. The aim of this retrospective study was to study the impact of concomitant metformin use with the type of systemic therapy (chemotherapy or ICIs) in CRC. Methods: CRC tumors were analyzed by NGS of DNA (592-gene) and RNA (whole transcriptome) at Caris Life Sciences. MSS was assessed by NGS, with MSI-H and MSS patients subcategorized by concomitant metformin use with ICI or with chemotherapy, respectively. Real-world OS, derived from insurance claims data, was calculated from the time of CRC sample collection to the last contact. Hazard ratio (HR) was calculated using the Cox proportional hazards model, with p values calculated using the log-rank test. Results: The study cohort included 31,083 CRC cases (male: female = 1.2, age: 80% > = 50 years). Common molecular alterations included TP53 (73.6%), APC (77.6%), KRAS (47.4%), CTNNB1 (2.0%), and HER2 amplification (1.7%). 1,503 patients were MSI-H (4.83%), 21,330 as MSS (68.6%), and 8,250 indeterminate. MSI-H patients receiving concurrent ICI/metformin had similar OS (52.7 vs. 55.4 months, HR = 0.78, 95% CI: 0.41 – 1.51, p = 0.462). Conversely, OS was increased in MSS patients on concomitant chemotherapy and metformin (38.1 vs 31.1 months, HR = 0.77, 95% CI: 0.69 – 0.86, p < 0.00001). In comparing real-world OS between left-sided and right-sided colorectal cancer (CRC), regardless of metformin use, multivariate analysis incorporating molecular alterations revealed longer OS for left-sided MSS CRC compared to right-sided (HR = 0.80, 95% CI: 0.76 – 0.84, p < 0.00001). However, further improvement in OS was associated with concurrent metformin use in both right-sided and left-sided tumors. Conclusions: In this retrospective analysis of real-world clinical outcomes, patients receiving concomitant metformin and cytotoxic chemotherapy had improved clinical outcomes in MSS CRC compared to those not on concurrent metformin. However, the concurrent use of metformin with ICIs in MSI-H CRC did not show an impact on clinical outcomes given low sample size. These results add to the existing body of literature on the potential benefits of metformin in the context of MSS CRC and underscore the importance of a prospective controlled study of concurrent metformin use with systemic chemotherapy in metastatic CRC. Limitations of this study involves the potential inclusion of cases with prior metformin use, small sample size, retrospective analyses, and the lack of control for the patients with type II DM.