Stable Colorectal Cancer Research Articles

Nivolumab plus ONC201 plus in microsatellite stable (MSS) metastatic colorectal cancer (mCRC) patients: a Brown University Oncology Research Group phase Ib/II study (BrUOG379).

Immune checkpoint inhibitors alone, or in combination with chemotherapy failed to provide meaningful clinical activity for patients with microsatellite stable (MSS) colorectal cancer (CRC). ONC201 is a small molecule that inactivates AKT and ERK signaling and actives the TRAIL pathway. Preclinical studies indicated potential benefits of combining ONC201 with checkpoint inhibitors. This is a phase Ib/II trial of ONC201 plus nivolumab for patient with MSS CRC who progressed on standard treatment. Enrolled patients received ONC201 plus nivolumab in a dose de-escalation fashion to determine the maximum tolerated dose (MTD). Additional patients were enrolled in the dose-expansion cohort. ONC201 at a dose of 625 mg was given orally at day -7 of cycle 1, followed by weekly dosing. Nivolumab was given every 2 weeks at 240 mg IV starting on day 1 of every cycle (cycle =28 days). The primary end point was dose-limiting toxicity (DLT) during the observation window (run-in dose day -7, cycle 1 to assessment pre-dosing cycle 2). The plan was to enroll 28 additional patients at the MTD so that a total of 34 patients would be treated at the MTD. Pharmacokinetics (PKs) and tumor biopsies were collected at several time points per study protocol. A total of 13 patients (8 patients in the dose escalation *6 evaluable*) were enrolled between December 4, 2019 and March 2021. All patients had received ≥2 previous lines of chemotherapy and had confirmed microsatellite stability or mismatch repair-proficient tumors. No DLTs were observed with 625 mg ONC201 in the first three patients. Three additional patients were enrolled at the same dose to confirm safety. Two patients progressed during the DLT period and had to be replaced. During the dose-expansion part, five patients were enrolled and none required dose reduction or modification. No objective tumor response was observed in the 13 treated patients. Disease progression was confirmed at the time of the first imaging evaluation at 8 weeks following cycle 2. Post discussion at the Data and Safety Monitoring Board (DSMB) on May 25, 2021, the principal investigator (PI) and Committee voted to close the study to new patient enrollment prior to reaching accrual of 34 patients, secondary to lack of efficacy. In this study of patients with advanced MSS CRC, combination ONC201/nivolumab was well-tolerated; objective responses to ONC201/nivolumab were not observed.

Analysis of the Treatment for Advanced Microsatellite Stable Colorectal Cancer in Middle-Aged and Elderly People

Microsatellite stabilized (MSS) rectal cancer is a highly prevalent cancer in the middle-aged and elderly population. There exists some expertise in detecting and evaluating the chemotherapy effect in advanced colorectal cancer patients. Multiple studies have combined targeted therapy, chemotherapy, and immunotherapy as a breakthrough. Programmed cell death protein 1 (PD-1) inhibitors have certain therapeutic effects in the treatment of MSS rectal cancer patients. The combination of PD-1 inhibitors and furoquinib can improve the disease control rate (DCR) and progression- free survival (PFS) in colorectal cancer (CRC) patients with advanced MSS, and the adverse reactions are controllable. The combination of erlotinib hydrochloride and Xindilizumab was more effective in treating MSS-type colorectal cancer patients than clinical standard treatment. This study analyzes the various treatment methods for MSS-type CRC in middle- aged and elderly people to provide a reference basis in clinical practice.

Abstract 5056: Molecular characterization of microsatellite stable (MSS) colorectal cancer (CRC) patients with a BRAF V600E mutation

Abstract Introduction: A BRAFV600E mutation is an unfavorable prognostic biomarker for CRC and is associated with short-lived treatment response to BRAF and EGFR blockade. Anti-PD-1 therapies are ineffective in MSS CRC but demonstrate efficacy in combination with BRAF + EGFR inhibition for MSS BRAFV600E CRC. A comprehensive characterization of MSS BRAFV600E CRC as an immunologically distinct subpopulation of MSS CRC has not been performed. Here, we characterize the clinicopathological and transcriptomic features of MSS BRAFV600E CRC patients, relative to BRAFWT MSS CRC patients.Methods: De-identified records of 12,009 MSS CRC patients were retrospectively analyzed from the Tempus clinicogenomic database. CMS subtypes were derived using the CMScaller algorithm. Survival was assessed using Kaplan-Meier survival analysis with risk set adjustment (RSA) method; log-rank test was used to compare survival. Categorical and continuous variables were compared using chi-squared test and Wilcoxon rank sum test, respectively. Results: BRAFV600E was found in 5% (n=630) of the patients. Demographics and clinicopathological features varied significantly between the BRAFWT and BRAFV600E cohorts (Table 1). Relative to the BRAFWT cohort, BRAFV600E tumors were enriched for the immune-activated CMS1 signature (53% vs 11%, p< .001), PD-L1 detection (9.5% vs 3%, p<0.001), and CD8+ T-cell infiltration (4.6% vs. 2.5%, p<0.001). CMS4 was linked to an unfavorable prognosis in the BRAFWT cohort (HR=0.58 compared to CMS1, p=0.026), yet this was not observed in the BRAFV600E cohort (HR=2.60 compared to CMS1, p=0.083). Conclusions: BRAFV600E MSS CRC exhibited immune activation characteristics that were not observed in the BRAFWT group, including greater CMS1 status, PD-L1 expression, and CD8-T cell infiltration. Our findings support investigation of novel immune-based therapeutic strategies of MSS BRAFV600E CRC as an immunologically distinct subpopulation of MSS CRC. Cohort characteristics BRAFWT BRAFV600E P-value Demographics Age (median) 60 (51,69) 65 (55,74) <0.001 Ethnicity Not Hispanic or Latino 3,444 (83%) 200 (93%) 0.001 Hispanic or Latino 699 (17%) 16 (7.4%) Gender Female 4,771 (42%) 345 (55%) <0.001 Male 6,608 (58%) 285 (45%) Race White 5,050 (75%) 318 (87%) <0.001 Black or African American 888 (13%) 19 (5.2%) Other 517 (7.7%) 20 (5.4%) Asian 297 (4.4%) 10 (2.7%) Clinicopathology TNM stage I 61 (0.7%) 0 (0%) 0.1 II 388 (4.6%) 17 (3.2%) III 1,130 (13%) 88 (17%) IV 6,941 (81%) 424 (80%) Laterality Right 777 (15%) 89 (39%) <0.001 Transverse 193 (3.8%) 27 (12%) Left 2,326 (46%) 73 (32%) Rectum 1,761 (35%) 37 (16%) CMS CMS1 1,077 (11%) 307 (53%) <0.001 CMS2 3,067 (31%) 6 (1%) CMS3 1,777 (18%) 71 (12%) CMS4 3,920 (40%) 199 (34%) Immunology CD8 T-cell infiltration (median, IQR) 2.5% (0.0, 7.7) 4.6% (0.0, 9.4) <0.001 PD-L1 127 (3%) 22 (9.5%) <0.001 Citation Format: Ymke van der Pol, Binyam Yilma, Van K. Morris, Calvin Chao, Michelle Harris, Justin Guinney, Scott Kopetz. Molecular characterization of microsatellite stable (MSS) colorectal cancer (CRC) patients with a BRAF V600E mutation [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 5056.

Abstract 2505: Immune dynamics and response predictors to regorafenib, ipilimumab, and nivolumab (RIN) treatment in chemotherapy resistant microsatellite stable (MSS) metastatic colorectal cancer (mCRC)

Abstract Background: In our previous study, RIN therapy in MSS mCRC patients, excluding those with liver metastases, yielded a notable 36.4% objective response rate. RIN treatment was observed to remodel the local tumor microenvironment by enhancing immune infiltration, especially in responders. Systemic immunity, as assessed through peripheral blood analysis, showed that responders had lower baseline immune activity, which increased markedly post-treatment. Patients with liver metastases demonstrated a reduced CD4/CD8 T cell ratio, indicative of immunosenescence. Methods: Building on our prior research, we analyzed blood samples from 29 chemotherapy-resistant MSS mCRC patients undergoing RIN therapy, collected at baseline and after two consecutive treatment cycles. Two 28-marker spectral flow cytometry panels were used to assess immune senescence and cytokine production in lymphocytes. Additionally, T cell receptor repertoires and transcriptional profiles were analyzed using RNA sequencing from sorted non-naïve T cells. Results: The study unveiled distinct immunological profiles in MSS mCRC patient responders vs non-responders to RIN treatment. Responders demonstrated increased numbers of newly expanding CD8 T cell clones on-treatment, indicative of an active immune responses. In contrast, non-responders displayed evidence of increased DNA damage in circulating lymphocytes (T, B, and NK cells), as marked by elevated γ-H2AX levels. We also identified divergent cytokine production profiles patterns at baseline: T cells in non-responders demonstrated increased capacity for production of IL-2, IL-10, IL-4/-13, and IL-22, while production of IFN-γ in both T cells and NK cells correlated with improved outcomes. Notably, patients with liver metastases exhibited greater frequencies of senescent T cells, characterized by increased expression of senescence markers (SA-βGal, CD57, Granzyme B/K) and a reduction in CD27 and CD28 in CD4 T cells, along with an accumulation of terminally differentiated CD8 T cells both pre- and post-treatment. Additionally, RNA sequencing revealed an upregulation of FOXO1 related genes in non-naive T cells of responder patients, which is consistent with its important role for this pathway in attenuating senescence and maintenance of stem-like properties in T cells. Conclusions: The effectiveness of RIN therapy is associated with baseline functional potential of circulating T cells and an on-treatment expansion of T cell clonotypes. Pre-existing and persistent features of senescence and DNA damage in circulating lymphocytes may be a major barrier to RIN treatment efficacy in MSS colorectal cancer and should be validated further in future studies. Citation Format: Jian Ye, Chongkai Wang, Colt Egelston, Weihua Guo, Peter Lee, Marwan Fakih. Immune dynamics and response predictors to regorafenib, ipilimumab, and nivolumab (RIN) treatment in chemotherapy resistant microsatellite stable (MSS) metastatic colorectal cancer (mCRC) [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 2505.

Abstract 4049: The beta-carboline Harmine sensitives microsatellite stable colorectal cancer mouse model to anti-PD-1 through upregulating MHC-I dependent antigen presentation machinery and improving the infiltration of CD8 T cells into the tumor microenvironment

Abstract Colorectal cancer (CRC) is the most common gastrointestinal cancer and the third leading cause of cancer-related death in both sexes. Mismatch repair deficient/microsatellite instability-high (MSI) colorectal cancer patients benefit from immune checkpoint blockades (ICBs)-based immunotherapy; however, this benefit has not been translated into microsatellite stable (MSS) colorectal cancer. It has been proposed that the loss of major histocompatibility complex class I (MHC-I) in tumor cells limits the use of ICB in colorectal cancer. Harmine displays a number of biological and pharmacological properties affecting the immune response and tumor phenotype. Here, we assessed the impact of combining harmine, also referred to as ACB1801 molecule, on improving the responsiveness of ICB in the mouse MSS CRC model. Our results showed that combining ACB1801 significantly improves the therapeutic benefit of anti-PD-1 in the CT26 CRC mouse model by decreasing tumor growth and improving the survival of tumor-bearing mice. The improvement of anti-PD-1-based therapy by combining ACB-1801 was associated with a modification of the immune landscape of tumors as evidenced by an increase in the CD8+ T cells and a decrease in the Treg infiltrations into the tumor microenvironment. By profiling the cytokine/chemokine network, we showed that ACB1801 induced the expression and the release of the proinflammatory chemokine CXCL10 by CRC tumor cells in vitro, which could be responsible for driving CD8+ T cells to the tumor microenvironment. Mechanistically, we showed that ACB1801 increased the expression of MHC-I genes, including TAP1 and TAPASIN, and improved the antigen loading on MHC-I in CRC cells in vitro. Overall, our study highlights the value of combining ACB1801 and anti-PD-1 as a therapeutic opportunity to convert MSS colorectal cancer into an “immune hot” cancer, which may define the future treatment paradigm of colorectal cancer for which there is a great unmet need. Citation Format: Ruize Gao, Kris Van Moer, Coralie Pulido, Anaïs Oudin, Diane Murera, Teresa L. Ramos, Margaux Poussard, Andreas Schläpfer, Annette Ives, Christian Auclair, Bassam Janji. The beta-carboline Harmine sensitives microsatellite stable colorectal cancer mouse model to anti-PD-1 through upregulating MHC-I dependent antigen presentation machinery and improving the infiltration of CD8 T cells into the tumor microenvironment [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 4049.

Abstract 900: Development and validation of an artificial-intelligence-based pathomics biomarker to predict resistance to first-line treatment in metastatic colorectal cancer

Abstract Background: The backbone chemotherapy of first-line standard of care (SOC) for microsatellite stable (MSS) metastatic colorectal cancer (mCRC) combines 5-Fluorouracil to oxaliplatin and/or irinotecan. There are no biomarkers to predict response, which is complete or long-lasting (CR/LLR) in 20-25% of patients, while 10-15% are primary refractory. The aim of this work is to develop a predictive biomarker, based on digital pathology images, that can help stratify patients according to their risk of resistance. Methods: We trained a supervised bag-of-words artificial intelligence (AI)-based model on a cohort of response outliers mCRC patients classified as “really sensitive” (RS) if they achieved CR or LLR >10 months to any SOC, or “really resistant” (RR) if progression occurred at first disease reassessment. Whole-slide Imaging (WSI) of the resected primary tumors were tiled into patches of 224 × 224 pixel (0.5μm/pixel). First-order and texture features were subsequently extracted from all tumoral tiles and grouped into homogenous clusters through a k-means algorithm (k=6). For each patient, the percentage of tiles belonging to each tiles’ cluster was computed to represent new features (called bag of words) with whom different machine learning classifiers were trained. Main clinicopathological features were matched to treatment response by Fisher’s exact test. Results: To date, we analyzed 82 response outlier patients, of whom 35 were classified as RS and 46 RR. Of them, patients identified at Italian centers were used as construction cohort (N=47; 27 RR and 20 RS) and those identified at Spanish centers were used a validation cohort (N=35; 19 RR and 16 RS). The best result was obtained using a stepwise logistic regression, reaching a negative predictive value (NPV) of 90% (18/20; 95% CI=70-97%) and 71% (10/14; 95% CI=49-87%), in the construction and validation sets. No standard clinicopathological features (including stage, RAS/BRAF status, histology and sidedness) was associated with the chance of being RR. Conclusions: We demonstrated that a pathomics signature has the potential to predict resistance to SOC in MSS mCRC. Further validation of these preliminary findings on a larger cohort of response outlier patients is ongoing. Citation Format: Luca Lazzari, Gianluca Mauri, Valentina Giannini, Debora Cafaro, Giulia Nicoletti, Caterina Marchiò, Andrea Sartore-Bianchi, Federica Marmorino, Maria Nieva Munoz, Nadia Saoudi Gonzalez, Alberto Puccini, Chiara Cremolini, Clara Montagut, Elena Elez, Stefania Sciallero, Enrico Berrino, Martina Carullo, Pietro Paolo Vitiello, Maria Costanza Aquilano, Martina Di Como, Emanuela Bonoldi, Salvatore Siena, Alberto Bardelli, Daniele Regge, Silvia Marsoni. Development and validation of an artificial-intelligence-based pathomics biomarker to predict resistance to first-line treatment in metastatic colorectal cancer [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 900.

Abstract 6727: QL335, a novel T cell engager with enhanced safety profile targeting MSS colorectal cancer

Abstract Colorectal cancer (CRC) is the fourth leading cause of death in the world with a global mortality rate of over 700,000 annually. While recent advancement in diagnosis and prevention has reduced the overall number of deaths, the lack of effective and durable treatment has not significantly improved survival rate. Emerging modalities in immunotherapy such as T cell engagers have paved new ways for disease management. However, a common challenge to advancing T cell engagers in the clinic is poor target specificity and high toxicity associated with non-specific T cell activation and cytokine release. To overcome these shortcomings, we have developed QL335, a bispecific antibody bridging LY6G6D antigen on tumor cells and CD3 on T cells. LY6G6D or Lymphocyte Antigen 6 Family Member G6D, is a GPI-anchored membrane protein that, according to TCGA RNAseq data, is highly expressed in microsatellite stable (MSS) colorectal cancer but not in normal tissues. We analyzed LY6G6D expression by IHC in over 200 CRC patient tumors and 40+ normal tissue samples from different organs, confirming its highly restricted CRC expression pattern. We constructed our bispecific antibody in a 2+1 format, comprising two high affinity LY6G6D binding domains and one reduced affinity CD3 binding domain. Such format allows for higher tumor accumulation while mitigating non-specific T cell activation in the periphery. In vitro evaluation showed that QL335 induced potent yet highly specific T cell activation and killing of only target cells expressing LY6G6D. QL335 demonstrated robust anti-tumor efficacy in vivo against human colon cancer xenograft models with human PBMC reconstitution. Importantly, QL335 was well tolerated in cynomolgus monkeys dosed up to 50 mg/kg, supporting the enhanced safety profile by design of this therapeutic candidate. QL335 is currently being evaluated in a phase 1 clinical trial. Citation Format: Shenda Gu, Guangjie Guo, Helena Yee, Hieu V. Tran, Shihao Chen, Chuanzeng Cao, Ke Liu, Chunyan Jin, Weihua Lun, Ruixue Ma, Lele Ren. QL335, a novel T cell engager with enhanced safety profile targeting MSS colorectal cancer [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 6727.

Abstract 4095: Targeting alternative classes of tumor antigens to improve immunotherapy for microsatellite stable, mismatch repair-proficient colorectal cancer

Abstract Background: The clinical outcomes of immunotherapy for microsatellite stable (MSS) colorectal cancer (CRC) remains unsatisfactory. The development of more effective immunotherapies is hampered by limited knowledge of the appropriate target antigens, also considering the low burden of mutations generating neoantigens of these tumors. Alternative classes of MSS CRC antigens may provide actionable epitopes. Methods: We have characterized the immunopeptidome of 2 MSS CRC cell lines derived from the primary tumor (SW480) and a metastatic lymph node (SW620) of the same patient. Given the low mutational burden of MSS, we focused on peptides belonging to alternative sources of HLA-A*02 epitopes, such as proteasomal cis-spliced peptides derived from cancer testis antigens and linear epitopes derived from Human Endogenous Retroviruses (HERVs), which are frequently reactivated in CRC. Peptide immunogenicity was functionally validated based on their ability to expand HLA-A*02+ donor T cells (>3) specifically killing targets cells presenting the relevant peptides or HLA-matched CRC cell lines. In vivo epitope immunogenicity was verified by analyzing polyfunctional responses of human CD8+ T cells obtained from NSG-A2 mice humanized with A*02+ donor PBMCs and immunized with epitope-loaded nano-emulsions selectively targeting human Clec9A+ dendritic cells. Therapeutic exploitation of the novel epitopes was investigated by adoptive transfer of epitope-specific T cells in NSG-A2 mice injected with SW620 cells. Epitope-specific T cell responses were investigated in PBMCs from 10 MSS CRC patients and 7 donors. Results: Among >50,000 HLA-bound peptides identified by in depth immunopeptidomics analysis, 11 cis-spliced peptides and 5 HERV peptides were functionally validated as immunogenic. Epitope-specific T cells showed specific cytotoxic activity against SW480 and SW620 CRC cells. Globally, cis-spliced peptides were more immunogenic in vitro than HERV-derived peptides. The immunogenicity of both groups of peptides was confirmed in vivo in humanized mice. Adoptively transferred HERV or cis-spliced epitope specific T cells significantly inhibited the growth of SW620 cells in vivo, with cis-spliced epitope-specific T cells being more effective. Therapeutic efficacy of Clec9A-targeting vaccines exploiting these epitopes is currently under investigation. T cell responses specific for cis-spliced and HERV epitopes were detected in PBMCs from 3/4 MSS CRC HLA-A*02+ patients but in none of 4 HLA-A*02- MSS CRC patients or 7 healthy donors (4 HLA-A*02+ and 3 HLA-A*02-). Conclusions: We have identified and validated a new class of immunogenic HLA-A*02 epitopes derived from alternative classes of MSS CRC antigens. We also provide the proof of principle supporting their therapeutic exploitation to improve the management of these poorly immunogenic tumors. Citation Format: Riccardo Dolcetti, Bijun Zeng, Jingran Ye, Tima Shamekhi, Madeleine Harpur, Davide Moi, Sara Roth, Rob Ramsay, Divya Duscharla, Antony W. Purcell, Pouya Faridi, Roberta Mazzieri. Targeting alternative classes of tumor antigens to improve immunotherapy for microsatellite stable, mismatch repair-proficient colorectal cancer [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 4095.

Abstract 1517: Tc1 cytotoxic T-cells and Th1 helper T-cell rich tumor microenvironment is a hallmark of MSI colorectal cancers

Abstract Background: Microsatellite instability (MSI-h) is a strong biomarker to predict response to immune checkpoint therapy and patient’s outcome in colorectal cancer. Although enrichment of distinct T-cell subpopulations, such as type 1 cytotoxic T-cells (Tc1) or type 1 helper T-cells (Th1), have been identified to impact patient’s outcome and response to immune checkpoint therapy, only little is known about the underlying changes in the composition of the immune tumor microenvironment. Material and Methods: To assess the density, composition, degree of functional marker expression, and spatial interplay of T-cell subpopulations in 93 MSI and 1153 microsatellite stable (MSS) colorectal cancers, a tissue microarray as well as large sections were stained with antibodies directed against CD3, CD4, CD8, FOXP3, T-bet, GATA3, RORyT, BCL6, CD27, CD56, TIM3, PD-1, CTLA-4 Granzym B, and Ki67 using our BLEACH&STAIN multiplex fluorescence immunohistochemistry approach. A deep learning-based framework comprising two different convolutional neuronal networks (U-Net and DeepLabv3+) was used for image analysis. Results: The composition of Type 1 (T-bet+), Type 2 (GATA3+), Type 17 (RORyT+), NKT-like (CD56+), regulatory (FOXP3+), and follicular (BCL6+) cytotoxic (CD3+CD8+) or helper (CD3+CD4+) T-cells showed marked differences between MSI and MSS patients. For instance, the density and proportion of Tc1 as well as Th1 was significantly higher (p<0.001 each) in MSI compared to MSS patients. In contrast, the density and composition of Tregs, Th2, Th17 T-cells was significantly lower in MSI compared to MSS patients in the intraepithelial tumor compartment. In addition, among the MSI colorectal cancers, NKT-cells showed the highest level of Granzyme B that was significantly higher than in MSS patients (p<0.05). The degree of immune checkpoint expression (i.e., TIM3, PD-1, CTLA-4) was significantly higher in MSI compared to MSS patients for most T-cell subpopulations (p<0.05 each). The additional analysis of 10 large sections confirmed the observations from the TMA analysis and revealed that the alterations in the T-cell composition seen in the center of the tumor largely reflect the T-cell composition at the invasive margin in both MSI and MSS patients. Conclusion: This study identified a higher proportion of Tc1 cytotoxic T-cells and Th1 helper T-cells accompanied by a paucity of regulatory T-cell subpopulations, Th17 and Th2 T- helper cells in MSI colorectal cancers compared to MSS patients. Citation Format: Tim Mandelkow, Zhihao Huang, Elena Bady, Jan H. Müller, Guido Sauter, Julia Ebner, Maximilian Lennartz, Natalia Gorbokon, Ronald Simon, Martina Kluth, Claudia Hube-Magg, Sarah Minner, Niclas C. Blessin. Tc1 cytotoxic T-cells and Th1 helper T-cell rich tumor microenvironment is a hallmark of MSI colorectal cancers [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 1517.

A new strategy for immunotherapy of microsatellite-stable (MSS)-type advanced colorectal cancer: Multi-pathway combination therapy with PD-1/PD-L1 inhibitors.

Colorectal cancer (CRC) is a frequent gastrointestinal malignancy with high rates of morbidity and mortality; 85% of these tumours are proficient mismatch repair (pMMR)-microsatellite instability-low (MSI-L)/microsatellite stable (MSS) CRC known as 'cold' tumours that are resistant to immunosuppressive drugs. Monotherapy with programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) inhibitors is ineffective for treating MSS CRC, making immunotherapy for MSS CRC a bottleneck. Recent studies have found that the multi-pathway regimens combined with PD-1/PD-L1 inhibitors can enhance the efficacy of anti-PD-1/PD-L1 in MSS CRC by increasing the number of CD8+ T cells, upregulating PD-L1 expression and improving the tumour microenvironment. This paper reviews the research progress of PD-1/PD-L1 inhibitors in combination with cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitors, oncolytic virus, intestinal flora, antiangiogenic agents, chemotherapy, radiotherapy and epigenetic drugs for the treatment of pMMR-MSI-L/MSS CRC.

Abstract 904: AI platform provides an EDGE and enables state-of-the-art identification of peptide-HLAs for the development of T cell inducing vaccines

Abstract T cell inducing vaccines are key for the development of effective therapies against cancer and infectious diseases. Peptides, presented by Human Leukocyte Antigens (HLAs), are the targets of T cells, and their identification is therefore critical to the development of such vaccines. Here, we present the latest improvement in our EDGETM (Epitope Discovery for GEnomes) platform to address this critical need. EDGE is comprised of AI models that can predict peptide presentation by HLA class I and class II. Although the models are trained primarily using immunopeptidomics data, EDGE scores are predictive of peptide-HLA immunogenicity. There are three class I presentation models in EDGE: an allele-specific model, a pan-specific model, and a model specific for infectious diseases. The allele-specific model is applicable to a large but pre-defined set of HLA alleles. On a large test dataset, the allele-specific model achieved an average precision (AP) of 63% (PPV40=79%) compared to the AP of a standard best-available public model of 21% (PPV40=28%). A Ph1/2 clinical study of personalized cancer vaccines encoding neoantigens predicted from the allele-specific model demonstrated a ~50% molecular response (defined as >=30% reduction in circulating tumor DNA relative to baseline) rate with associated extended overall survival (vs non-responders) in metastatic, microsatellite stable colorectal cancer patients. We observed that >50% of the mutations were able to elicit T cell responses. The pan-specific class I model uses HLA sequences as input feature when training and, therefore, is applicable to any HLA. On the same test dataset as above, it achieved an AP of 65% (PPV40=81%) and performed better on average for ~40 less-common HLA alleles. Prediction of viral peptide presentation by HLA class I is challenging due to the lack of immunopeptidomics data. The class I model for infectious diseases was specifically optimized to predict for viral peptides and, therefore, performed better than available class I models on published HIV and Influenza A datasets. Prediction of peptide presentation by HLA class II is challenging due to the flexibility in how the longer peptides interact with open HLA grooves as well as the lack of immunopeptidomics data as compared to the class I peptides. The class II model in EDGE, EDGE-II, uses the latest developments in protein large language models, a novel learned HLA allele-deconvolution strategy, and in-house immunopeptidomics data, resulting in improved prediction of peptide presentation by HLA class II and immunogenicity driven by CD4+ T cells. On a benchmark validation dataset, EDGE-II achieved an AP of 71% as compared to AP of 62% of a leading published model. In summary, EDGETM provides a comprehensive state-of-the-art platform for the development of vaccines that can induce both CD8+ and CD4+ T cell responses to provide durable benefit to patients. Citation Format: Joshua Klein, Daniel Sprague, Monica Lane, Meghan Hart, Olivia Petrillo, Italo Faria do Valle, Matthew Davis, Andrew Ferguson, Andrew Allen, Karin Jooss, Ankur Dhanik. AI platform provides an EDGE and enables state-of-the-art identification of peptide-HLAs for the development of T cell inducing vaccines [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 904.

Abstract 6139: Genetic modifiers of KRAS-mutant colorectal cancer

Abstract Somatic driver mutations in KRAS are found in ~40% of colorectal cancers (CRCs) and are associated with worse outcomes. In the US, individuals of African ancestry with CRC are more likely to have tumors with KRAS mutations than individuals of European ancestry, even after considering differences in socioeconomic status and other risk factors. Germline variants can influence somatic mutation events and may provide a selective advantage such that a mutated cell is more likely to progress to a cancer. Germline variants have been found to associate with somatic mutations in a variety of tumors. Based on ancestry differences in KRAS tumor mutation frequency, we hypothesized that germline variants altering molecular pathways that support KRAS tumorigenesis will associate with KRAS-mutant status. To test this, we performed a two-step association study using KRAS mutation status as our phenotype in individuals with microsatellite stable CRC. First, we conducted a discovery analysis using genome-wide germline genotypes and somatic mutation data from 6,386 non-Hispanic White CRC patients from the Cancer Genome Atlas and the GECCO consortium, considering sex and tumor location as covariates. To select variants for additional study, we used in-silico tools, such as expression quantitative trait loci (eQTL), to predict how associated variants may alter gene expression and chromatin. After reviewing relevant genomic features, excluding SNVs with minor allele frequency <10% and pruning SNVs in linkage disequilibrium (r2>0.75), we identified candidate loci for a multi-ancestry independent validation analysis. In the discovery analysis, no variants met genome-wide significance thresholds (p-value <5x10-8), though 3 SNVs associated with KRAS mutation status with p-values <1x10-6 and 50 with p-values <1x10-5. Of these 50 SNVs, 21 met criteria for validation. We chose 80 additional SNVs (p-value <1x10-4) with predicted functional effect, for a total of 101 SNVs for analyses. In our preliminary validation analysis of 1,487 individuals grouped by self-reported race, no variants had a significant association in all populations. The rs726800 variant showed nominal association with KRAS mutation status (p-value=0.028) in self-reported non-Hispanic White individuals. This variant is within potential cis-regulatory distance of HS3ST3A1 and COX10-DT, both of which have been reported as cancer biomarkers. Nominal associations were identified in self-reported Black (n=7 SNVs) and Hispanic or Latino (n=3 SNVs) individuals, though both groups had limited sample sizes. Additional validation samples are being analyzed, as well as analysis with adjustment for genetic ancestry. Future functional studies of top candidate variants will add mechanistic insight. In summary, we identified germline SNVs that associate with KRAS somatic mutations in CRC and may inform the cellular context that supports development of colorectal tumors with KRAS mutations. Citation Format: Nijole P. Tjader, Johnny Ramroop, Tanish Gandhi, Peter T. Campbell, Andrew T. Chan, Steven Gallinger, Graham G. Giles, Marc J. Gunter, Tabitha A. Harrison, Michael Hoffmeister, Polly A. Newcomb, Shuji Ogino, Amanda I. Phipps, Conghui Qu, Robert E. Schoen, Andrew Pellatt, Michael O. Woods, Bethany Van Guelpen, Patrick Stevens, Heather Hampel, Ulrike Peters, Joseph P. McElroy, Amanda E. Toland. Genetic modifiers of KRAS-mutant colorectal cancer [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 6139.

Abstract 2796: Colibactin mutation signatures are associated with younger age of onset in colorectal cancer

Abstract Escherichia coli (E. coli) strains carrying the polyketide synthase (pks) island produce the genotoxin colibactin associated with pathogenesis of colorectal cancer (CRC). Colibactin binds directly to DNA, causing alkylating damage leading to double stranded DNA breaks. Colibactin mutagenesis results in mutations in hexameric A-T rich DNA motifs that can be identified by whole genome sequencing (WGS) as mutation signatures SBS28 and SBS88 (SBS-pks). Since WGS is not routinely used clinically, we developed an approach to specifically identify SBS-pks using a clinical targeted exon capture assay, MSK-IMPACT. In an institutional pan-cancer clinical cohort of 78,905 tumors we identified 15,967 samples with > 20 mutations detected by MSK-IMPACT. We identified 150/1,884 samples with microsatellite stable CRC with >10% mutations attributable to SBS-pks. Examining clinicopathologic and genomic features of SBS-pks+ compared to SBS-pks- CRC tumors, we found that SBS-pks+ signatures are significantly enriched in younger age of onset compared to SBS-pks- CRC patients (median age 55 vs median age 59, p= 0.0028). SBS-pks+ CRC exhibit similar driver mutations and tumor mutational burden (TMB) compared to SBS-pks- CRC but are associated with increased fraction of genome altered (FGA). To mechanistically investigate colibactin-associated damage we developed a novel long-term human colon organoid-microbe co-culture model system that takes advantage of reversal of polarity in suspension culture. WGS of human CRC organoids co-cultured for three months with pks+ and delta-pks E. coli NC101 confirmed induction of pks+ specific SBS-pks signatures, validating the model. Leveraging this model and a biobank of normal colon organoids from donors aged </> 50 years, we are investigating the molecular mechanisms underlying the association of SBS-pks with younger age of onset CRC. Citation Format: Stefanie Gerstberger, Melissa Lumish, Saskia Hartner, Farheen Shah, Seongmin Choi, Anisha Luthra, Qingwen Jiang, Hyung Jun Woo, Ahmed Mahmoud, Henry Walch, Simran Asawa, Mark Donoghue, Andrea Cercek, Rona Yaeger, Andrew McPherson, Francisco Sanchez-Vega, Karuna Ganesh. Colibactin mutation signatures are associated with younger age of onset in colorectal cancer [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 2796.

Abstract 1168: Anti-CSF1R antibody monotherapy inhibits minimal residual disease in a novel immunocompetent murine colorectal cancer metastasis model

Abstract Background: The high mortality rate of colorectal cancer (CRC) is primarily due to metastatic disease. Microscopic minimal residual disease (MRD) detected in patients by circulating tumor DNA (ctDNA) may remain radiographically undetectable and persist following therapeutic intervention, resulting in recurrence. Preclinical models are urgently needed to elucidate the mechanisms whereby MRD escapes anti-tumor immune surveillance. We have developed and characterized a preclinical liver MRD model that recapitulates immune response, with the goal to discover novel strategies to improve outcomes for patients with ctDNA+ MRD. Methods: We generated genetically engineered mouse colonic organoids CDX2 CRE; Rosa26 LSL-CAS9-GFP with APC and TP53 mutation and performed intrasplenic injection in syngeneic C57BL/6 mice to recapitulate microsatellite stable CRC metastasis to the liver. Mice were monitored for radiographically detectable disease using MRI. Tumors were resected at various timepoints, with comparison between metastases at various sizes of development (dichotomized as micro- and macro-metastases). We performed multiplex immunofluorescence staining, single cell RNA seq, and spatial transcriptomic analysis to delineate the immune compartments of micro- and macro-metastasis in untreated mice. After identifying colony stimulating factor -1 receptor (CSF1R) as a target, we performed a vehicle controlled anti-CSF1R monotherapy study for 4 weeks. Treatment began at either 7 days before metastasis is radiographically detectable to mimic treatment of MRD, or 21 days post tumor engraftment. Results: Immune exclusion was confirmed through intratumoral CD45+ leukocyte densities, with transition to an immune excluded phenotype occurring above 0.5mm in size (defined as macro-metastases). In our preclinical model, immunosuppressive populations of macrophages (IBA1+CD163+) dominated in the micro-metastases. This observation was confirmed in 13 patients with paired micro- and macro-metastases to the liver (p=0.0065). Strikingly, anti-CSF1R treatment in the murine model eradicated IBA1+CD163+ macrophages (p<0.0001) and induced complete remission of liver micro-metastasis (0/7 mice with residual disease), in contrast to limited efficacy for macro-metastases (5/5 mice with progressed radiographically disease). Additionally, a long-term study confirmed 100% survival (5/5 mice) and no evidence of disease via MRI at 6 months upon anti-CSF1R treatment compared to vehicle (0/5 mice). Conclusions: We established a reproducible preclinical model of MRD with similar immunopathological features of MRD in patients. Our data further identified infiltration of immunosuppressive macrophage populations as an early determinant of MRD progression and indicated CSF1R as a potential new clinical therapeutic target for ctDNA+-defined MRD patients. Citation Format: Alaa M. Mohamed, Kyung Serk Cho, Melinda Soeung, Amanda Anderson, Jumanah Alshenaifi, Ganiraju Manyam, Oscar Villareal, Jennifer Davis, Will Norton, Sisi Gao, Christopher Bristow, Federica Carbone, Stefania Napolitano, Oluwadara Coker, Isha Khanduri, Justin Huang, Dipen M. Maru, Mauro Di Pilato, Ryan Sun, Linghua Wang, David G. Menter, Giannicola Genovese, Giulio Draetta, Natalie Fowlkes, Scott Kopetz. Anti-CSF1R antibody monotherapy inhibits minimal residual disease in a novel immunocompetent murine colorectal cancer metastasis model [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 1168.

RNA methylation-related genes INHBB and SOWAHA are associated with MSI status in colorectal cancer patients and may serve as prognostic markers for predicting immunotherapy efficacy.

The role of RNA methylation is vital in the advancement and spread of tumors. However, its exact role in microsatellite instability in colorectal cancer (CRC) is still not fully understood. To address this gap in knowledge, this study investigated the impact of genes associated with RNA methylation on the prognosis and response to immunotherapy in individuals diagnosed with low microsatellite instability (MSI-L) or microsatellite stable (MSS) CRC. The differentially expressed genes (DEGs) in two groups of patients: those with high microsatellite instability (MSI-H) and those with MSI-L/MSS was thoroughly investigated and compared with aims of exploring the association between them and the 60 RNA methylation regulators. We employed these genes and developed an MSI-RMscore to establish a risk signature capable of forecasting patient outcomes. Furthermore, an investigation of the immunophenotypic traits was conducted encompassing patients categorized as high-risk and low-risk. By combining the MSI-RMscore and clinicopathological features, a predictive nomogram was developed, which was subsequently validated using the GEO database. Furthermore, immunohistochemistry was employed to establish the correlation between INHBB and SOWAHA and the MSI status, as well as patient prognosis. Our findings indicated that the high-risk subgroup exhibited unfavorable overall survival rates, reduced responsiveness to immune checkpoint blockers, elevated estimate scores, and increased infiltration of macrophages and fibroblasts. We also confirmed that INHBB and SOWAHA were associated with CRC patient prognosis and MSI status, as well as immunotherapy response. These findings suggest that targeting INHBB and SOWAHA could be a promising strategy to enhance patient responsiveness to immunotherapy.

Multiplexed analysis of macrophage polarisation in pulmonary metastases of microsatellite stable colorectal cancer

Tumour-associated macrophages (TAMs) express a continuum of phenotypes ranging from an anti-tumoural M1-like phenotype to a pro-tumoural M2-like phenotype. During cancer progression, TAMs may shift to a more M2-like polarisation state, but the role of TAMs in CRC metastases is unclear. We conducted a comprehensive spatial and prognostic analysis of TAMs in CRC pulmonary metastases and corresponding primary tumours using multiplexed immunohistochemistry and machine learning-based image analysis. We obtained data from 106 resected pulmonary metastases and 74 corresponding primary tumours. TAMs in the resected pulmonary metastases were located closer to the cancer cells and presented a more M2-like polarised state in comparison to the primary tumours. Higher stromal M2-like macrophage densities in the invasive margin of pulmonary metastases were associated with worse 5-year overall survival (HR 3.19, 95% CI 1.35–7.55, p = 0.008). The results of this study highlight the value of multiplexed analysis of macrophage polarisation in cancer metastases and might have clinical implications in future cancer therapy.

Interferon Gamma Induces Higher Neutrophil Extracellular Traps Leading to Tumor-Killing Activity in Microsatellite Stable Colorectal Cancer.

Many patients with colorectal cancer do not respond to immune checkpoint blockade (ICB) therapy, highlighting the urgent need to understand tumor resistance mechanisms. Recently, the link between the IFNγ signaling pathway integrity and ICB resistance in the colorectal cancer tumor microenvironment has been revealed. The immunosuppressive microenvironment poses a significant challenge to antitumor immunity in colorectal cancer development. Tumor-associated neutrophils found in tumor tissues exhibit an immunosuppressive phenotype and are associated with colorectal cancer patient prognosis. Neutrophil extracellular traps (NET), DNA meshes containing cytotoxic enzymes released into the extracellular space, may be promising therapeutic targets in cancer. This study showed increased NETs in tumor tissues and peripheral neutrophils of high levels of microsatellite instability (MSI-H) patients with colorectal cancer compared with microsatellite stable (MSS) patients with colorectal cancer. IFNγ response genes were enriched in MSI-H patients with colorectal cancer compared with patients with MSS colorectal cancer. Co-culturing neutrophils with MSI-H colorectal cancer cell lines induced more NET formation and higher cellular apoptosis than MSS colorectal cancer cell lines. IFNγ treatment induced more NET formation and apoptosis in MSS colorectal cancer cell lines. Using subcutaneous or orthotopic CT-26 (MSS) tumor-bearing mice models, IFNγ reduced tumor size and enhanced PD-1 antibody-induced tumor-killing activity, accompanied by upregulated NETs and cellular apoptosis. These findings suggest that IFNγ could be a therapeutic strategy for MSS colorectal cancer.

The Efficacy of Immune Checkpoint Inhibitors in Microsatellite Stable Colorectal Cancer: A Systematic Review.

The use of immune checkpoint inhibitors (ICIs) has revolutionized cancer care, particularly in immune-inflamed tumors and tumors with a high mutational burden, like microsatellite instable colorectal cancer (CRC). However, their effectiveness in microsatellite stable (MSS) CRC is limited. This systematic review aims to evaluate the efficacy of ICIs in MSS CRC and explore promising combination strategies. A comprehensive search from the Web of Science, Medline, and Embase databases, for studies published until 14 November 2022, identified 53 clinical trials included in the review. ICI monotherapy or ICI-ICI combinations demonstrated limited clinical activity for patients with MSS CRC, with overall response rates below (ORR) 10% in most studies. The ICI and tyrosine kinase inhibitor (TKI) garnered ORRs ranging from 10% to 40% and indicated a higher benefit for patients, particularly those without active liver metastases. The combination of ICIs with anti-VEGF agents showed modest ORRs, especially in the earlier treatment lines and in combination with chemotherapy. While these combinations could lead to modest improvements, well-defined biomarkers for long-term benefit are yet to be delineated. Combinations involving BRAF inhibitors with ICIs were studied, showing promising responses with combination approaches in molecularly defined subgroups. In conclusion, while ICI monotherapy has limited efficacy in MSS CRC, combination strategies hold promise to enhance survival outcomes. Further research is necessary to identify optimal combination approaches, predictive biomarkers for treatment response, as well as enrollment according to tumor molecular characteristics.

Multiomic molecular characterization of the response to combination immunotherapy in MSS/pMMR metastatic colorectal cancer

BackgroundImmune checkpoint inhibitor (ICI) combinations represent an emerging treatment strategies in cancer. However, their efficacy in microsatellite stable (MSS) or mismatch repair-proficient (pMMR) colorectal cancer (CRC) is variable. Here, a...

Integrating clinical and cross-cohort metagenomic features: a stable and non-invasive colorectal cancer and adenoma diagnostic model.

Background: Dysbiosis is associated with colorectal cancer (CRC) and adenomas (CRA). However, the robustness of diagnostic models based on microbial signatures in multiple cohorts remains unsatisfactory. Materials and Methods: In this study, we used machine learning models to screen metagenomic signatures from the respective cross-cohort datasets of CRC and CRA (selected from CuratedMetagenomicData, each disease included 4 datasets). Then select a CRC and CRA data set from the CuratedMetagenomicData database and meet the requirements of having both metagenomic data and clinical data. This data set will be used to verify the inference that integrating clinical features can improve the performance of microbial disease prediction models. Results: After repeated verification, we selected 20 metagenomic features that performed well and were stably expressed within cross-cohorts to represent the diagnostic role of bacterial communities in CRC/CRA. The performance of the selected cross-cohort metagenomic features was stable for multi-regional and multi-ethnic populations (CRC, AUC: 0.817-0.867; CRA, AUC: 0.766-0.833). After clinical feature combination, AUC of our integrated CRC diagnostic model reached 0.939 (95% CI: 0.932-0.947, NRI=30%), and that of the CRA integrated model reached 0.925 (95%CI: 0.917-0.935, NRI=18%). Conclusion: In conclusion, the integrated model performed significantly better than single microbiome or clinical feature models in all cohorts. Integrating cross-cohort common discriminative microbial features with clinical features could help construct stable diagnostic models for early non-invasive screening for CRC and CRA.