pMMR Colorectal Cancer Research Articles

Mismatch Repair Status and Surgical Outcomes in Localized Colorectal Cancer: A Nationwide Cohort Study

Objective: This study examined the association between deficient mismatch repair (dMMR) versus proficient MMR (pMMR) status and overall survival and disease-free survival in patients with localized colorectal cancer. Background: Several distinctions exist between patients with dMMR and pMMR colorectal cancer. However, the impact on prognosis is yet to be investigated in large-scale cohort studies. Methods: In this cohort study, we included patients who underwent curative-intent surgery for localized colorectal cancer between 2009 and 2020. Patients were identified in the Danish Colorectal Cancer Group database and patient-level data were extracted from 6 registry databases. After inclusion, patients with dMMR status were matched 1:1 to patients with pMMR status using an estimated propensity score. Results: After matching, 5994 patients were included. The patients had a median age of 74 years and a median follow-up of 4.1 years. There was no significant association between mismatch repair (MMR) status and overall survival (hazard ratio, 0.91; 95% confidence interval [CI], 0.81–1.03) or disease-free survival (hazard ratio, 0.89; 95% CI, 0.78–1.01). However, the restricted 5-year mean disease-free survival time, calculated due to violation of the proportional hazards assumption, showed a significant absolute difference of 0.13 years (95% CI, 0.03–0.23; P = 0.01) in favor of the dMMR group. Conclusions: No significant association with overall survival was found according to MMR status. dMMR status was, however, found to be associated with marginally improved disease-free survival compared to pMMR status in patients with localized colorectal cancer undergoing curative-intent surgery.

Neoadjuvant immunotherapy for dMMR and pMMR colorectal cancers: therapeutic strategies and putative biomarkers of response.

Approximately 15% of locally advanced colorectal cancers (CRC) have DNA mismatch repair deficiency (dMMR), resulting in high microsatellite instability and a high tumour mutational burden. These cancers are frequently sensitive to therapy with immune-checkpoint inhibitors (ICIs) in the metastatic setting. This sensitivity seems to be even more pronounced in locally advanced disease, and organ preservation has become a realistic aim in ongoing clinical trials involving patients with dMMR rectal cancer. By contrast, metastatic CRCs with proficient DNA mismatch repair (pMMR) are generally resistant to ICIs, although a proportion of locally advanced pMMR tumours seem to have a high degree of sensitivity to ICIs. In this Review, we describe the current and emerging clinical evidence supporting the use of neoadjuvant ICIs in patients with dMMR and pMMR CRC, and the potential advantages (based on a biological rationale) of such an approach. We discuss how neoadjuvant 'window-of-opportunity' trials are being leveraged to progress biomarker discovery and we provide an overview of potential predictive biomarkers of response to ICIs, exploring the challenges faced when evaluating such biomarkers in biopsy-derived samples. Lastly, we describe how these discoveries might be used to drive a rational approach to trialling novel immunotherapeutic strategies in patients with pMMR CRC, with the ultimate aim of disease eradication and the generation of long-term immunosurveillance.

T cell factor 1 (TCF-1) defines T cell differentiation in colorectal cancer

The presence of precursor to exhausted (Tpex) CD8+ Tcells is important to maintain robust immunity following treatment with immune checkpoint inhibition (ICI). Impressive responses to ICI are emerging in patients with stage II-III mismatch repair (MMR)-deficient (dMMR) colorectal cancer (CRC). We found 64% of dMMR and 15% of mismatch repair-proficient (pMMR) stage III CRCs had a high frequency of tumor infiltrating lymphocytes (TIL-hi). Furthermore, expression of TCF-1 (Tcf7) by CD8+ Tcells predicted improved patient prognosis and Tpex cells (CD3+CD8+TCF-1+PD-1+) were abundant within lymphoid aggregatesof stage III CRCs. In contrast, CD3+CD8+TCF-1-PD-1+ cells were more abundant at the invasive front and tumor core, while γδ Tcells were equally abundant in all tumor areas. Interestingly, no differencesin the frequency of Tpex cells were observed between TIL-hi dMMR and TIL-hi pMMR CRCs. Therefore, Tpex cell function and ICI response rates in TIL-hi CRC warrants further investigation.

Sporadic deficient mismatch repair in colorectal cancer increases the risk for non-colorectal malignancy: A European multicenter cohort study.

Disparities between tumors arising via different sporadic carcinogenetic pathways have not been studied systematically. This retrospective multicenter cohort study evaluated the differences in the risk for non-colorectal malignancy between sporadic colorectal cancer (CRC) patients from different DNA mismatch repair status. A retrospective European multicenter cohort study including in total of 1706 CRC patients treated between 1996 and 2019 in three different countries. The proficiency (pMMR) or deficiency (dMMR) of mismatch repair was determined by immunohistochemistry. Cases were analyzed for tumor BRAFV600E mutation, and BRAF mutated tumors were further analyzed for hypermethylation status in the promoter region of MLH1 to distinguish between sporadic and hereditary cases. Swedish and Finish patients were matched with their respective National Cancer Registries. For the Czech cohort, thorough scrutiny of medical files was performed to identify any non-colorectal malignancy within 20 years before or after the diagnosis of CRC. Poisson regression analysis was performed to identify the incidence rates of non-colorectal malignancies. For validation purposes, standardized incidence ratios were calculated for the Swedish cases adjusted for age, year, and sex. Of the 1706 CRC patients included in the analysis, 819 were female [48%], median age at surgery was 67 years [interquartile range: 60-75], and sporadic dMMR was found in 188 patients (11%). Patients with sporadic dMMR CRC had a higher incidence rate ratio (IRR) for non-colorectal malignancy before and after diagnosis compared to patients with a pMMR tumor, in both uni- (IRR = 2.49, 95% confidence interval [CI] = 1.89-3.31, p = 0.003) and multivariable analysis (IRR = 2.24, 95% CI = 1.67-3.01, p = 0.004). This association applied whether or not the non-colorectal tumor developed before or after the diagnosis of CRC in both uni- (IRR = 1.91, 95% CI = 1.28-2.98, p = 0.004), (IRR = 2.45, 95% CI = 1.72-3.49, p = 0.004) and multivariable analysis (IRR = 1.67,95% CI = 1.05-2.65, p = 0.029), (IRR = 2.35, 95% CI = 1.63-3.42, p = 0.005), respectively. In this retrospective European multicenter cohort study, patients with sporadic dMMR CRC had a higher risk for non-colorectal malignancy than those with pMMR CRC. These findings indicate the need for further studies to establish the need for and design of surveillance strategies for patients with dMMR CRC.

Association of class II and III BRAF mutations with EGFR blockade therapy response and representation of molecularly distinct subgroups of BRAF mutations in MMR proficient CRC.

168 Background: BRAF mutations (mts) represent a highly heterogeneous group of molecular alterations seen in colorectal cancer (CRC). Class I BRAF mts (V600) render aggressive biology to CRC and poor response to anti EGFR inhibitors. Currently there are limited data on clinical and molecular features of class II and III BRAF mts. In this study, we investigated the clinical and molecular characteristics of BRAF mutation classes and their impact on clinical outcomes in a large cohort of patients (pts) with mismatch proficient (pMMR) CRC. Methods: A total of 18575 pMMR CRC specimens were profiled by next-generation sequencing (592-gene, NextSeq; WES, WTS NovaSeq) (Caris Life Sciences, Phoenix, AZ). BRAF mutations were detected by NGS and classified using published literature (Sahin et al. JCO OP 2021). Interferon gamma signature and MAPK pathway activity score (MPAS) were calculated using RNA expression data (TPM). Real-world overall survival information was obtained from insurance claims and calculated from tissue collection to last contact, while post-treatment survival from first of treatment to last contact. KM estimates were calculated for molecularly defined cohorts. Significance was determined as p values of <0.05. Results: A total of 930,105, and 262 pts with class I, II, and III BRAF mts were identified. Pts with class III BRAF mts were significantly more common among younger pts (age<45) compared to class I and class II (8.8% vs. 4.8% vs. 1.0 % respectively; P<0.05). Class I BRAF mts were significantly enriched with consensus molecular subtype 1 (CMS1) (Class I, II and III: 44% vs. 17% vs. 18%) while class II and III BRAF mts were more often CMS2 subtype (canonical) compared to class I (2%, 30% and 35%, p<0.05). Class I BRAF and KRAS/ NRAS mts were mutually exclusive, while KRAS mts incidences were 15% and 22% for class II and class III, and NRAS mts incidence were 8% and 12%, respectively. MPAS score was significantly lower for class III (-0.32, arbitrary unit) compared to Class I (p<0.05), but similar MPAS scores were seen in class I and II (1.3 versus 1.38). No difference in interferon gamma signature was noted between classes. Pts with class II and III mts had significantly better overall survival compared to patients with class I mts (HR=0.69 CI: 0.597-0.804 p<0.0001) and slightly worse overall survival compared to wild-type BRAF pts. (HR; 0.85 CI: 0.74-0.96 P=0.011). Among pts treated with anti-EGFR, pts with class II and III BRAF mts had significantly better post-anti-EGFR survival compared to class I BRAF mts (HR 0.498 CI 0.32-0.766 P=0.001 and similar survival compared to those with BRAF wild-type (P=0.21). Conclusions: Class II and III BRAF mts are associated with improved outcomes with EGFR blockade and represent a distinct biological subgroup of pMMR CRC. Class III BRAF mts have lower MAPK activation, consistent with the pattern of kinase-dead mutations.

Mitochondrial DNA copy number plays opposing roles in T-lymphocyte infiltration of colorectal cancer based on mismatch repair status: new directions for immunotherapy?

Researchers have previously reported that mitochondrial DNA copy number (mtDNA-CN) can play different roles in microsatellite instable/mismatch repair-deficient (MSI/dMMR) and microsatellite stable/mismatch repair-proficient (MSS/pMMR) colorectal cancer (CRC). To support malignancy, dMMR CRC relies on glycolysis, while pMMR CRC favors oxidative phosphorylation. However, it is unclear whether mtDNA-CN changes are related to T cell infiltration in CRC. The mtDNA-CN was detected by qRT-PCR in 532 patients, and the expression of CD3 and CD8 in 485 patients was detected by immunohistochemistry. The correlation between mtDNA-CN and the prognosis of CRC patients was further analyzed, and the correlation between mtDNA-CN and T lymphocyte infiltration was also analyzed. Biopsy specimens from the immune checkpoint inhibitors (ICIs) treatment cohort were obtained to verify the correlation between mtDNA-CN and the efficacy of ICIs. The effects of mtDNA-CN and MMR status on gene expression were analyzed by RNA-seq. Our results show that mtDNA-CN has inverse relationships to CRC prognosis in cases with different MMR statuses, potentially inducing the U-shaped association in CRC. The opposing correlations between mtDNA-CN and T lymphocyte infiltration in cases of dMMR CRC and pMMR CRC further suggest that mtDNA-CN might play an important role in CRC development. More importantly, cases of pMMR CRC with lower mtDNA-CN and of dMMR CRC with higher mtDNA-CN can benefit more dramatically from ICIs. Furthermore, RNA-seq revealed a link between the level of mtDNA-CN and T lymphocyte infiltration in CRC cases with different MMR statuses. Our study found a potential relationship between mtDNA-CN and CRC development that differs by MMR status, potentially providing a rationale for the use of mtDNA-CN as both a predictive biomarker and a therapeutic target for ICIs.

RNA Expression-Based Analysis to Predict Response in Patients with Metastatic Mismatch Repair Proficient Colorectal Cancer Treated with Regorafenib and Nivolumab

Introduction: We previously conducted a phase I/Ib study (NCT03712943) with regorafenib and nivolumab in patients with refractory metastatic mismatch repair proficient (pMMR) colorectal cancer (CRC). This study aimed to investigate the role of Xerna™ TME Panel in predicting the treatment response. Methods: Twenty-two archival pretreatment tumor samples were subjected to the Xerna™ TME Panel, a machine learning-based RNA-sequencing biomarker assay. The Xerna tumor microenvironment (TME) subtypes were evaluated for correlation with overall survival (OS), progression-free survival (PFS), disease control rate (DCR), and other biomarkers including KRAS, PD-L1, CD8 expression, and Treg cells in TME. Results: Based on Xerna™ TME Panel, 4 patients with immune-active (IA) subtype and 6 patients with immune-suppressed subtype were classified as biomarker-positive, and five with angiogenic (A) subtype and seven with immune desert subtype were biomarker-negative. While not reaching statistical significance, Xerna TME biomarker-positive patients seemed to have longer median PFS (7.9 vs. 4.1 months, p = 0.254), median OS (15.75 vs. 11.9 months, p = 0.378), and higher DCR (70% vs. 58%, p = 0.675). The IA subtype in our cohort had higher levels of CD4+ FOXP3+ Treg cells, whereas the A subtype showed lower levels of Treg cells. Conclusion: Xerna™ TME Panel analysis in patients with refractory metastatic pMMR CRC who were treated with regorafenib plus nivolumab might be of value for predictive clinical benefit. Further studies are needed to evaluate the predictive role of Xerna™ TME Panel analysis in patients with refractory metastatic pMMR CRC.

Characterization of colorectal cancer by hierarchical clustering analyses of five immune cell markers.

The present study analyzed the expression of five independent immunohistochemical markers, CD4, CD8, CD66b, CD68, and CD163, on immune cells within the colorectal cancer (CRC) tumor microenvironment (TME). Using hierarchical clustering, patients were successfully classified according to significant associations with clinicopathological features and/or survival. Patients with mismatch repair-proficient (pMMR) CRC were categorized into four groups with survival differences (p = 0.0084): CD4Low , CD4High , MΦHigh , and CD8Low . MΦHigh tumors showed significantly higher expression of CD47 (p < 0.0001), a phagocytosis checkpoint molecule. These tumors contained significantly greater numbers of PD-1+ (p < 0.0001), TIM-3+ (p < 0.0001), and SIRPA+ (p < 0.0001) immune cells. Notably, 10% of the patients with pMMR CRC expressed PD-L1 (CD274) on tumor cells with significantly worse survival (p = 0.00064). The Cox proportional hazards model identified MΦ High (hazard ratio [HR] = 2.02, 95%, p = 0.032), CD8Low (HR = 2.45, p = 0.011), and tumor PD-L1 expression (HR = 2.74, p = 0.0061) as potential risk factors. PD-L1-PD-1 and/or CD47-SIRPA axes targeting immune checkpoint therapies might be considered for patients with pMMR CRC according to their tumor cells and tumor immune microenvironment characteristics.

Immune checkpoint status and oncogenic mutation profiling of rectal cancer after neoadjuvant chemotherapy (KSCC1301-A2).

Immune checkpoint inhibitors (ICIs) are less effective in mismatch repair (MMR)-proficient (pMMR) colorectal cancers (CRCs) than in MMR-deficient CRCs. Here, we investigated changes in the tumor microenvironment after neoadjuvant chemotherapy (NAC) without radiotherapy in locally advanced rectal cancer (LARC) and the potential of ICIs as therapeutic agents for pMMR CRCs. This was an ad hoc analysis of a KSCC1301 randomized phase II trial in which patients with untreated resectable LARC were randomly assigned to receive S-1 and oxaliplatin or folinic acid, 5-fluorouracil, and oxaliplatin as NAC. Forty-nine patients were studied in this ad hoc analysis. As a reference cohort, we assessed 25 rectal cancer patients who underwent surgery without NAC outside the randomized trial. Immune checkpoint molecules (ICMs; PD-1, PD-L1, CTLA-4, LAG3), tumor-infiltrating lymphocytes (TILs; CD8, FOXP3), and other related proteins were evaluated by immunohistochemistry. Next-generation sequencing (NGS) using Oncomine™ Comprehensive Assay version 3 was conducted in 23 patients. The expression levels of PD-1, CTLA-4, and LAG3 in the NAC group were significantly higher than in reference patients (p < 0.001). Additionally, the infiltration of CD8+ and FOXP3+ T cells, and the CD8/FOXP3 ratio were significantly higher in the NAC group than in reference patients (p < 0.0001). NGS analysis revealed no specific gene alteration related to TILs or ICMs. We demonstrated changes in the tumor immune microenvironment after NAC in pMMR rectal cancer. NAC was associated with increased expression of ICMs and TILs. Rectal cancer could be susceptible to combined immunotherapy with chemotherapy.

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

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

Heterogeneous expression of mismatch repair proteins and interpretation of immunohistochemical results in colorectal cancer and endometrial cancer

To investigate the heterogeneous expression patterns of four mismatch repair (MMR) proteins in colorectal cancer (CRC) and endometrial cancer (EC), and their effects on the interpretation of immunohistochemical (IHC) results. A total of 1636 CRC and EC specimens were collected from two hospitals. Seventy-eight cases had heterogeneous expression of MMR proteins, including 49 CRC and 29 EC cases. Polymerase chain reaction-capillary electrophoresis (PCR-CE) was then performed to detect the microsatellite instability (MSI) status, and 44 cases were further verified by targeted next-generation sequencing (NGS). Heterogeneous expression of MMR proteins was observed in 66 cases (66/78, 84.6%) of proficient MMR (pMMR) and 12 cases (12/78, 15.4%) of deficient MMR (dMMR). The proportion of heterogeneous MMR protein expression in EC (12.0%) was higher than that in CRC (3.5%). The heterogeneous expression patterns were divided into focal clonal heterogeneity (6/78, 7.7%) and glandular mosaic heterogeneity (72/78, 92.3%). Surprisingly, three pMMR CRC cases showed isolated small focal clonal heterogeneity of mutS homologue 6 (MSH6), with < 15% positive tumour cells, which was validated as high MSI (MSI-H) with PCR-CE and NGS. However, the other three EC pMMR cases with > 50% focal clonal heterogeneity of MMR proteins were verified as microsatellite stable (MSS) or low MSI (MSI-L). Fifteen EC cases with glandular mosaic heterogeneous expression of MMR proteins included two MSI-H cases, which were validated using PCR-CE and NGS. Among the dMMR cases, only two EC cases with mutL homologue 1 (MLH1)/PMS1 homologue 2, mismatch repair system component (PMS2) loss and MSH2/MSH6 mosaic heterogeneous expression were confirmed as MSS using PCR-CE and NGS, which may be related to the mechanism of MLH1 promoter methylation. Thus, in CRC, only cases with small focal clonal heterogeneous expression of MSH6 have a high likelihood of MSI-H, and further PCR-CE or NGS testing is recommended. The possibility of MSI-H cannot be ruled out in EC cases with glandular mosaic heterogeneous expression of MMR proteins; PCR-CE or NGS detection is therefore necessary.

Addressing Resistance to Therapy in Colorectal Cancer

Addressing Resistance to Therapy in Colorectal Cancer

An open-label clinical trial of RP2 and RP3 oncolytic immunotherapy in combination with atezolizumab and bevacizumab for the treatment of patients with advanced colorectal carcinoma.

TPS3628 Background: Colorectal cancer (CRC) is the third most frequently diagnosed cancer and the second leading cause of cancer-related mortality worldwide. PD-1/PD-L1 inhibition, in combination with other modalities, has demonstrated significant benefit in patients (pts) with microsatellite instability-high or mismatch repair deficient CRC. However, these agents have limited, if any, clinical benefit in pts with microsatellite stable (MSS) or mismatch repair proficient (pMMR) CRC. RP2 is an enhanced potency oncolytic herpes simplex virus type 1 which expresses the fusogenic gibbon ape leukemia virus glycoprotein with the R sequence deleted (GALV-GP-R–), granulocyte-macrophage colony-stimulating factor (GM-CSF), and an anti–CTLA-4 antibody-like molecule; RP3 additionally expresses 4-1BB and CD40 activating ligands, but does not express GM-CSF. Both agents have demonstrated preliminary safety and efficacy in pts with solid tumors. This study will evaluate the safety and efficacy of RP2 and RP3 in combination with atezolizumab (Atezo) and bevacizumab (Bev) in pts with advanced MSS and pMMR CRC. Methods: Pts with a histologic diagnosis of unresectable and/or metastatic CRC, with documented MSS or pMMR status, and previously treated with up to 3 standard-of-care systemic regimens will be enrolled in the RP2 + Atezo + Bev or RP3 + Atezo + Bev treatment groups (30 pts per group). Further key inclusion criteria include Eastern Cooperative Oncology Group performance status 0–1 and adequate hepatic, renal, and hematologic function. RP2/RP3 will be injected into tumors by direct (including via colonoscope) or image-guided injection. Pts will receive 8 total doses of up to 10 mL of RP2 or RP3, with a first dose concentration of 1 × 106 plaque-forming units (PFU)/mL, followed by 3 doses of 1 × 107 PFU/mL every 2 weeks, and then 4 doses of 1 × 107 PFU/mL every 3 weeks. Pts may receive a second course of up to 8 injections of RP2/RP3 if study criteria are met. Bev will be administered starting on day 1 (with first dose of RP2/RP3); Atezo will be administered starting week 7 on the same days as RP2/RP3. The primary endpoint is objective response rate; secondary endpoints are safety, overall survival, progression-free survival, duration of response, and complete response rate. Antitumor activity will be evaluated using Response Evaluation Criteria in Solid Tumors version 1.1 as modified for this study. Safety will be assessed by physical examination, clinical laboratory evaluations, vital signs, and monitoring for adverse events (AEs; including serious AEs). Clinical trial information: NCT05733611 .

The Impact of Tumor Cell-Intrinsic Expression of Cyclic GMP-AMP Synthase (cGAS)-Stimulator of Interferon Genes (STING) on the Infiltration of CD8+ T Cells and Clinical Outcomes in Mismatch Repair Proficient/Microsatellite Stable Colorectal Cancer.

The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway plays a crucial role in activating immune cells in the tumor microenvironment, thereby contributing to a more favorable response to immune checkpoint inhibitors (ICI) in colorectal cancer (CRC). However, the impact of the expression of cGAS-STING in tumor cells on the infiltration of CD8+ T cells and clinical outcomes in mismatch repair proficient/microsatellite stable (pMMR/MSS) CRC remains largely unknown. Our findings reveal that 56.8% of all pMMR CRC cases were cGAS-negative/STING-negative expressions (cGAS-/STING-) in tumor cells, whereas only 9.9% of all pMMR CRC showed cGAS-positive/STING-positive expression (cGAS+/STING+) in tumor cells. The frequency of cGAS+/STING+ cases was reduced in the advanced stages of pMMR/MSS CRC, and histone methylation might be involved in the down-regulation of STING expression in tumor cells. Since the expression level of cGAS-STING in tumor cells has been associated with the infiltration of CD8+ and/or CD4+ T cells and the frequency of recurrence in pMMR/MSS CRC, decreased expression of cGAS-STING in tumor cells might lead to poor immune cell infiltration and worse prognosis in most pMMR/MSS CRC patients. Our current findings provide a novel insight for the treatment of patients with pMMR/MSS CRC.

China special issue on gastrointestinal tumor: Regulatory-immunoscore-A novel indicator to guide precision adjuvant chemotherapy in colorectal cancer.

Novel biomarkers are essential to improve the treatment efficacy and overall survival of stage II and III colorectal cancer (CRC), allowing for personalized treatment decisions. Here, the densities of CD8+ and FOXP3+ T cells in the tumor and invasive margin were processed by immunohistochemistry and digital pathology to form a scoring system named regulatory-Immunoscore (RIS). Cox proportional hazards regression models were used to determine the risk factors associated with time to recurrence. Harrell's concordance index and the time-dependent area under the curve were used to assess model performance. A total of 1213 stage I-III DNA mismatch repair-proficient colorectal cancer (pMMR CRC) patients were randomly assigned to a training set (n = 642) and a validation set (n = 571). From the Cox multivariable analysis, the association of RIS with survival was independent of patient age, sex and anatomy-based tumor risk parameters (P < .0001). For stage II patients, chemotherapy was significantly associated with better recurrence time in patients with low (95% confidence interval [CI]: 0.11-0.54, P = .001) and intermediate (95% CI = 0.25-0.57, P < .001) RIS values. In stage III patients treated with adjuvant chemotherapy, a treatment duration of 6 or more months was significantly associated with better recurrence time in patients with intermediate RIS values (95% CI = 0.38-0.90, P = .016) when compared with duration under 6 months. Therefore, these findings suggest that RIS is reliable for predicting recurrence risk and treatment responsiveness for patients with stage I-III pMMR CRC.

The Gut Microbiome, Microsatellite Status and the Response to Immunotherapy in Colorectal Cancer.

There is increasing evidence in a range of cancer types that the microbiome plays a direct role in modulating the anti-cancer immune response both at the gut level and systemically. Differences in the gut microbiota have been shown to correlate with differences in immunotherapy responses in a range of non-gastrointestinal tract cancers. DNA mismatch repair-deficient (dMMR) colorectal cancer (CRC) is radically different to DNA mismatch repair-proficient (pMMR) CRC in clinical phenotype and in its very good responses to immunotherapy. While this has usually been thought to be due to the high mutational burden in dMMR CRC, the gut microbiome is radically different in dMMR and pMMR CRC in terms of both composition and diversity. It is probable that differences in the gut microbiota contribute to the varied responses to immunotherapy in dMMR versus pMMR CRC. Targeting the microbiome offers a way to boost the response and increase the selection of patients who might benefit from this therapy. This paper reviews the available literature on the role of the microbiome in the response to immunotherapy in dMMR and pMMR CRC, explores the potential causal relationship and discusses future directions for study in this exciting and rapidly changing field.

Biomarker analysis to predict response in patients with metastatic mismatch repair proficient colorectal cancer treated with regorafenib and nivolumab.

228 Background: We previously conducted a phase I/Ib study with regorafenib and nivolumab in patients with refractory metastatic mismatch repair proficient (pMMR) colorectal cancer (CRC). This study aimed to investigate the biomarkers that predict the treatment response. Methods: Out of the 51 patients who received regorafenib and nivolumab, 22 archival pretreatment tumor samples were subjected to the Xerna TME Panel, a machine learning-based RNA-sequencing biomarker assay and were classified into one of four TME biomarker subtypes: Angiogenesis (A), Immune Active (IA), Immune Desert (ID), or Immune Suppressed (IS). Potential predictive biomarkers including the TME subtypes, KRAS (wild type vs mutant), PD-L1 (negative vs. positive, samples with &gt; 1% tumor cells for PD-L1 were considered positive), CD8 expression (low vs. high), and Treg cells (low vs. high) in tumor microenvironment were evaluated for correlation with overall survival (OS), progression free survival (PFS) and disease control rate (DCR, defined as complete response + partial response + stable disease). Results: Among the 22 patients, 16 (72.7%) had liver metastasis and 15 (68.2%) had lung metastasis. KRAS mutation was found in 16 (68.2%) patients. 11/21 (52.4%) were positive for PD-L1. 12 (54.5%) had high CD8 expression, whereas 9/21 (42.9%) had high Treg cells in tumor microenvironment. Ten (45.5%) patients were classified as biomarker-positive (IA + IS subtypes) and 12 (54.5%) were biomarker-negative (A + ID) based on Xerna TME panel. Two (9.1%) patients achieved partial response, 12 (54.5%) had stable disease, and five (22.7%) developed progressive disease. The median PFS was 5.6 months and median OS was 13.1 months. No significant correlation was observed between RAS mutation (p = 0.664, p = 0.609), PD-L1 expression (p = 0.287, p = 0.173), CD8 (p = 0.152, p = 0.456) and PFS or OS. Low Treg was found to be associated with prolonged PFS (median: 9.8 vs. 1.9 months, p = 0.011) but not OS (p = 0.280). Similarly, only low Treg level was related with DCR (83.3% vs. 33.3%, p = 0.032). While not reaching statistical significance, Xerna TME biomarker-positive patients showed trends for higher median PFS (7.9 months vs. 4.1 months, p = 0.254), median OS (15.75 months vs. 11.9 months, p = 0.378), and higher DCR (70% vs. 58%, p = 0.675) compared to biomarker-negative patients. Additionally, the two patients with partial responses were Xerna TME biomarker-positive. Conclusions: Our study demonstrated that low Treg in tumor microenvironment is correlated with better prognosis in patients with refractory metastatic pMMR CRC who were treated with regorafenib plus nivolumab. Xerna TME panel analysis of these patients also showed trends for predictive clinical benefit. Prospective and larger cohort studies are needed to better define predictive biomarkers for this combination in the future.

Soluble PD-L1: a potential dynamic predictive biomarker for immunotherapy in patients with proficient mismatch repair colorectal cancer

BackgroundCirculating soluble programmed death ligand 1 (sPD-L1) can negatively regulate T-cell function and serve as a prognostic or predictive marker in a variety of cancers. However, rare studies have evaluated the potential roles of sPD-L1, and no study has estimated its predictive value for the efficacy of immune treatment in colorectal cancer (CRC).MethodsPlasma samples from 192 CRC patients were used to estimate correlations between clinicopathological features and sPD-L1, secreted PD-L1 (secPD-L1) and exosomal PD-L1 (exoPD-L1). Baseline and posttreatment sPD-L1 levels were also investigated in 55 patients with metastatic CRC (mCRC) treated with chemotherapy ± targeted therapy and 40 patients with proficient mismatch repair (pMMR) mCRC treated with combination immunotherapy. Both sPD-L1 and secPD-L1 were quantified by enzyme-linked immunosorbent assay, while exoPD-L1 was analyzed using flow cytometry.ResultssecPD-L1 was the major component and positively correlated with sPD-L1 in CRC, while exoPD-L1 was almost undetectable. Higher levels of sPD-L1 were detected in patients with distant metastasis, especially those with distant lymph node metastasis and tissue combined positive score (CPS) instead of tumor proportion score (TPS). Chemotherapy or targeted therapy did not significantly impact sPD-L1 concentration. Progressive disease on combination immunotherapy was associated with an increase in sPD-L1 level, whereas no significant change was observed in patients with durable clinical benefit.ConclusionsPD-L1 mainly consisted of secPD-L1, and its level was higher in patients with distant metastasis, especially distant lymph node metastasis and positive CPS. sPD-L1 is a potential dynamic marker to identify rapid progression on combination immunotherapy and avoid ineffective treatment for pMMR CRC.

Identification of genes and cellular response factors related to immunotherapy response in mismatch repair-proficient colorectal cancer: a bioinformatics analysis

Mismatch repair-proficient (pMMR) colorectal cancers (CRCs) are thought to be primarily resistant to immune checkpoint inhibitor (ICI) monotherapy. However, recent clinical trials have reported that early-to-mid stage (non-metastatic) CRC responds well to ICI monotherapy. We hypothesized that the efficacy of immunotherapy is linked to a series of gene expression profiles that can characterize the pMMR CRC disease stage. Using The Cancer Genome Atlas (TCGA) CRC data sets, we first investigated transcriptomic features that continuously changed (were continuously upregulated or downregulated) with pMMR CRC disease-stage progression. We defined these gene sets as stage-associated genes. The deconvolution algorithm then enriched these genes with the dynamic changes in the cell type populations of the CRC tumor microenvironment (TME). Finally, the stage-associated genes were cross-referenced to the current transcriptome profile data on ICI treatment of pMMR CRC, which revealed the gene set specifying an effective pMMR tumor response. In total, 774 genes were found to increase in expression and 845 genes to decrease in expression as the stage increased. Using deconvolution methods, we discovered 2 major disease stage-associated alterations in the cellular composition of pMMR CRCs, including changes in cell types involved in host immune responses and tumor cell metastasis. The central memory CD8+ T cell population decreased as the pMMR CRC disease stage increased, but the endothelial cell populations associated with proliferation and metastasis increased. Using a different cell type annotation set (LM22), we discovered that as the disease progressed, M1 macrophages and CD8+ T cells decreased in the TME. In mismatch repair-deficient patients with CRC, however, such a decrease was not observed. Finally, we identified 27 signature genes that can be used to assess ICI efficacy in treatment-naïve patients with pMMR CRC. The current study sought to identify the underlying molecular mechanisms, pathways, and cell landscapes that explain why early-to-mid stage pMMR CRC responds well to ICI treatment. This analysis might be valuable for the selection of patients who might benefit from immunotherapeutic strategies.

Abstract IA012: Sulindac modulates the response of proficient MMR colorectal cancer to immune checkpoint inhibitors

Abstract Immune checkpoint inhibitors (ICIs) therapy has been widely used to treat different human cancers, particularly advanced solid tumors. However, clinical studies have reported that ICI immunotherapy benefits only ~15% of colorectal cancer (CRC) patients, specifically those with tumors characterized by microsatellite instability (MSI), a molecular marker of defective DNA mismatch repair (dMMR). For the majority of CRC patients who carry proficient MMR (pMMR), ICIs have shown little clinical benefit. In this study, we examined the efficacy of sulindac to enhance the response of pMMR CRC to anti-PD-L1 immunotherapy. We first utilized CT26 and CMT93 syngeneic mouse tumor models to compare the inhibitory effects of PD-L1 antibody (Ab), sulindac, and their combination on pMMR CRC tumor growth. We found that mice treated with combination therapy showed a significant reduction in tumor volume, along with increased infiltration of CD8+ T lymphocytes in the tumor tissues. We also demonstrated that sulindac could downregulate PD-L1 by blocking NF-κB signaling, which in turn led to a decrease in exosomal PD-L1. Notably, PD-L1 Ab can be bound and consumed by exosomal PD-L1 in the blood circulation. Therefore, in combination therapy, sulindac downregulating PD-L1 leads to increased availability of PD-L1 Ab, which potentially improves the overall efficacy of anti-PD-L1 therapy. We validated all these results using humanized Patient-Derived Xenografts (PDX) animal models. Moreover, we demonstrated the safety of low-dose sulindac as it did not have a systemic inhibitory effect on prostaglandin E2 (PGE2). In conclusion, our findings provide unique insights into the mechanism of action and efficacy for sulindac as an immunomodulatory agent for the ICI therapy and immunoprevention of pMMR CRC. Citation Format: Yaguang Xi. Sulindac modulates the response of proficient MMR colorectal cancer to immune checkpoint inhibitors [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr IA012.