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

Objectives: Mismatch repair deficient (dMMR) colorectal cancer (CRC) is caused by inactivation of the MMR DNA repair system, most commonly via epigenetic inactivation of the MLH1 gene, and these tumors occur most frequently in the right colon. The objective was to determine whether cholecystectomy (CCY) increases the risk of a dMMR CRC by comparing CCY incidence in patients with dMMR CRC and proficient MMR (pMMR) CRC to unaffected controls.Materials and methods: All patients diagnosed with CRC in Iceland from 2000 to 2009 (n = 1171) were included. They had previously been screened for dMMR by immunohistochemistry (n = 129 were dMMR). Unaffected age- and sex-matched controls (n = 17,460) were obtained from large Icelandic cohort studies. Subjects were cross-referenced with all pathology databases in Iceland to establish who had undergone CCY. Odds ratios were calculated using unconditional logistic regression.Results: Eighteen (13.7%) dMMR CRC cases and 90 (8.7%) pMMR CRC cases had undergone CCY compared to 1532 (8.8%) controls. CCY-related odds ratios (OR) were 1.06 (95% CI 0.90–1.26, p = .577) for all CRC, 1.16 (95% CI 0.66–2.05 p = .602) for dMMR CRCand 1.04 (95% CI 0.83–1.29, p = .744) for pMMR CRC. Furthermore, OR for dMMR CRC was 0.51 (95% CI 0.16–1.67, p = .266), 2.04 (95% CI 0.92–4.50, p = .080) and 1.08 (95% CI 0.40–2.89, p = .875) <10 years, 10–20 years and >20 years after a CCY, respectively.Conclusions: There was no evidence of increased risk of developing dMMR CRC after CCY although a borderline significantly increased 2-fold risk was observed 10–20 years after CCY. Larger studies are warranted to examine this further.

Mismatch repair–deficient colorectal cancer: a model of immunogenic and immune cell–rich tumor despite nonsignificant programmed cell death ligand-1 expression in tumor cells

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...

BackgroundLinks between colorectal cancer (CRC) and the gut microbiome have been established, but the specific microbial species and their role in carcinogenesis remain an active area of inquiry. Our understanding would be enhanced by better accounting for tumor subtype, microbial community interactions, metabolism, and ecology.MethodsWe collected paired colon tumor and normal-adjacent tissue and mucosa samples from 83 individuals who underwent partial or total colectomies for CRC. Mismatch repair (MMR) status was determined in each tumor sample and classified as either deficient MMR (dMMR) or proficient MMR (pMMR) tumor subtypes. Samples underwent 16S rRNA gene sequencing and a subset of samples from 50 individuals were submitted for targeted metabolomic analysis to quantify amino acids and short-chain fatty acids. A PERMANOVA was used to identify the biological variables that explained variance within the microbial communities. dMMR and pMMR microbial communities were then analyzed separately using a generalized linear mixed effects model that accounted for MMR status, sample location, intra-subject variability, and read depth. Genome-scale metabolic models were then used to generate microbial interaction networks for dMMR and pMMR microbial communities. We assessed global network properties as well as the metabolic influence of each microbe within the dMMR and pMMR networks.ResultsWe demonstrate distinct roles for microbes in dMMR and pMMR CRC. Bacteroides fragilis and sulfidogenic Fusobacterium nucleatum were significantly enriched in dMMR CRC, but not pMMR CRC. These findings were further supported by metabolic modeling and metabolomics indicating suppression of B. fragilis in pMMR CRC and increased production of amino acid proxies for hydrogen sulfide in dMMR CRC.ConclusionsIntegrating tumor biology and microbial ecology highlighted distinct microbial, metabolic, and ecological properties unique to dMMR and pMMR CRC. This approach could critically improve our ability to define, predict, prevent, and treat colorectal cancers.

To identify exonic markers that could improve analytic performance characteristics of next-generation sequencing (NGS) in detecting mismatch repair deficiency (dMMR) using colorectal cancer (CRC) as a model. Coding sequences of a target NGS panel (~1.13 megabase) were compared between dMMR CRC and mismatch repair-proficient (pMMR) CRC in a training cohort (41 dMMR CRCs and 213 pMMR CRCs) and a validation cohort (33 dMMR CRCs and 307 pMMR CRCs) with documented mismatch repair status by immunohistochemical and/or microsatellite instability assays. The dMMR CRC cases showed significantly higher insertion/deletion (indel) mutations within exonic homopolymers (homo-indels), occurring predominantly within longer repeats of 5 to 10 nucleotides (92%, P < .0001), rather than shorter repeats of 2 to 4 nucleotides seen in pMMR CRC (62%). Homo-indels in dMMR CRC were not random. Hotspot loci were consistent between the training and validation cohorts. The dMMR defined by indels within homopolymers of 5 or more nucleotides, homopolymers of 7 or more nucleotides, or a panel of hotspots all showed 100% sensitivity and specificity with a range of cutoffs. We propose that this approach allows one to identify highly sensitive and specific markers for detecting dMMR CRC by NGS alone. Further studies are warranted to test whether these markers are applicable to non-CRC neoplasms.

Background Immune checkpoint-inhibitors targeting the PD-1/PD-L1 system are FDA approved in microsatellite instable (MSI) or mismatch repair deficient (dMMR) colorectal cancer (CRC). PD-L1 expression is tightly linked to features connected to immune checkpoint inhibitor response, but studies on large subsets of cancers analyzing the correlation between different status of MSI/dMMR, tumor infiltrating lymphocytes and PD-L1 expression are still lacking. Methods More than 1800 CRC were analyzed for PD-L1 by immunohistochemistry in a tissue microarray format. Data were compared to MMR, the number of intratumoral CD8+ cytotoxic T-cells, and adverse clinico-pathological parameters. Different cutoff levels for defining PD-L1 positivity in tumor cells (1%, 5%, 10%, and 50%) yielded comparable results. Results At a cutoff level of 5%, PD-L1 positivity was seen in 5.1% of tumors. PD-L1 was more often positive in dMMR (18.6%) than in MMR proficient (pMMR) cancers (4.1%; p < 0.0001). The number of intratumoral CD8+ lymphocytes was strikingly higher in PD-L1 positive (939.5 ± 118.2) than in PD-L1 negative cancers (310.5 ± 24.8). A higher number of intratumoral CD8+ lymphocytes was found in dMMR CRC (PD-L1 positive: 1999.7 ± 322.0; PD-L1 negative: 398.6 ± 128.0; p < 0.0001) compared to pMMR CRC (PD-L1 positive: 793.2 ± 124.8; PD-L1 negative: 297.2 ± 24.2; p < 0.0001). In dMMR and pMMR CRC, PD-L1 expression in tumor cells was unrelated to tumor stage, lymph node status or lymphatic/venous invasion. PD-L1 positivity in tumor associated immune cells was seen in 47.5% of cases and was significantly linked to high numbers of tumor infiltrating CD8+, low tumor stage, and absence of lymph node metastasis and lymphatic/venous invasion (p < 0.0001 each). Conclusion The data support the previously suggested fact that PD-L1 expression in tumor cells is driven by extensive cytotoxic T-cell infiltration in highly immunogenic dMMR and pMMR CRC. Frequent and intense PD-L1 expression in tumor cells of dMMR CRC may contribute to the high response rates of dMMR CRC to immune checkpoint-inhibitors.

Purpose: Mismatch repair-deficient (dMMR) colorectal cancer (CRC) is associated with increased local immune response as compared with mismatch repair-proficient (pMMR) CRC. We evaluated the relationship between MMR status and systemic inflammatory factors, including neutrophil lymphocyte ratio (NLR) and C-reactive protein (CRP). We also assessed the prognostic value of these parameters.Methods and materials: We analysed the relationship between MMR status (obtained by histochemical analysis), neutrophil and lymphocyte counts, NLR, and CRP level. The impact of systemic inflammatory factors on survival was also evaluated in dMMR and pMMR CRC patients.Results: A total of 1353 male and 892 female patients were eligible for analysis, of which, 253 patients (11.3%) were found to have dMMR status. Patients with dMMR status presented with increased neutrophil counts, and higher NLR and CRP levels in early stage CRC. In stage IV CRC patients, no correlation between MMR status and systemic inflammatory factors was found. Lymphocyte counts did not correlate with MMR status. High NLR was a prognostic factor for poor survival in pMMR CRC. However, NLR was not a prognostic factor in dMMR CRC.Conclusions: Our results suggest that dMMR CRC correlates with higher neutrophil count, NLR and CRP levels only in non-metastatic patients, and NLR has prognostic value only in pMMR CRC.

45 Background: Patients with hereditary non polyposis colorectal cancer (HNPCC) diagnosed with CRC have an elevated risk of a second primary CRC (SPCRC) and of rapid primary cancer development. This informs both initial surgical approach and endoscopic surveillance intervals. A feature of HNPCC is dMMR, also found in 15% of sporadic CRC, where the risk of SPCRC has yet to be defined. Methods: We examined a multi-site comprehensive CRC database (Melbourne, Australia), where prospectively collected data includes family history (including HNPCC), histology, surgery performed and incidence of second primary cancer. Sporadic dMMR included any case with a BRAF V600E mutation, confirmed hypermethylation, negative germline testing, or age over 60 years. We explored the incidence and timing of SPCRC in patients with early stage sporadic dMMR (or MSI-H) versus pMMR cancers. Results: From February 2004 to December 2019, 7442 patients diagnosed with stage I-III CRC were recorded. MMR status was known in 4079 (54.8%), including 714 with dMMR colorectal cancer (17.4%) of which 575 (14.6%) were deemed sporadic dMMR. Sporadic dMMR patients were older (mean 76.2 years vs 65.9 years, p = &lt; 0.001), more likely to be female (65.2% vs 42%, p = &lt; 0.001) and have a right sided primary (80.9% vs 32.8%, p = &lt; 0.001), compared to patients with pMMR CRC. A SPCRC was diagnosed in 11/575 patients (1.91%) with sporadic dMMR CRC versus 27/3365 (0.83%) patients with pMMR CRC (HR = 2.57, 95% CI 1.28 - 5.17, p = 0.008). Median time to SPCRC was 1.13 years vs 2.38 years (p = 0.49). The SPCRC diagnosed in sporadic dMMR CRC vs pMMR CRC were more likely to be dMMR ((72.7%) vs (25.9%), p = 0.03), but a similar number were stage I or II ((81.8%) vs (81.5%)) and a similar number were surveillance detected ((72.7%) vs (77.8%). Conclusions: Patients with sporadic dMMR appear to have a significantly elevated risk of SPCRC compared to the pMMR population and were diagnosed at a shorter interval. The SPCRC is also more likely to also be dMMR. Increased colonoscopic surveillance of patients presenting with an initial sporadic dMMR cancer should be considered where clinically appropriate.

A constitutive interferon-high immunophenotype defines response to immunotherapy in colorectal cancer.

While mismatch repair-deficient (dMMR) colorectal cancers (CRCs) exhibit strong immunogenicity and better response to immune checkpoint inhibitors, the more prevalent mismatch repair-proficient (pMMR) CRCs typically show poor T- cell infiltration and inferior outcomes. Profiling the limited tumor-infiltrating CD8+ T cells helps identify responders and guides new strategies to enhance their infiltration and function in pMMR CRC tumors. Our study reveals that the proportion and number of CD103−CD8+ T (CD103N) cells are significantly increased in pMMR CRC tissue compared to adjacent non-tumor tissue. Distinguish from CD103+CD8+ T (CD103P) cells with elevated TEX markers, these CD103N cells display a precursor exhausted T cells (TPEX) phenotype with elevated stemness properties, reduced exhaustion markers, and retained functional capacity to secrete anti-tumor mediators. Moreover, CD103N cells in pMMR CRC shared a substantial number of identical TCR clonotypes with both CD103P cells in tumor and CD103N cell in peripheral blood. In dMMR CRC patients, enrichment of TPEX-like CD103N cells is associated with a favorable prognosis following anti-PD-1 therapy, suggesting their association with clinical outcome. Our findings identify an expanded population of CD103N cells exhibiting a TPEX phenotype with anti-tumor potential in pMMR CRC, highlighting their promise as therapeutic targets for recruitment into the tumor microenvironment to enhance the efficacy of immunotherapy.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00262-025-04214-w.

Phase I Clinical Trial of Autologous Ascites-derived Exosomes Combined With GM-CSF for Colorectal Cancer

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.

3558 Background: Dostarlimab is a programmed death 1 (PD-1) inhibitor approved in the US as a monotherapy in patients (pts) with dMMR advanced/recurrent endometrial cancer that has progressed on or after prior treatment with a platinum-containing regimen or in pts with dMMR solid tumors that have progressed on or after prior treatment, with no satisfactory alternative treatment options. Here we report on patient-reported outcomes (PROs) in pts with dMMR CRC, a post hoc subgroup analysis. Methods: GARNET is a multicenter, open-label, single-arm phase 1 study. Cohort F enrolled pts with dMMR/microsatellite instability–high or POLε-mutated non-endometrial solid tumors. Pts with CRC must have received prior fluoropyrimidine, oxaliplatin, and irinotecan. Pts received 500 mg of IV dostarlimab Q3W for 4 cycles, then 1000 mg Q6W until discontinuation. PRO assessment, an exploratory endpoint, was measured using the EORTC-QLQ-C30. PROs were collected at baseline, at each dose cycle, and after discontinuation. Multi-item and item-level analyses were collected and scored; improvement was defined as mean change from baseline (directional change dependent on item). A 10-pt change in mean from baseline was considered clinically significant for the analysis. Results: PRO data at baseline were available for 96 pts with CRC who received ≥1 dose of dostarlimab. Improvements from baseline for quality of life (QOL) and most gastrointestinal symptom scales were observed and maintained from cycle 2 through 7 (Table). Stability in diarrhea was seen from cycles 2 through 7. Conclusions: PROs from the GARNET trial in pts with dMMR CRC show that QOL and disease-related gastrointestinal symptoms were improved or maintained while on dostarlimab treatment. These data, with the previously reported efficacy and safety profile, support the use of dostarlimab in pts with dMMR CRC. Clinical trial information: NCT02715284. [Table: see text]

Immune checkpoint inhibitors (ICI) have become the standard of care for patients with mismatch repair-deficient/microsatellite instability-high (dMMR/MSI-H) colorectal cancer. However, biomarkers of response to ICI are still lacking. Forty-two patients with dMMR colorectal cancer treated with neoadjuvant PD-1 blockade were prospectively enrolled. To identify biomarkers of pathologic complete response (pCR) to neoadjuvant therapy, we analyzed genomic and transcriptomic profiles based on next-generation sequencing, and immune cell density based on multiplex immunofluorescence (mIF) staining. An integrated analysis of single-cell RNA sequencing from our previous study and GSE178341, as well as mIF was performed to further explore the significance of the tumor microenvironment (TME) on pCR response. The tumor mutation burden of both tumor tissue and plasma blood samples was comparable between the pCR and non-pCR groups, while HLA-DQA1 and HLA-DQB1 were significantly overexpressed in the pCR group. Gene signature enrichment analysis showed that pathways including T-cell receptor pathway, antigen presentation pathway were significantly enriched in the pCR group. In addition, higher pre-existing CD8+ T-cell density was associated with pCR response (767.47 per.mm2 vs. 326.64 per.mm2, P = 0.013 Wilcoxon test). Further integrated analysis showed that CD8+ T cells with low PD-1 expression (PD-1lo CD8+ T cells) expressing high levels of TRGC2, CD160, and KLRB1 and low levels of proliferated and exhausted genes were significantly associated with pCR response. Immune-associated transcriptomic features, particularly CD8+ T cells were associated with pCR response to ICI in dMMR colorectal cancer. Heterogeneity of TME within dMMR colorectal cancer may help to discriminate patients with complete response to neoadjuvant ICI.

Identifying the target of natural killer (NK) cells in colorectal cancer (CRC) is critical for optimising the clinical use of NK cell-mediated immunotherapy. Mismatch repair deficiency (dMMR) is associated with high immune cell infiltration and MHC Class I defects. Whether dMMR CRC responses to NK cell therapy remains unclear. MLH1, DR4, and DR5 knockout cell lines were established using CRISPR-Cas9 system. NK92-MI or NK cell isolated from BABL/C mice were used as effector cells against tumour cells. Inflammatory cytokines secretion by CRC cells was assessed via cytokine analysis. NK-cell-deficient/proficient animal models were used to validate the NK cell sensitivity. We observed that dMMR CRC cells were more sensitive to NK cell-mediated cytotoxicity than were mismatch-repair-proficient (pMMR) CRC cells. In dMMR CRC, Death receptor (DR)4/5 was upregulated and mediated sensitivity to NK cell-mediated cytotoxicity. DR4/5-mediated secretion of interleukin -12 sustained NK cell viability in dMMR CRC. NK cell depletion induced dMMR CRC tumour growth, and NK cell transfer inhibited lung metastasis of dMMR CRC with DR4/5 expression in vivo. TP53 upregulated DR4/DR5 expression in dMMR CRC. dMMR associated with increased sensitivity to NK cell-mediated cytotoxicity in CRC. DR4/DR5 sensitise dMMR CRC to NK cell-mediated cytotoxicity.