pMMR Tumors Research Articles

Integrating spatial profiles and cancer genomics to identify immune-infiltrated mismatch repair proficient colorectal cancers.

pMMR tumors with high T cell infiltration and active immunosuppression are identifiable with a mid-plex imaging assay whose clinical deployment might double the number of treatment-naïve CRCs eligible for ICIs. Moreover, the low tumor mutational burden in tipMMR CRC shows that MMR status is not the only factor promoting immune infiltration.

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.

Upfront Fluorouracil, Leucovorin, Oxaliplatin, and Irinotecan Plus Bevacizumab With or Without Atezolizumab for Patients With Metastatic Colorectal Cancer: Updated and Overall Survival Results of the ATEZOTRIBE Study.

Clinical trials frequently include multiple end points that mature at different times. The initial report, typically based on the primary end point, may be published when key planned co-primary or secondary analyses are not yet available. Clinical Trial Updates provide an opportunity to disseminate additional results from studies, published in JCO or elsewhere, for which the primary end point has already been reported.We report 4-year results of the phase II randomized AtezoTRIBE study. Eligible patients with metastatic colorectal cancer (mCRC) received first-line fluorouracil, leucovorin, oxaliplatin, and irinotecan (FOLFOXIRI)/bevacizumab (control group, n = 73) or FOLFOXIRI/bevacizumab plus atezolizumab (experimental group, n = 145). We present overall survival (OS) and updated outcomes according to tumor immune-related biomarkers, both in the intention-to-treat (ITT) population and the cohort of patients with proficient mismatch repair (pMMR) tumors. Median follow-up was 45.2 months (IQR, 42.6-49.2). In the ITT population, median OS was 33.0 and 27.2 months for experimental and control groups, respectively (hazard ratio [HR], 0.78 [80% CI, 0.61 to 0.98]; P = .084). An interaction effect between Immunoscore Immune-Checkpoint (IC) and treatment arm was observed (Pint, .089), with higher benefit from atezolizumab in the Immunoscore IC-high group. In the pMMR cohort (N = 202), median OS was 30.8 and 29.2 months for experimental and control groups, respectively (HR, 0.80 [80% CI, 0.63 to 1.02]; P = .117). Interactions between treatment group and tumor mutational burden (TMB) and Immunoscore IC were reported (Pint, .043 and .092, respectively), with patients bearing TMB-high and Immunoscore IC-high tumors deriving higher benefit from the addition of atezolizumab. First-line FOLFOXIRI/bevacizumab plus atezolizumab improves OS in patients with mCRC. In the pMMR group, patients with Immunoscore IC-high and/or TMB-high tumors are identified as a subgroup of interest to further develop this treatment.

Integrated analysis of two phase 2 trials of nivolumab in metastatic rare solid cancer with dMMR or pMMR (MASTER KEY sub-studies: ROCK and ROLL trial).

e14603 Background: Immune checkpoint inhibitors play a critical role in mismatch repair deficient (dMMR) / microsatellite instability-high (MSI-H) cancers, but has yet to investigate the activity of nivolumab in a subset of rare cancers (incidence < 6 / 100,000/year), that are MMR proficient (pMMR). Methods: We conducted two single arm phase 2 trials to investigate the efficacy and safety of nivolumab monotherapy in patients with advanced rare tumors with either pMMR (jRCT2091220345) or dMMR (jRCT2091220344). dMMR was defined by the complete loss of at least one of the following proteins: MLH1, MSH2, MSH6, PMS2. pMMR was defined when all four proteins were retained. Primary endpoint was independently reviewed response rate (RR). Secondary endpoints included progression free survival (PFS), overall survival (OS), and adverse events (AE). The dMMR/MSI-H trial enrolled 5 to 15 patients, based on a Bayesian design with a threshold RR of 5% and an expected RR of 30%, requiring ≥2 responders to achieve a posterior probability of ≥95% that the true RR of nivolumab is at least 5%. The pMMR trial was designed to explore the clinical activity in patients with pMMR, but also to confirm the clinical significance of “dMMR/MSI-H” as a predictive marker. If the upper limit of 95% confidence interval of the objective RR in the pMMR trial is below 30%, it implies that dMMR/MSI-H serves as a predictive marker. To evaluate this hypothesis at one-sided significance level of 5% with 90% power, a sample size of 54 patients with pMMR is necessary, assuming that the expected RR of nivolumab to be 15%. Results: Between May 2018 and March 2021, 11 and 57 patients received nivolumab treatment in the dMMR/MSI-H trial and pMMR trial, respectively. 10 and 54 patients in the respective trials were included in the full analysis set. The objective RR was 60% (95% CI 26.2-87.8) and 5.6% (95% CI 1.2-15.4) in the dMMR/MSI-H trial and pMMR trial, respectively. Median PFS was 10.1 months (95% CI 0.9-NE), and median OS was not yet reached in the dMMR/MSI-H trial, while the median PFS was 2.5 months (95% CI 1.9-4.8), and median OS was 12.7 months (95% CI 8.1-18.5) in the pMMR trial. Histology of the three responders in the pMMR trial were adnexal carcinoma of skin (2/4, 50%), and soft tissue sarcoma (1/14, 7.1%). In dMMR/MSI-H patients with tumor mutation burden (TMB) data, 5 of 6 were TMB ≥ 10/Mb, all of whom showed response. In contrast, in the pMMR group, two of the 22 patients with TMB data had TMB > 10/Mb, but neither showed response. Treatment-related grade 3 or higher AEs were observed in 0 and 8 (14%) patients in the dMMR/MSI-H and pMMR trials, respectively. Conclusions: Clinical benefit of nivolumab in pMMR advanced rare cancer patients was limited in contrast to dMMR/MSI-H patients, confirming the clinical significance of dMMR/MSI-H as a predictive marker. Further predictive biomarker studies are needed for pMMR tumors. Clinical trial information: jRCT2091220345 , jRCT2091220344 .

Abstract 5153: Pyramidobacter-DMMR synergy in colorectal cancer: Unveiling prognostic impact, mechanistic insights, and tumor microenvironment dynamics

Abstract Background: Colorectal cancer (CRC) exhibits a heightened presence of intratumoral bacteria, and deficient DNA mismatch repair (dMMR) contributes to nucleotide mismatch accumulation, fostering mutagenesis. However, the combined impact of intratumoral bacteria and dMMR on the tumor immune microenvironment (TIME) in CRC, and its consequential influence on patient prognosis, remains elusive. Methods: We utilized samples from the Cancer Microbiome Atlas project (TCMA), collecting tumor and matched paracancer tissues from colorectal cancer patients. Phyloseq in R was employed to scrutinize microbial abundance differences. The effects of microbial enrichment on prognosis were assessed through survival analysis, and GSEA enrichment analysis provided mechanistic insights. Results: A total of 173 samples, including 28 dMMR tumors, 112 proficient MMR (pMMR) tumors, and 22 paired para-cancer tissues, were collected. Following stringent filtration, we analyzed 230 bacterial genera. Notably, 26 genera, including Fusobacterium and Streptococcus, showed significant enrichment in dMMR tumors compared to para-cancer tissues. Furthermore, 18 genera were enriched in dMMR tumors compared to pMMR tumors. Of these, 8 genera like Desulfovibrio and Pyramidobacter consistently exhibited enrichment in dMMR CRC compared to either para-cancer tissues or pMMR tumors. Survival analysis highlighted Pyramidobacter as the only genus significantly associated with prognosis in dMMR CRC. Increased Pyramidobacter abundance correlated with decreased overall survival (OS) (P value: 0.049). Stratifying dMMR CRC patients based on Pyramidobacter abundance further emphasized the significantly worse survival in the Pyramidobacter-High group (HR 0.93, 95%CI 1.03-9.60, log-rank p = 0.015). Exploring the impact of Pyramidobacter infection on tumor progression, we assessed TIME and oncogenic pathways between Pyramidobacter-Low and Pyramidobacter-High groups. While overall immune infiltration was higher in the Pyramidobacter-High group, it primarily stemmed from increased B cell and naive CD8+ T cell infiltration. Importantly, pathways enriched in the Pyramidobacter-High group included inflammatory response, epithelial-mesenchymal transition (EMT), and KRAS signaling, while those enriched in the Pyramidobacter-Low group encompassed IFNα response, IFNγ response, and DNA damage repair. These findings suggest Pyramidobacter's role in inducing inflammation, promoting tumor proliferation and metastasis, and suppressing interferon response. Conclusion: Pyramidobacter emerges as a key player in the development of dMMR CRC, exerting a pivotal influence on patient prognosis through the induction of inflammatory responses and EMT. Citation Format: Shisen LI, Yunlong LI, Hongjiang Ma, Shihao Qin, Feilong Zhao, Jipeng Li. Pyramidobacter-DMMR synergy in colorectal cancer: Unveiling prognostic impact, mechanistic insights, and tumor microenvironment dynamics [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 5153.

Abstract 700: Impact of heterogeneity for mismatch repair activity on colon tumor development and therapeutic response

Abstract Background: Colorectal cancers (CRC) that are deficient for DNA mismatch repair (dMMR) proteins have high levels of genetic instability and consequently high mutational burden, creating numerous neoantigens that can elicit an immune response. Therefore, dMMR CRC are the best candidates for immune checkpoint inhibition (ICI) compared to proficient DNA mismatch repair (pMMR) CRC. Early clinical trials revealed that only 30-55% metastatic dMMR CRC responded to ICI. Recent studies utilizing allograft models have demonstrated that tumor immune landscape can be primed with radiopharmaceutical therapy (RPT). Although early results have been quite promising, allograft models often fail to accurately predict efficacy for many reasons, but most often are due to a lack of heterogeneity. Methods: To better understand how heterogenous MMR expression impacts tumor development and response to ICI alone or in combination with RPT, we developed a new mouse model that develops three tumor types: (1) homotypic dMMR tumors that express green fluorescent protein, (2) homotypic pMMR tumors that express red fluorescent protein, and (3) heterotypic tumors with a mixture of dMMR and pMMR cells. Surveillance bright field and fluorescent colonoscopies allow us to assess treatment response by measuring tumor size and proportion of dMMR and pMMR tumor cells in real-time. These outcomes are verified through ex vivo imaging and histological analysis. Results: Tumor response to anti-PD-L1 ICI was dependent on MMR status. Homotypic dMMR tumors exhibited the strongest response: significantly smaller in treated mice than controls, exhibited areas of vascular congestion and increased CD8+ cytotoxic T-cells infiltration. Response of heterotypic MMR tumors varied depending on percentage of dMMR cells; tumors with a high percentage of dMMR cells remained small, whereas tumors with a low percentage of dMMR had an outgrowth of pMMR cells. Treated and untreated homotypic pMMR tumors were indistinguishable. To enhance the response of all three tumor types, mice were treated with dual ICI treatment, anti-PD-L1 and anti-CTLA-4, or RPT. Animals administered the tri-therapy (dual ICI + RPT) survived significantly longer with fewer tumors than animals treated with either dual ICI or RPT alone. Conclusions: Our innovative mouse model allows for the highly detailed characterization of tumor response to therapies. Simplistic allograft models may not adequately account for the dynamic effects that neighboring cell death may have on radio-resistant cells, on tumor vascular perfusion and oxygenation, or on tumor immune infiltration and adaptive immune recognition of remaining cells. We begin to fill these critical gaps in knowledge and therefore the results from our studies will help guide the translation and integrated use of ICIs and RPTs in clinic. Citation Format: Santina M. Snow, Dawn Albrecht, Paul A. Clark, Caroline P. Kerr, Joseph J. Grudzinski, Justin J. Jeffery, Hansel Comas Rojas, Reinier Hernandez, Kristina A. Matkowskyj, Jamey P. Weichert, Zachary S. Morris, Richard B. Halberg. Impact of heterogeneity for mismatch repair activity on colon tumor development and therapeutic response [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 700.

Efficacy and safety of PD-1 blockade plus long-course chemoradiotherapy in locally advanced rectal cancer (NECTAR): a multi-center phase 2 study

Adding PD-1 blockade in the neoadjuvant regimens for locally advanced rectal cancer (LARC) patients with microsatellite stable (MSS) / mismatch repair-proficient (pMMR) tumors is an attractive, but debatable strategy. This phase 2, multicenter, prospective, single-arm study enrolled patients from 6 centers from June 2021 to November 2022. Locally advanced rectal cancer (LARC, cT3-4aN0M0 and cT1-4aN1-2M0) patients aged ≥18 years with the distance from distal border of tumor to anal verge ≤10 cm (identified by Magnetic Resonance Imaging) were qualified for inclusion. The patients received long-course radiotherapy (50 Gy/25 fractions, 2 Gy/fraction, 5 days/week) and three 21-day cycles capecitabine (850–1000 mg/m2, bid, po, day1–14) and three 21-day cycles tislelizumab (200 mg, iv.gtt, day8) as neoadjuvant. Total mesorectal excision (TME) was 6–12 weeks after the end of radiotherapy to achieve radical resection. A total of 50 patients were enrolled in this study. The pathological complete response rate was 40.0% [20/50, 95% confidence interval (CI): 27.61–53.82%], while 15 (30.0%, 95% CI: 19.1–43.75%), 9 (18.0%, 95% CI: 9.77–30.8%), 2 (4.0%, 95% CI: 1.10–13.46%) patients respectively achieved grade 1, 2, and 3 tumor regression. Treatment-related adverse events (TRAEs) occurred in 28 (56.0%) LARC patients, including 26(52.0%) with grade I-II and 2 (4.0%) with grade III (1 with grade 3 immune-related colitis and 1 with grade 3 rash). PD-1 blockade plus long-course chemoradiotherapy (CRT) showed promising therapeutic effects according to pathological complete response rate and is well-tolerated in LARC patients. A larger randomized controlled study is desired to further validate the above findings.

Clinicopathological features of colon cancer depending on the dMMR status of the tumor

Aim. To conduct a clinical and morphological assessment of the characteristics of colon cancer depending on the dMMR / pMMR status of the tumor.Materials and methods. A retrospective study included 66 patients with operable colorectal cancer (CRC) (T1-4bN0-2bM1), who were treated at Cancer Research Institute of Tomsk National Research Medical Center (NRMC). The average age of the patients was 64.4 ± 12.8 years. All patients underwent hemicolectomy or colon resection, as well as intraoperative resection of distant metastases, if present.Results. We determined that in CRC patients with pMMR tumors, hematogenous metastases were detected in 27.3% of cases, while in patients with dMMR tumors, hematogenous metastases were detected only in 6.1% of cases (p = 0.021). A comparative analysis of dMMR and pMMR tumors also allowed to establish higher frequency of perineural invasion among the pMMR subgroup of carcinomas (p = 0.039). The sign of tumor budding was found both in dMMR carcinomas (36%) and in pMMR tumors (45%). This sign was associated with damage to regional lymph nodes (p = 0.0017). A more detailed analysis of the tumor budding phenomenon showed that in dMMR tumors, Bd1 low-grade budding (83%) predominated. In pMMR tumors, Bd2 intermediate-grade budding (33%) and Bd3 high-grade budding (26.7%) prevailed. Bd2 and Bd3 tumor budding types were associated with hematogenous metastasis (p < 0.001).Conclusion. The obtained data demonstrate the differences in such pathomorphological parameters as perineural invasion and the degree of tumor budding depending on the dMMR / pMMR status of the tumor. These histologic parameters in tumor tissue are also associated with higher incidence of distant metastasis in patients with pMMR carcinomas as opposed to patients with dMMR tumors.

Immune checkpoint blockade combined with targeted therapies to provide disease control in mismatch repair-proficient colorectal cancer with peritoneal metastases.

133 Background: Metastatic colorectal adenocarcinoma (mCRC) with proficient mismatch repair (pMMR) is associated with poor response to cytotoxic chemotherapy in the refractory setting, with a median overall survival (OS) of 10.8 months. Recent immunotherapy (IO) regimens utilizing a combination of immune checkpoint blockade (ICB) and tyrosine kinase inhibitors (TKIs) that suppress tumor-associated macrophages (TAMs) have shown promise for extra-hepatic, but not hepatic metastases. However, few studies include patients with peritoneal metastases (PMs) and the response rate of PMs to these therapies is unknown. We sought to examine the disease control rates (DCR) and OS of patients treated with PM who were treated with ICB regimens. Methods: Patients with pMMR tumors treated with ICB and TKI after progression on 1st and 2nd-line therapy from 2015 to 2022 were identified. Date of start of therapy, end of therapy, last follow up and vital status were determined from the medical record. Radiographic disease response within each metastasis site at 6 months and at last follow-up on therapy was determined for each patient. Overall survival was estimated using the Kaplan-Meier method. Results: A total of 93 patients treated with an ICB and TKI after at least two lines of cytotoxic systemic therapy were identified. Five were excluded due to dMMR/MSI-H status and eight due to lack of follow-up. A total of 33 patients with PM, with or without other metastases, had a median follow-up on therapy of 6.1 months and median follow-up until death or censoring of 9.8 months. Median OS was 15.1 (9.7-29.5) months, and 18-month survival was 43% (95%CI: 24-62%). OS was not associated with either BRAF (HR 0.98, 0.38-2.6, p=0.98) or RAS (HR 1.50, 0.62- 3.63, p=0.37) mutation status. Partial response of PM was noted in 5 patients (15%), stable disease in 14 (42%) and progressive disease in 14 (42%), resulting in DCR of 57%. Disease control for all metastatic sites was noted in 9 (27%) of patients, and median OS was not reached at 2 years. Median OS for patients without peritoneal progression was 22.0 months [12.3-NR], and PM progression was associated with worse OS (HR 0.66, p=0.024). A total of 32 patients with any metastatic site were treated for at least 6 months. Median follow-up was 16.1 months for this cohort. DCR at 6 months for CRLM was 23% (3/13), PM was 58% (14/24, p=0.08 vs CRLM) and for LM was 62% (8/13, p=0.11 vs CRLM). Conclusions: Response to ICB and targeted therapy combinations can vary widely by metastatic site, with liver trending towards less response than peritoneum or lung. Disease control in the peritoneum can be achieved in the majority of patients, and median OS compares favorably to standard-of-care therapy in the refractory setting. Patients with peritoneal disease should be included in future trials of ICBs as treatment options are otherwise limited.

Statement of the Uterus Commission of the Gynecological Oncology Working Group (AGO) on the Use of Primary Chemoimmunotherapy to Treat Patients with Locally Advanced or Recurrent Endometrial Cancer.

The publication of two large randomized studies - the ENGOT-EN-6-NSGO/GOG-3031/RUBY trial and the NRG-GY018 trial - which investigated combining chemotherapy with immunotherapy to treat patients with primary advanced or recurrent endometrial cancer (EC) has transformed the clinical study landscape in terms of first-line therapy for affected patients and has set a new standard of therapy. In the ENGOT-EN-6-NSGO/GOG-3031/RUBY trial, the addition of dostarlimab to standard chemotherapy with carboplatin and paclitaxel resulted in a significant and clinically relevant improvement of progression-free survival and overall survival in the overall population, a significant and clinically relevant improvement of progression-free survival and overall survival in the subgroup with dMMR/MSI-high tumors, and a significant and clinically relevant improvement of progression-free survival in the subgroup with pMMR/MSI-low tumors. In the NRG-GY018 trial, the addition of pembrolizumab to standard chemotherapy with carboplatin and paclitaxel resulted in a significant and clinically relevant improvement of progression-free survival in the group with dMMR tumors, and a significant and clinically relevant improvement of progression-free survival in the group with pMMR tumors. As expected, the effect in both trials was much more pronounced in the group of patients with dMMR/MSI-high tumors. According to the assessment of the Uterus Organ Commission of the AGO, all patients with dMMR/MSI-high tumors should receive chemoimmunotherapy and all patients with pMMR/MSI-low tumors who meet the inclusion criteria of the two trials discussed here may have chemoimmunotherapy. For dostarlimab this means: patients with EC recurrence who will not undergo surgery or radiotherapy, patients with stage IIIA, IIIB or IIIC1 disease and a measurable lesion postoperatively, patients with stage IIIA, IIIB or IIIC1 disease with histological findings of serous EC, clear-cell EC or carcinosarcoma with or without a measurable lesion postoperatively, and patients with stage IIIC2 or IV disease with or without a measurable lesion postoperatively. For pembrolizumab this means: patients with EC recurrence (except carcinosarcoma) who will not undergo surgery or radiotherapy, and patients with stage III or IVA disease (except carcinosarcoma) and a measurable lesion postoperatively or with stage IVB disease with or without a measurable lesion.

Survival of Patients with Deficient Mismatch Repair Versus Proficient Mismatch Repair Metastatic Colorectal Cancer Receiving Curative-Intent Local Treatment of Metastases in a Nationwide Cohort

BackgroundIt is unclear whether curative-intent local therapy of metastases is of similar benefit for the biological distinct subgroup of patients with deficient mismatch repair (dMMR) metastatic colorectal cancer (mCRC) compared with proficient mismatch repair (pMMR) mCRC.Patients and MethodsIn this nationwide study, recurrence-free (RFS) and overall survival (OS) were analyzed in patients with dMMR versus pMMR mCRC who underwent curative-intent local treatment of metastases between 2015 and 2018. Subgroup analyses were performed for resection of colorectal liver metastases (CRLM) and cytoreductive surgery ± hyperthermic intraperitoneal chemotherapy (CRS ± HIPEC). Multivariable regression was conducted.ResultsMedian RFS was 11.1 months [95% confidence interval (CI) 8.5–41.1 months] for patients with dMMR tumors compared with 8.9 months (95% CI 8.1–9.8 months) for pMMR tumors. Two-year RFS was higher in patients with dMMR versus pMMR (43% vs. 21%). Results were similar within subgroups of local treatment (CRLM and CRS ± HIPEC). Characteristics differed significantly between patients with dMMR and pMMR mCRC; however, multivariable analysis continued to demonstrate dMMR as independent factor for improved RFS [hazard ratio (HR): 0.57, 95% CI 0.38–0.87]. Median OS was 33.3 months for dMMR mCRC compared with 43.5 months for pMMR mCRC, mainly due to poor survival of patients with dMMR in cases of recurrence in the preimmunotherapy era.ConclusionPatients with dMMR eligible for curative-intent local treatment of metastases showed a comparable to more favorable RFS compared with patients with pMMR, with a clinically relevant proportion of patients remaining free of recurrence. This supports local treatment as a valuable treatment option in patients with dMMR mCRC and can aid in shared decision-making regarding upfront local therapy versus immunotherapy.

The impact of mismatch repair status and systemic inflammatory markers on radiological staging in colon cancer.

Mismatch repair (MMR) deficient (dMMR) colon cancer (CC) is distinct from MMR proficient (pMMR) CC, yet the impact of MMR status on radiological staging is unclear. The purpose of this study was to investigate how MMR status impacts CC CT staging. We retrospectively compared CT staging accuracy between dMMR and pMMR CC patients undergoing curative resection. Accuracy was assessed as individual tumour (T)/nodal (N) stages and as dichotomous "statuses" (T1/2 vs T3/4; N0 vs N1/2). Patient characteristics were analysed for factors to support staging. There was no significant difference in overall staging accuracy between the dMMR (44 patients) and pMMR (57 patients) groups. dMMR tumours with incorrect N stage/"status" were more likely to be overstaged than pMMR tumours (90% vs 59%; p = 0.023 for "N status"). Platelet count, CRP and neutrophil count (AUC 0.76 (p = 0.0078), 0.75 (p = 0.034) and 0.70 (p = 0.044), respectively) were associated with "N status" in dMMR tumours. Whilst overall staging accuracy was similar between groups, incorrectly N staged dMMR tumours were more likely to be overstaged than pMMR tumours, risking inappropriate surgical or neoadjuvant treatment. We describe novel relationships between several inflammatory markers and pathological "N status" in dMMR CC, which if integrated into routine practice may improve CT staging accuracy. Compared to pMMR CC, dMMR CC is at significant risk of N overstaging. Platelet count, CRP and neutrophil count are higher in dMMR CC patients with nodal metastases than those without, and their role in refining clinical staging requires further investigation.

Prognostic value of Lynch syndrome, BRAFV600E , and RAS mutational status in dMMR/MSI-H metastatic colorectal cancer in a pooled analysis of Dutch and French cohorts.

Current knowledge on prognostic biomarkers (especially BRAFV600E /RAS mutations) in metastatic colorectal cancer (mCRC) is mainly based on mCRC patients with proficient mismatch repair (pMMR) tumors. It is uncertain whether these biomarkers have the same prognostic value in mCRC patients with deficient mismatch repair (dMMR) tumors. This observational cohort study combined a population-based Dutch cohort (2014-2019) and a large French multicenter cohort (2007-2017). All mCRC patients with a histologically proven dMMR tumor were included. In our real-world data cohort of 707 dMMR mCRC patients, 438 patients were treated with first-line palliative systemic chemotherapy. Mean age of first-line treated patients was 61.9 years, 49% were male, and 40% had Lynch syndrome. BRAFV600E mutation was present in 47% of tumors and 30% harbored a RAS mutation. Multivariable regression analysis on OS showed significant hazard rates (HR) for known prognostic factors as age and performance status, however showed no significance for Lynch syndrome (HR: 1.07, 95% CI: 0.66-1.72), BRAFV600E mutational status (HR: 1.02, 95% CI: 0.67-1.54), and RAS mutational status (HR: 1.01, 95% CI: 0.64-1.59), with similar results for PFS. BRAFV600E and RAS mutational status are not associated with prognosis in dMMR mCRC patients, in contrast to pMMR mCRC patients. Lynch syndrome is also not an independent prognostic factor for survival. These findings underline that prognostic factors of patients with dMMR mCRC are different of those with pMMR, which could be taken into consideration when prognosis is used for clinical decision-making in dMMR mCRC patients and underline the complex heterogeneity of mCRC.

Artificial intelligence–derived immune phenotypes for prediction of prognosis in patients with stage III colon cancer (NCCTG N0147; Alliance).

3542 Background: Solid tumors can be characterized by distinct immune phenotypes based on the level of T cell infiltration in the tumor microenvironment (TME) which may provide prognostic information and also inform strategies to restore the anti-tumor immune response. Immune phenotypes were determined using an artificial intelligence (AI) algorithm in digitized whole-slide images (WSI) of tumors with deficient (d) mismatch repair (MMR) vs proficient (p) MMR. Methods: Stage III colon carcinomas (N = 401; 393 met QC) from participants in a phase III trial of FOLFOX-based adjuvant chemotherapy were analyzed including all available tumors with dMMR (n = 196) and a randomly selected cohort of pMMR tumors (n = 195). Using an AI algorithm (Lunit SCOPE) previously trained on solid tumors, digitized tumors were categorized into three immune phenotypes based on epithelial and stromal TIL data-driven cutpoints in dMMR and pMMR (inflamed: TIL high in epithelium (> upper 25%); excluded: TIL high in stroma (>lower 25%), low in epithelium; and desert: TIL low in epithelium and stroma). Phenotypes were then examined in relationship to disease-free survival (DFS) using Kaplan-Meier methodology. Results: Of the 3 immune phenotypes in dMMR tumors, 25% were inflamed, 50% were immune-excluded, and 25% were immune-desert. Based on univariate results, multivariable modeling was performed incorporating immune phenotype in addition to age, histological grade, N stage, T stage, performance status, treatment arm, BRAF and KRAS status and identified immune phenotypes and N stage to be significantly associated with DFS. Among dMMR tumors, immune desert phenotype was associated with the poorest DFS (Desert: HR adj 1.95, 95%CI (1.01, 3.80); Excluded: HR adj 0.89, 95%CI (0.47, 1.69); Inflamed: ref; p:0.01). When compared to immune desert tumors, immune-excluded tumors had significantly better DFS (HR adj 0.49, 95%CI (0.27, 0.88), p:0.01). At a median follow-up of 60 months, 3-year DFS of patients with dMMR was 71.2% with Immune inflamed, 78.5% with Excluded, and 54.5% with Desert phenotypes. In contrast to dMMR, univariate analyses of data-driven cutpoints in pMMR tumors was not prognostic. Using a different data-driven cutpoint (15%), the revised immune phenotypes remained non-significant for DFS within pMMR tumors. Conclusions: Distinct AI-derived immune phenotypes in the TME were identified that were significantly prognostic in patients with dMMR, but not pMMR colon cancers. A data-driven immune-desert phenotype was identified in dMMR tumors that was associated with significantly poorer survival. Further investigation of the potential predictive utility of these phenotypes for immunotherapy are planned. Support: https://acknowledgments.alliancefound.org . ClinicalTrials.gov Identifier: NCT00079274 .

Pathological analysis of tumor microenvironmental features using deep learning to predict survival of colon carcinomas.

3527 Background: Advances in deep learning may improve the ability to evaluate and quantify pathological features in solid tumors to improve prediction of patient disease-free survival (DFS). We used an artificial intelligence (AI) algorithm to quantify morphological features in the tumor microenvironment of stage III colon cancers. Methods: We analyzed digitized stage III colon carcinomas (N = 402; 382 met QC) from participants in a phase III trial of FOLFOX-based adjuvant chemotherapy that included all available tumors with dMMR and a randomly selected cohort of pMMR tumors (median follow-up 60 months) [NCCTG N0147; Alliance for Clinical Trials in Oncology]. Fifteen morphological features were extracted using the QuantCRC algorithm that segments images into carcinoma (low-grade, high-grade, signet ring cell), stroma (immature, mature, and inflammatory), mucin, tumor/stroma ratio, tumor budding/poorly differentiated clusters (TB/PDC), necrosis, smooth muscle, fat, and tumor-infiltrating lymphocytes (TILs) and tumor area (mucin, epithelium and TB/PDC). Analysis of AI-derived features with clinical variables, molecular alterations ( BRAF/KRAS), and DFS were examined using Kaplan-Meier methodology and multivariable Cox regression. Results: Among the 15 AI-derived morphological features, the following differed significantly between dMMR (n = 191) and pMMR (n = 189) whereby dMMR tumors had lower mature stroma, but higher inflammatory stroma and tumor, higher tumor grade and increased mucin, TB/PDCs, signet ring cells, and TILs (all p< 0.05). Among dMMR tumors, multivariable analysis revealed that tumor area [mucin, epithelium and TB/PDC] (HR adj 0.45, 95%CI (0.24, 0.84), p:0.01, [Q2/3 vs Q1]) and N stage were the only significant and independent prognostic variables. Among pMMR tumors, multivariable analysis identified that tumor budding/poorly differentiated clusters (TB/PDCs) (HR adj 0.20, 95%CI (0.06, 0.61), p:0.01, [Q1 vs Q4]) was the strongest prognostic variable and the only morphological feature that was significantly associated with DFS along with age, N stage and T stage. Conclusions: Using AI, we can extract and quantify distinct morphological features in tumor sections that differ between dMMR and pMMR and multivariately, can significantly and robustly enhance prognostication within each MMR group. Among pMMR tumors, tumor budding/poorly differentiated clusters was the strongest predictor of DFS. Support: NIH U10CA180821, U10CA180882, U24CA196171, R01 CA210509 (to FAS). Study NCCTG N0147 received funds from Sanofi https://acknowledgments.alliancefound.org . ClinicalTrials.gov Identifier: NCT00079274 Key words Artificial Intelligence; Tumor Microenvironment; Colonic Neoplasms; Disease-free survival.

FOLFOXIRI plus bevacizumab and atezolizumab as upfront treatment of unresectable metastatic colorectal cancer (mCRC): Updated and overall survival results of the phase II randomized AtezoTRIBE study.

3500 Background: AtezoTRIBE (NCT03721653) is a phase II randomized trial in which unresectable mCRC pts were randomized 1:2 to 1st-line FOLFOXIRI/bev [arm A] or FOLFOXIRI/bev/atezo [arm B]. Adding atezo to FOLFOXIRI/bev was safe and improved PFS (primary endpoint), with a modest benefit also among pts with pMMR tumors. Subgroup analyses suggest that TMB and Immunoscore IC (IS IC) -an IHC biomarker measuring CD8 and PD-L1 cell densities and their proximity- may identify pts with pMMR tumors deriving benefit from adding atezo to FOLFOXIRI/bev. Methods: The study had 85% power to detect a HR for PFS (time from randomization to 1st PD or death [PD1]) of .66 in favor of arm B with 1-sided α error of .10. Secondary endpoints included PFS2 (time from randomization to PD on any treatment given after PD1 or death [PD2]), 2nd PFS (time from PD1 to PD2), and OS. MMR, TMB, IS IC were correlated to clinical outcome. Results: 218 pts (arm A/B:73/145) were enrolled. Main pts’ characteristics were right-sided 44%/45%, RAS mut 71%/74%, BRAF mut 14%/8%, dMMR 7%/6%, high TMB 10%/12%, high IS IC 32%/32%. At a median follow-up of 37.0 mos, 175 (80%, arm A/B: 64/111) PD1, 150 (69%, arm A/B: 53/97) PD2, and 118 (54%, arm A/B: 43/75) OS events were collected. Out of 175 pts with a PD1 event, 135 (77%, arm A/B:50/85) received a subsequent treatment; among them, 121 pts (arm A/B: 43/78) had a PD2 event. PFS, PFS2, 2nd PFS and OS results in the intention-to-treat (ITT) population and the pMMR group are listed in the Table. In the ITT population, significant interactions between treatment and MMR status (Pint .011), TMB (Pint .008), and IS IC (Pint .037) were reported in terms of PFS. Only IS IC was associated with a differential OS benefit (Pint .065), with pts bearing IS IC-high tumors deriving benefit from adding atezo (HR 0.43, 95%CI 0.19-1.00), differently than those with IS IC-low tumors (HR 1.09, 95%CI 0.65-1.83). In the pMMR group, significant interactions between treatment and TMB and IS IC were reported in terms of PFS (Pint .016 and .051, respectively) and OS (Pint .043 and .063, respectively). Pts bearing IS IC-high tumors derived higher OS benefit from adding atezo (HR 0.44, 95%CI 0.19-1.03), than those with IS IC-low tumors (HR 1.15, 95% CI 0.67-1.97). Conclusions: Pts with IS IC-high and/or TMB high pMMR mCRC seem to derive a survival benefit from adding atezo to FOLFOXIRI/bev as upfront treatment. These findings deserve confirmation in a properly designed phase III trial. Clinical trial information: NCT03721653 . [Table: see text]

Modified FOLFOXIRI plus cetuximab and avelumab as initial therapy in RAS wild-type unresectable metastatic colorectal cancer: Results of the phase II AVETRIC trial by GONO.

3575 Background: The modified schedule of FOLFOXIRI (mFOLFOXIRI) in combination with an anti-EGFR agent showed a manageable safety profile and remarkable activity in RAS wild-type (wt) metastatic colorectal cancer (mCRC). The association of an active cytotoxic regimen with cetuximab (cet) may increase the exposure of tumour-associated neoantigens and induce immunogenic cell death and antibody-dependent cell-mediated cytotoxicity thus enabling the effect of immune checkpoint inhibitors. The AVETRIC study aimed at exploring the efficacy and safety of first-line mFOLFOXIRI plus cet and avelumab (ave) in RAS wt mCRC patients (pts). Methods: AVETRIC is a prospective, open label, multicenter, phase II, single arm trial in which initially unresectable and previously untreated RAS wt mCRC pts received mFOLFOXIRI (irinotecan 150 mg/sqm, oxaliplatin 85 mg/sqm, folinate 200 mg/sqm leucovorin [LV], and 5-fluorouracil [5FU] 2400 mg/sqm) plus cet (500 mg/sqm) and ave (800 mg) every 2 weeks up to 12 cycles followed by maintenance with 5FU/LV plus cet and ave until disease progression. A safety run-in phase including the first 6 enrolled pts was planned. Due to the occurrence of grade 3-4 diarrhoea in 2 (33%) pts, the protocol study was amended to reduce the irinotecan dose to 130 mg/sqm. Primary endpoint was Progression Free Survival (PFS). Fifty-eight pts were needed to detect an increase in median PFS (mPFS) from 10.0 to 19.4 months (mos), setting one-sided α and β errors at 0.05 and 0.10, respectively. The trial is registered at Clinicaltrial.gov, NCT04513951. Results: Between Jun 2020 and Dec 2021, 62 pts were enrolled in 16 Italian centres. Main pts' characteristics were: median age 56 yrs, ECOG PS 0 87%, synchronous metastases 94%, liver-only disease 42%, left-sided primary tumour 89%; all pts had BRAF wt and proficient MMR (pMMR) tumours. The primary endpoint was met. At a median follow-up of 16.0 months, 39 (63%) events were recorded and mPFS was 14.1 months (90% CI 12.0-16.7, Brookmeyer-Crowley test p < 0.001). Response rate and disease control rate were 82% and 98%, respectively, and R0 resection rate was 21% (27% in liver-only subgroup). Early tumour shrinkage was achieved in 74% pts and median deepness of response was 56%. In pts treated after study amendment (n = 56), main grade 3-4 adverse events were neutropenia (27%), diarrhoea (27%), skin rash (14%), asthenia (14%), nausea (11%), stomatitis (7%), febrile neutropenia (2%). Grade 3-4 immune-related adverse events occurred in 6% of pts. Overall survival results are still immature. Conclusions: AVETRIC study met its primary endpoint, showing that combining mFOLFOXIRI plus cet and ave achieves promising results in terms of PFS as well as response rate, in pts with pMMR RAS and BRAF wt mCRC. Translational analyses to evaluate the impact of immune-related biomarkers are ongoing. Clinical trial information: NCT04513951 .

Gut microbiome composition as predictor of the efficacy of adding atezolizumab to first-line FOLFOXIRI plus bevacizumab in metastatic colorectal cancer: A translational analysis of the AtezoTRIBE study.

3534 Background: Gut microbiome has emerged as a biomarker of clinical benefit to immune-checkpoint inhibitors (ICI) but no data are available in metastatic colorectal cancer (mCRC). The AtezoTRIBE study demonstrated that the addition of atezolizumab (atezo) to FOLFOXIRI plus bevacizumab (bev) prolongs progression-free survival (PFS), but this benefit is limited for patients with proficient mismatch repair (pMMR) tumors. Here, we aimed at investigating the potential predictive role of microbiome in identifying mCRC patients able to achieve benefit from ICI. Methods: AtezoTRIBE was a phase II trial in which 218 mCRC patients, unselected for MMR status, were randomized 1:2 to receive first-line FOLFOXIRI/bev (arm A) or FOLFOXIRI/bev/atezo (arm B). Stools were prospectively collected. Metagenomic (MG) data from whole genome sequencing (WGS) at level of species genome bins (SGBs) were analysed by linear models corrected for clinical and tumor-related parameters and fold-ratios. We defined as responders (R) those patients who experienced a PFS ≥ 12 months. Results: Stool samples were collected at baseline for 171 (78%) patients (55 in arm A and 116 in arm B) but only 163 were available for MG. Patients with deficient MMR (dMMR) tumors (N = 10) showed significantly lower MG diversity than pMMR ones (N = 148) harboring oral bacteria and pathobionts whose intrinsic immunogenicity has not been demonstrated. Regarding pMMR mCRC patients, baseline microbiome composition was not significantly different according to the treatment arm. The microbiome diversity was not significantly different between R and not-R in both arms, but specific immunogenic SGBs (Lachnospiraceae family members) were over-represented in R treated in arm B. Veillonellaceae and pathobionts were associated with poor prognosis and/or differential benefit from the addition of atezo. Fusobacterium nucleatum was associated with a poor prognosis, also in arm B. Conclusions: This is the largest prospective analysis showing that SGBs may be useful as a biomarker of potential benefit or detrimental effect from atezo in pMMR mCRC patients. Our results prompt the design of microbiota-centered diagnostic tests to identify pMMR mCRC patients more likely to benefit from ICI-based therapeutic strategies. Clinical trial information: NCT03721653 .

KRT17 Promotes T-lymphocyte Infiltration Through the YTHDF2-CXCL10 Axis in Colorectal Cancer.

Poor infiltration of T lymphocytes has been regarded as a crucial mechanism of tumor immune escape. Here, we demonstrate a protective role of KRT17 in colorectal cancer, where KRT17 reversed the tumor immunosuppressive microenvironment by increasing T-lymphocyte infiltration. High-throughput RNA sequencing suggested that KRT17 was significantly upregulated in deficient mismatch repair (dMMR) tumors compared with proficient mismatch repair (pMMR) tumors. In a colorectal cancer cohort of 446 cases, KRT17 expression positively correlated with better clinical outcomes. Krt17 overexpression decreased xenograft tumor growth in immune-competent mice. T-cell depletion in a murine model showed that the presence of T lymphocytes was necessary for Krt17-mediated disruption of tumorigenesis. Mass spectrometry and coimmunoprecipitation assays suggested KRT17 caused YTHDF2 degradation through the ubiquitin-proteasome system. Through high-throughput RNA immunoprecipitation sequencing, we found that CXCL10 was the target gene of the N6-methyladenosine (m6A) "reader" YTHDF2. KRT17 synergized with anti-PD-1 for better tumor control in an immunotherapy-resistant murine model. In a cohort of patients with colorectal cancer receiving pembrolizumab, high KRT17 expression was found within the tumors of responders. Collectively, we elucidated a critical role of KRT17 in colorectal cancer to prevent immune escape. These findings present new insights into potential therapeutic strategies and effective markers of immunotherapy reactivity against pMMR tumors.

BRD4 inhibition impairs DNA mismatch repair, induces mismatch repair mutation signatures and creates therapeutic vulnerability to immune checkpoint blockade in MMR-proficient tumors

BackgroundMismatch repair deficiency (dMMR) is a well-recognized biomarker for response to immune checkpoint blockade (ICB). Strategies to convert MMR-proficient (pMMR) to dMMR phenotype with the goal of sensitizing tumors to...