Background Several studies have established a correlation between the VEGF–VEGFR2 axis and an immunosuppressive microenvironment; this immunosuppression can be overcome by anti-angiogenic reagents, such as ramucirumab (RAM). However, little is known about the immunological impact of anti-angiogenic reagents within the tumor microenvironment in human clinical samples. This study aimed at investigating the effects of RAM on the tumor microenvironmental immune status in human cancers. Methods We prospectively enrolled 20 patients with advanced gastric cancer (GC) who received RAM-containing chemotherapy. We obtained paired samples from peripheral blood mononuclear cells (PBMCs) and tumor-infiltrating lymphocytes (TILs) in primary tumors both pre- and post-RAM therapy to assess immune profiles by immunohistochemistry and flow cytometry. Results Within the tumor microenvironment, both PD-L1 expression and CD8⁺ T-cell infiltration increased after RAM-containing therapies. In addition, CD45RA⁻FOXP3^highCD4⁺ cells (effector regulatory T cells [eTreg cells]) and PD-1 expression by CD8⁺ T cells were significantly reduced in TILs compared with PBMCs after RAM-containing therapies. Patients with partial response and longer progression-free survival had significantly higher pre-treatment eTreg frequencies in TILs than those with progressive disease. In in vitro analysis, VEGFR2 was highly expressed by eTreg cells. Further, VEGFA promoted VEGFR2⁺ eTreg cell proliferation, and this effect could be inhibited by RAM. Conclusions This study suggests that the frequency of eTreg cells in TILs could be a biomarker for stratifying clinical responses to RAM-containing therapies. Further, we propose that RAM may be employed as an immuno-modulator in combination with immune checkpoint blockade.

Background Clinicopathological and molecular features of responders to nivolumab for advanced gastric cancer (AGC) are not well understood. Methods Patients (pts) with AGC who were treated with nivolumab after two or more chemotherapy regimens in a single institution from September 2017 to May 2018 were enrolled in this study. PD-L1 expression in tumor cells (TC) and mismatch repair (MMR) were analyzed by immunohistochemistry. Epstein-Barr virus (EBV) was detected by in situ hybridization. Cancer genome alterations were evaluated by a next-generation sequencing-based panel. High tumor mutation burden (TMB) was defined as more than 10 mutations/megabase. Results A total of 80 pts were analyzed in this study. Tumor response was evaluated in 72 pts with measurable lesions and 14 pts (19%) had an objective response. Overall response rate (ORR) was significantly higher in pts with ECOGPS 0 in those with PS 1 or 2, MMR-deficient (MMR-D) in those with MMR-proficient (MMR-P), PD-L1+ in TC in those with PD-L1- in TC and PIK3CA mutation in those with PIK3CA wild-type. ORR was 31% in pts with at least one of the following factors; MMR-D, high TMB, EBV+ and PD-L1+ in TC vs. 0% in those without these factors. Progression-free survival was significantly longer in pts with PS 0 than in those with PS 1 or 2, MMR-D than in those with MMR-P, and PD-L1+ in TC than in those with PD-L1- in TC. Conclusions Some features were associated with favorable response to nivolumab for AGC. Combining these features might be useful to predict efficacy.

Purpose: This study performed a comprehensive molecular characterization of microsatellite instability–high (MSI-H)/mismatch repair–deficient (dMMR) gastrointestinal (GI) tumors to elucidate predictors of response to PD-1 blockade. Experimental Design: Forty-five patients with MSI-H/dMMR GI tumors, including gastric cancer, colorectal cancer, cholangiocarcinoma, small intestine cancer, pancreatic cancer, and duodenal cancer, receiving PD-1 blockade were analyzed. We conducted the genomic profiling of GI tumors by whole-exome sequencing or targeted next-generation sequencing. The tumor microenvironment was evaluated by transcriptomic analysis and multiplex fluorescence IHC. Results: Patients with low tumor mutational burdens (TMBs) had lower objective response rates (ORRs; 0% vs. 48.8%) and a significantly shorter progression-free survival (PFS; 2.3 vs. 15.6 months; HR, 6.20; P = 0.002) than those with high TMBs. Among common gene alterations in GI tumors, only PTEN mutations, which were mutually exclusive with a low TMB, were significantly associated with a lower ORRs than wild-type PTEN (21.4 vs. 54.8%; odds, 4.45; P = 0.045). Compared with wild-type PTEN, PTEN mutations in the phosphatase domain were associated with significantly lower ORRs (12.5 vs. 54.8%; P = 0.049), shorter PFS (2.6 vs. 15.6 months; HR, 5.04; P < 0.001), lower intratumoral CD8+ T-cell levels, higher intratumoral CD204+ macrophage levels, and PI3K/AKT/mTOR pathway enrichment, whereas PTEN mutations in the C2 domain were not. Conclusions: Low TMBs and PTEN mutations, especially mutations in the phosphatase domain associated with an immunosuppressive environment, were mutually exclusive and might be negative predictors of PD-1 blockade responses in patients with MSI-H/dMMR GI tumors.

Purpose: This is a phase I/II trial to assess the efficacy and safety of napabucasin plus pembrolizumab for metastatic colorectal cancer (mCRC). Patients and Methods: Phase I was conducted to determine the recommended phase 2 dose (RP2D) in a dose escalation design of napabucasin (240 to 480 mg twice daily) with 200 mg pembrolizumab every 3 weeks. Phase II included cohort A (n = 10, microsatellite instability high, MSI-H) and cohort B (n = 40, microsatellite stable, MSS). The primary endpoint was immune-related objective response rate (irORR). PD-L1 combined positive score (CPS), genomic profiles, and the consensus molecular subtypes (CMS) of colorectal cancer were assessed. Results: A total of 55 patients were enrolled in this study. In phase I, no patients experienced dose-limiting toxicities, and napabucasin 480 mg was determined as RP2D. The irORR was 50.0% in cohort A and 10.0% in cohort B. In cohort B, the irORR was 0%, 5.3%, and 42.9% in CPS < 1, 1≤ CPS <10, and CPS ≥ 10, respectively. Patients with objective response tended to have higher tumor mutation burden than those without. Of evaluable 18 patients for CMS classification in cohort B, the irORR was 33.3%, 0%, 33.3%, and 33.3% in CMS1, CMS2, CMS3, and CMS4, respectively. The common grade 3 or higher treatment-related adverse events included fever (10.0%) in cohort A and decreased appetite (7.5%) and diarrhea (5.0%) in cohort B. Conclusions: Napabucasin with pembrolizumab showed antitumor activity with acceptable toxicities for patients with MSS mCRC as well as MSI-H mCRC, although it did not meet the primary end point. The impact of related biomarkers on the efficacy warrants further investigations in the additional cohort. See related commentary by Nusrat, p. 5775

Purpose: We evaluated the association between molecular subtypes of advanced gastric cancer (AGC) and the efficacy of standard chemotherapy or immune checkpoint inhibitors. Experimental Design: Patients with AGC who received systemic chemotherapy from October 2015 to July 2018 with available molecular features were analyzed. We investigated the efficacy of standard first- (fluoropyrimidine + platinum ± trastuzumab) and second-line (taxanes ± ramucirumab) chemotherapy, and subsequent anti–PD-1 therapy in patients with four molecular subtypes: MMR-D (mismatch repair deficient), EBV+, HER2+, and all negative. Results: 410 patients were analyzed: MMR-D 5.9%, EBV+ 4.1%, HER2+ 13.7%, and all negative 76.3%. In 285 patients who received standard first-line chemotherapy, the median progression-free survival (PFS) times were 4.2, 6.0, 7.5, and 7.6 months and the objective response rates (ORR) were 31%, 62%, 60%, and 49% in MMR-D, EBV+, HER2+, and all-negative subtypes, respectively. Multivariate analysis showed shorter PFS in MMR-D versus all-negative patients [HR, 1.97; 95% CIs, 1.09–3.53; P = 0.022]. In second-line setting, there were no significant differences in efficacy. In 110 patients who received anti–PD-1 therapy, median PFS times were 13.0, 3.7, 1.6, and 1.9 months and the ORRs were 58%, 33%, 7%, and 13%, respectively. Twelve patients with MMR-D received subsequent anti–PD-1 therapy and showed longer PFS compared with that in 10 (83%) patients who received earlier-line chemotherapy. Conclusions: MMR-D might result in shorter PFS with first-line chemotherapy for AGC. Subsequent anti–PD-1 therapy achieved higher ORR and longer PFS than prior chemotherapy in most patients with MMR-D, supporting the earlier use of immune checkpoint inhibitors.