Gut-microbiota modulation shows promise in improving immune-checkpoint blockade (ICB) response; however, precision biomarker-driven, placebo-controlled trials are lacking. We performed a multicenter, randomized placebo-controlled, biomarker-stratified phase I trial in patients with ICB-naïve metastatic melanoma using SER-401, an orally delivered Firmicutesenriched spore formulation. Fecal microbiota signatures were characterized at baseline; patients were stratified by high versus low Ruminococcaceae abundance prior to randomization to the SER-401 arm (oral vancomycin-preconditioning/SER-401 alone/nivolumab + SER-401), versus the placebo arm [placebo antibiotic/placebo microbiome modulation (PMM)/nivolumab + PMM (NCT03817125)]. Analysis of 14 accrued patients demonstrated that treatment with SER-401 + nivolumab was safe, with an overall response rate of 25% in the SER-401 arm and 67% in the placebo arm (though the study was underpowered related to poor accrual during the COVID-19 pandemic). Translational analyses demonstrated that vancomycin preconditioning was associated with the disruption of the gut microbiota and impaired immunity, with incomplete recovery at ICB administration (particularly in patients with high baseline Ruminococcaceae). These results have important implications for future microbiome modulation trials. Significance: This first-of-its-kind, placebo-controlled, randomized biomarker-driven microbiome modulation trial demonstrated that vancomycin + SER-401 and anti–PD-1 are safe in melanoma patients. Although limited by poor accrual during the pandemic, important insights were gained via translational analyses, suggesting that antibiotic preconditioning and interventional drug dosing regimens should be carefully considered when designing such trials.

Purpose: To characterize changes in the soft-tissue sarcoma (STS) tumor immune microenvironment induced by standard neoadjuvant therapy with the goal of informing neoadjuvant immunotherapy trial design. Experimental Design: Paired pre- and postneoadjuvant therapy specimens were retrospectively identified for 32 patients with STSs and analyzed by three modalities: multiplexed IHC, NanoString, and RNA sequencing with ImmunoPrism analysis. Results: All 32 patients, representing a variety of STS histologic subtypes, received neoadjuvant radiotherapy and 21 (66%) received chemotherapy prior to radiotherapy. The most prevalent immune cells in the tumor before neoadjuvant therapy were myeloid cells (45% of all immune cells) and B cells (37%), with T (13%) and natural killer (NK) cells (5%) also present. Neoadjuvant therapy significantly increased the total immune cells infiltrating the tumors across all histologic subtypes for patients receiving neoadjuvant radiotherapy with or without chemotherapy. An increase in the percentage of monocytes and macrophages, particularly M2 macrophages, B cells, and CD4+ T cells was observed postneoadjuvant therapy. Upregulation of genes and cytokines associated with antigen presentation was also observed, and a favorable pathologic response (≥90% necrosis postneoadjuvant therapy) was associated with an increase in monocytic infiltrate. Upregulation of the T-cell checkpoint TIM3 and downregulation of OX40 were observed posttreatment. Conclusions: Standard neoadjuvant therapy induces both immunostimulatory and immunosuppressive effects within a complex sarcoma microenvironment dominated by myeloid and B cells. This work informs ongoing efforts to incorporate immune checkpoint inhibitors and novel immunotherapies into the neoadjuvant setting for STSs.

Therapies targeting the RAF–MEK–ERK pathway are generally considered to have limited efficacy in KRAS-mutant cancers. However, specific KRAS mutants exhibit distinct behaviors. Notably, KRASG12R pancreatic ductal adenocarcinoma (PDAC) tumors have shown sensitivity to MEK inhibitors (MEKi) in combination with autophagy inhibitors, but a better understanding of the underlying mechanisms is needed to optimize this treatment strategy. Using a systems-level approach, we uncovered a mechanistic explanation for this phenomenon. Due to distinct biophysical properties, KRASG12R had an impaired ability to activate wild-type HRAS and NRAS (WT-RAS) compared with other KRAS mutants, such as KRASG12D. This reduced activation stemmed from the weaker interaction between KRASG12R and guanine exchange factors (SOS), as well as the tumor suppressor neurofibromin (NF1), crucial in regulating WT-RAS activity. The impaired ability to activate WT-RAS led to weaker holistic MAPK signaling in KRASG12R-driven tumors, which conferred increased sensitivity to MEKi. To substantiate the preclinical findings, the utility of MEKi in combination with the autophagy inhibitor hydroxychloroquine was analyzed in patients with KRASG12R-mutated metastatic PDAC. Five of the eight (62.5%) patients treated in first- or second-line settings had a progression-free survival exceeding 6 months. Three patients had impressive disease control: two had stable disease of 11 and 22.7 months, and one achieved a partial response with an 83% decrease in tumor size that lasted for 8.9 months. Overall, this work highlights how systems-based approaches in precision medicine can uncover mechanistic insights to guide the identification of patients with PDAC most likely to benefit from tailored therapeutic strategies. Significance: The unique sensitivity of KRASG12R-mutant cancers to MEK inhibitors offers a critical advancement in understanding MAPK signaling and paving the way for precision-targeted therapies in previously untreatable contexts. See related commentary by Tiriac and Engle, p. 1817 See related article by Burge et al., p. 1854 See related article by Burge et al., p. 1868

Background: Biliary tract cancers (BTCs) have distinct tumor biology but share a poor prognosis, with a 5-year-survival-rate of 5–19%. Surgical resection is the only potential cure, but recurrences are common. The role of adjuvant radiotherapy (XRT) remains unclear. Methods: Using the National Cancer Database (2006–2018), we analyzed resected non-metastatic BTCs. Patients who survived beyond 90 days post-surgery were included, while those with R2 resections or neoadjuvant therapy were excluded. Propensity matching was performed based on predictors of adjuvant radiation, age, and sex. Survival outcomes were compared between no adjuvant therapy, chemotherapy alone, and XRT ± chemotherapy. Results: Among 21,275 patients, including 5308 intrahepatic cholangiocarcinoma (IHC), 2689 perihilar cholangiocarcinoma (PHC), 3092 distal cholangiocarcinoma (DCC), and 10,186 gallbladder cancer (GBC) cases, adjuvant XRT did not improve survival for IHC. For PHC and DCC, XRT improved survival over no adjuvant therapy (PHC: 31.2 vs. 26.3 months, p = 0.004; DCC: 33.7 vs. 27.0 months, p = 0.015) but not over chemotherapy alone. For GBC, XRT significantly improved survival compared to both no adjuvant therapy and chemotherapy (30.2 vs. 26.6 and 24.6 months; p = 0.05 and p = 0.001). Conclusions: XRT provides a survival benefit for GBC, especially in node-positive and R1-resected patients. For PHC and DCC, XRT improves outcomes compared to no therapy, but its benefit over chemotherapy is uncertain. No benefit was observed for IHC.