Both targeted therapies and immunotherapies provide benefit in resected Stage III melanoma. We hypothesized that the combination of targeted and immunotherapy given prior to therapeutic lymph node dissection (TLND) would be tolerable and drive robust pathologic responses. In NeoACTIVATE (NCT03554083), a Phase II trial, patients with clinically evident resectable Stage III melanoma received either 12 weeks of neoadjuvant vemurafenib, cobimetinib, and atezolizumab ( BRAF -mutated, Cohort A, n = 15), or cobimetinib and atezolizumab ( BRAF -wild-type, Cohort B, n = 15) followed by TLND and 24 weeks of adjuvant atezolizumab. Here, we report outcomes from the neoadjuvant portion of the trial. Based on intent to treat analysis, pathologic response (≤50% viable tumor) and major pathologic response (complete or near-complete, ≤10% viable tumor) were observed in 86.7% and 66.7% of BRAF -mutated and 53.3% and 33.3% of BRAF -wild-type patients, respectively (primary outcome); these exceeded pre-specified benchmarks of 50% and 30% for major pathologic response. Grade 3 and higher toxicities, primarily dermatologic, occurred in 63% during neoadjuvant treatment (secondary outcome). No surgical delays nor progression to regional unresectability occurred (secondary outcome). Peripheral blood CD8 + T CM cell expansion associated with favorable pathologic responses (exploratory outcome).

Supplementary Figure 1. Gut microbial composition and diversity in patients with and without adenomas. A) Microbial composition was similar between groups across all taxonomic levels. The groups did not differ in terms of α-diversity: B) observed number of operational taxonomic units (OTUs) or C) the Shannon diversity index. The groups did not cluster by β-diversity: D) unweighted and E) weighted UniFrac distance metrics.

INTRODUCTION: The development of Clostridioides difficile infection (CDI) and response to treatment has previously been associated with fecal microbiome. The role of small intestinal microbiome in CDI remains unclear and as C. difficile primarily colonizes the colon, it is unclear if the small intestinal microbiota is affected by CDI. In this study we investigated changes in the small intestinal microbiota composition potentially related to CDI. METHODS: Duodenal aspirates were collected from patients with gastrointestinal symptoms being tested for small intestinal bacterial overgrowth (SIBO). A presumptive diagnosis of SIBO was made by aspirate culture ≥105 CFU/mL. Microbial DNA extraction from aspirates was done using phenol-chloroform and the bacterial 16S rRNA gene V4 region was amplified and sequenced using the MiSeq Illumina platform. Preprocessed reads were analyzed by closed reference picking at 97 percent identity. RESULTS: We included 126 individuals (mean age = 51.6 (SD = 17.8) years, 79.4% female, mean BMI = 27.2 (SD = 6.6)) for whom small bowel aspirates were available. The clinical indications for collection of aspirates were diarrhea (40.5%), abdominal pain (31.0%), bloating (15.1%), nausea/vomiting (0.6%), and weight loss (0.1%). Among the 126 individuals, 12 patients had history of CDI prior to collection of aspirates (mean time after CDI = 22.2 months, range = 6.8-43.0 months) and 5 patients developed CDI subsequently (mean time to CDI = 12.4 months, range = 0.4 to 51.3 months). SIBO was identified in 52.4% patients and was not related to CDI. The small intestinal microbial alpha diversity (species richness) was lower in patients with any instance of CDI (P = 0.06 for all instances of CDI (n = 17), P = 0.15 for CDI pre-aspirate and P = 0.19 for CDI post-aspirate (t-test)). The small intestinal microbial beta diversity (unweighted UniFrac) was significantly different in patients with CDI compared to those who did not have CDI (P = 0.02 for all instances of CDI, P = 0.04 for CDI post-aspirate (PERMANOVA); Figure 1). The relative abundance of two families, Clostridiales Family XIII and Erysipelotichacaceae, was significantly lower in those with any instance of CDI. CONCLUSION: The small intestinal microbial composition is altered in patients with CDI which may be a consequence of the infection and may also play a role in recurrent CDI. Further prospective studies are needed to better evaluate the role of small intestinal microbiome in CDI.Figure 1.: Microbiome compositonal profiles comparing CDI to non-CDI patients.