Anti-PD-1-based immunotherapy has had a major impact on cancer treatment but has only benefited a subset of patients. Among the variables that could contribute to interpatient heterogeneity is differential composition of the patients' microbiome, which has been shown to affect antitumor immunity and immunotherapy efficacy in preclinical mouse models. We analyzed baseline stool samples from metastatic melanoma patients before immunotherapy treatment, through an integration of 16 ribosomal RNA gene sequencing, metagenomic shotgun sequencing, and quantitative polymerase chain reaction for selected bacteria. A significant association was observed between commensal microbial composition and clinical response. Bacterial species more abundant in responders included ,, and Reconstitution of germ-free mice with fecal material from responding patients could lead to improved tumor control, augmented T cell responses, and greater efficacy of anti-PD-L1 therapy. Our results suggest that the commensal microbiome may have a mechanistic impact on antitumor immunity in human cancer patients.

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Purpose Tumor genotyping using cell free plasma DNA (cfDNA) has the potential to allow noninvasive assessment of tumor biology, yet many existing assays are cumbersome and vulnerable to false positive results. We sought to determine whether droplet digital PCR (ddPCR) of cfDNA would allow highly specific and quantitative assessment of tumor genotype. Experimental Design ddPCR assays for EGFR, KRAS, and BRAF mutations were developed using plasma collected from patients with advanced lung cancer or melanoma of a known tumor genotype. Sensitivity and specificity were determined using cancers with non-overlapping genotypes as positive and negative controls. Serial assessment of response and resistance was studied in EGFR-mutant lung cancer patients on a prospective trial of erlotinib. Results We identified a reference range for EGFR L858R and exon 19 deletions in specimens from KRAS-mutant lung cancer, allowing identification of candidate thresholds with high sensitivity and 100% specificity. Received operative characteristic (ROC) curve analysis of 4 assays demonstrated an area under the curve in the range of 0.80-0.94. Sensitivity improved in specimens with optimal cfDNA concentrations. Serial plasma genotyping of EGFR-mutant lung cancer on erlotinib demonstrated pretreatment detection of EGFR mutations, complete plasma response in most cases, and increasing levels of EGFR T790M emerging prior to objective progression. Conclusions Noninvasive genotyping of cfDNA using ddPCR demonstrates assay qualities that could allow effective translation into a clinical diagnostic. Serial quantification of plasma genotype allows noninvasive assessment of response and resistance, including detection of resistance mutations up to 16 weeks prior to radiographic progression.