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High-dimensional cytometry is routinely used to characterize patient responses to cancer immunotherapy and other treatments, producing a wealth of datasets ripe for exploration but whose biological and technical heterogeneity make them difficult to analyze with current tools. We introduce a new interpretable machine learning method for single-cell mass and flow cytometry studies, FAUST, that robustly performs unbiased cell population discovery and annotation and enables data integration across studies and platforms. FAUST returns biologically interpretable cell phenotypes that can be compared across studies, making it well-suited for the analysis and integration of complex datasets. We use FAUST to perform candidate biomarker discovery and validation by applying it to flow and mass cytometry datasets from melanoma anti-PD-1 trials in order to discover and validate new CD4+ and CD8+ effector-memory T cell correlates of outcome co-expressing PD-1, HLA-DR, and CD28. Existing state-of-the-art computational discovery approaches as well as prior manual analyses did not detect any statistically significant T cell correlates associated with anti-PD-1 treatment these data. We use FAUST to replicate the discovery of published myeloid cell correlates and validate these by identifying them de novo in independent trials. FAUST’s phenotypic annotations are used to perform cross-study integration diverse data sets, allowing hypothesis-driven inference about cell sub-populations through a Phenotypic and Functional Differential Abundance (PFDA) framework. We showcase this approach on data across multiple trials. These results establish FAUST as a powerful new approach for unbiased discovery in single-cell cytometry.

BackgroundMerkel-cell carcinoma is an aggressive skin cancer that is linked to exposure to ultraviolet light and the Merkel-cell polyomavirus (MCPyV). Advanced Merkel-cell carcinoma often responds to chemotherapy, but responses are transient. Blocking the programmed death 1 (PD-1) immune inhibitory pathway is of interest, because these tumors often express PD-L1, and MCPyV-specific T cells express PD-1.MethodsIn this multicenter, phase 2, noncontrolled study, we assigned adults with advanced Merkel-cell carcinoma who had received no previous systemic therapy to receive pembrolizumab (anti-PD-1) at a dose of 2 mg per kilogram of body weight every 3 weeks. The primary end point was the objective response rate according to Response Evaluation Criteria in Solid Tumors, version 1.1. Efficacy was correlated with tumor viral status, as assessed by serologic and immunohistochemical testing.ResultsA total of 26 patients received at least one dose of pembrolizumab. The objective response rate among the 25 patients with at least one evaluation during treatment was 56% (95% confidence interval [CI], 35 to 76); 4 patients had a complete response, and 10 had a partial response. With a median follow-up of 33 weeks (range, 7 to 53), relapses occurred in 2 of the 14 patients who had had a response (14%). The response duration ranged from at least 2.2 months to at least 9.7 months. The rate of progression-free survival at 6 months was 67% (95% CI, 49 to 86). A total of 17 of the 26 patients (65%) had virus-positive tumors. The response rate was 62% among patients with MCPyV-positive tumors (10 of 16 patients) and 44% among those with virus-negative tumors (4 of 9 patients). Drug-related grade 3 or 4 adverse events occurred in 15% of the patients.ConclusionsIn this study, first-line therapy with pembrolizumab in patients with advanced Merkel-cell carcinoma was associated with an objective response rate of 56%. Responses were observed in patients with virus-positive tumors and those with virus-negative tumors. (Funded by the National Cancer Institute and Merck; ClinicalTrials.gov number, NCT02267603.).

Frequency of tetramer+ CD8 T cells. Frequency of MCPyV tetramer positive CD8 T cells are reported in percent of all CD8s with background subtracted. Abbreviations for RECIST 1.1 response criteria are as follows: CR = complete response; PR = partial response; PD = progressive disease. (DOCX 69 kb)

Class I HLA Tetramers and MCPyV peptide pools. Description of data: A) Summary of HLA and peptide combinations used for CD8 class I tetramers including which position of the MCPyV oncoprotein (small, common, or large T antigen) the peptide corresponds to. B) Schematic of MCPyV peptide pools and locations of tetramer epitopes. Details of peptide pools are available in Iyer et al., 2011. (DOCX 364â kb)

Frequency of IFN-γ and/or IL-2 secreting CD8 T cells in response to Merkel polyomavirus peptide pools. IFN-γ and/or IL-2 in A) 2 of 13 VP-MCC responders and B) 1 of 4 VP-MCC non-responders was detectible via flow cytometry after a 16 h stimulation with MCPyV peptide pools. Dotted line represents background signal cutoff. *Post = first blood draw after initiation of treatment. (DOCX 425 kb)

Frequency of tetramer+ CD8 T cells. Frequency of MCPyV tetramer positive CD8 T cells are reported in percent of all CD8s with background subtracted. Abbreviations for RECIST 1.1 response criteria are as follows: CR = complete response; PR = partial response; PD = progressive disease. (DOCX 69 kb)

Class I HLA Tetramers and MCPyV peptide pools. Description of data: A) Summary of HLA and peptide combinations used for CD8 class I tetramers including which position of the MCPyV oncoprotein (small, common, or large T antigen) the peptide corresponds to. B) Schematic of MCPyV peptide pools and locations of tetramer epitopes. Details of peptide pools are available in Iyer et al., 2011. (DOCX 364â kb)

Serology results. Raw results for MCPyV-specific oncoprotein antibodies were reported as Standard Titer Units (STUs). Patient with a value of 75 or greater were considered oncoprotein antibody producers and values below 74 were considered negative. Abbreviations for RECIST 1.1 response criteria are as follows: CR = complete response; PR = partial response; PD = progressive disease. (DOCX 114 kb)

Serology results. Raw results for MCPyV-specific oncoprotein antibodies were reported as Standard Titer Units (STUs). Patient with a value of 75 or greater were considered oncoprotein antibody producers and values below 74 were considered negative. Abbreviations for RECIST 1.1 response criteria are as follows: CR = complete response; PR = partial response; PD = progressive disease. (DOCX 114 kb)

Frequency of IFN-γ and/or IL-2 secreting CD8 T cells in response to Merkel polyomavirus peptide pools. IFN-γ and/or IL-2 in A) 2 of 13 VP-MCC responders and B) 1 of 4 VP-MCC non-responders was detectible via flow cytometry after a 16 h stimulation with MCPyV peptide pools. Dotted line represents background signal cutoff. *Post = first blood draw after initiation of treatment. (DOCX 425 kb)