IFN, interferon; IL, interleukin; VEGF, vascular endothelial growth factor; VCAM-1, vascular cell adhesion molecule; MDC, macrophage-derived chemokine; SDF-1, stromal cell-derived factor 1; NSwM B cells, unswitched memory B cells; OS, overall survival; Tfh, T follicular helper; TNF, tumor necrosis factor. Correlation between circulating soluble factors and the tumor microenvironment Given the correlation of circulating NSwM B cells with both TLS TC21 and certain circulating soluble factors (BAFF, BCA-1/CXCL13) we then investigated the correlation between the 14 circulating soluble factors and the 2′-Deoxyguanosine presence of TLS. patients included in the translational program of the study was available. The association of blood and tissue-based biomarkers, with overall survival 2′-Deoxyguanosine (OS), progression-free survival (PFS) and response was analyzed. Results Among the 44 patients, baseline unswitched memory B cells (NSwM B cells) were enriched in responders (p=0.006) and associated with improved OS (HR=0.08, p=0.002) and PFS (HR=0.54, p=0.048). Responders were enriched in circulating T follicular helper (Tfh) (p=0.027) and tertiary lymphoid structures (TLS) (p=0.043). Circulating NSwM B cells positively correlated with Tfh (r=0.70, p 0.001). 2′-Deoxyguanosine Circulating NSwM B cells correlated positively with TLS and CD20 +B cells at the tumor center (r=0.59, p=0.044, and r=0.52, p=0.033) and inversely correlated with BCA-1/CXCL13 and BAFF (r=?0.55?and r=?0.42, p 0.001). Tfh cells also inversely correlated with BCA-1/CXCL13 (r=?0.61, p 0.001). IL-6, BCA-1/CXCL13 and BAFF significantly associated with worse OS in the discovery (n=40) and validation cohorts (n=313). Conclusion We report the first fresh blood immune-monitoring of patients with m-ccRCC treated with nivolumab. Baseline blood concentration of NSwM B cells was associated to response, PFS and OS in patients with m-ccRCC treated with nivolumab. BCA-1/CXCL13 and BAFF, inversely correlated to NSwM B cells, were both associated with worse OS in discovery and validation cohorts. Our data confirms a role for B cell subsets in the response to immune checkpoint blockade therapy in patients with m-ccRCC. Further studies are needed to confirm these findings. identified IgM +memory B cells as predictors of response to anti-programmed cell death protein-1 (PD-1) treatment in advanced NSCLC.30 The aim of our study was to determine whether specific immune cell populations and/or soluble factors at baseline were predictive of clinical outcomes to ICB in m-ccRCC. Therefore, we characterized immune-cell populations in fresh-whole-blood and performed circulating soluble factors quantification at baseline in patients with m-ccRCC treated with nivolumab within the phase II NIVOREN GETUG-AFU 26. Furthermore, tumor immune microenvironment (TIME) characterization was correlated with 2′-Deoxyguanosine blood-based biomarkers. Patients and methods Patient cohorts and sample collection This study was conducted as part of the translational program of the multicenter, prospective, phase II NIVOREN GETUG-AFU 26 trial (EudraCT n: 2015-004117-24/03013335) (online supplemental figure S1). Seven hundred and twenty-nine patients with m-ccRCC previously treated with at least one anti-angiogenic were included in this trial. Patients received nivolumab monotherapy 3?mg/kg every 2?weeks until death, disease progression, unacceptable toxicity, or withdrawal of the informed consent. Response was assessed every 12 weeks by RECIST V.1.1. Supplementary data jitc-2022-004885supp001.pdf Supplementary data jitc-2022-004885supp002.pdf Six hundred and seventeen patients out of the 729 patients included in this trial took part in the translational program. The study design included the collection of patients clinical data, blood samples at baseline, at 1?month and at end of treatment (EOT) (including fresh blood samples for the patients treated at the Institute Gustave Roussy (IGR)), and tumor tissue samples, after signature of informed consent. This study was conducted in accordance with ethical principles for medical research involving human subjects reported in the Declaration of Helsinki. For the fresh blood immune-phenotyping and soluble factors quantification analysis, all consecutive 44 patients of the trial treated at a single institution (IGR) and enrolled in the translational study were defined as the discovery cohort. For the immunohistochemistry (IHC) analysis, 324 patients with centrally confirmed clear cell renal cell carcinoma tumor tissue ( 50% tumor cells) were included (PRTK cohort). Seventeen patients were included in both the fresh blood immune-phenotyping and IHC analysis. The results of the soluble factor quantification analysis were confirmed in an independent validation cohort including 313 patients where plasma samples were available. Multicolor flow cytometry The procedures to perform blood immune phenotyping on fresh-whole-blood samples and flow cytometry antibodies and fluorochromes used are described in the online supplemental methods and table S1, respectively. Unsupervised analysis of.