Spatial assessment of stromal B cell aggregates predicts response to checkpoint inhibitors in unresectable melanoma

Quantitative assessment of multiplex immunofluorescence (mIF) data represents a powerful tool for immunotherapy biomarker discovery in melanoma and other solid tumors. In addition to providing detailed phenotypic information of immune cells of the tumor microenvironment, these datasets contain spatial information that can reveal biologically relevant interactions among cell types. To assess quantitative mIF analysis as a platform for biomarker discovery, we used a 12-plex mIF panel to characterize tumor samples collected from 50 patients with melanoma prior to treatment with immune checkpoint inhibitors (ICI). Consistent with prior studies, we identified a strong association between stromal B cell percentage and response to ICI therapy. We then compared pathologist assessment of lymphoid aggregates with a density based clustering algorithm, DBSCAN, to both automatically detect B cell aggregates and quantify their size, morphology, and distance to tumor. Spatial neighborhood analysis identified TCF1+ and LAG3- T cell subpopulations enriched near stromal B cells. These analyses provide a roadmap for the further development and validation of spatial immunotherapy biomarkers in melanoma and other diseases. ### Competing Interest Statement J.W.S. Research funding: IO Biotech (Inst), Regeneron (Inst), Daiichi Sankyo (Inst); Consulting or advisory role: IO Biotech; X.P. No disclosures; F.D.E. No disclosures; A.P.M. No disclosures; M.Y. No disclosures; C.M. No disclosures; N.A. No disclosures; R.V. No disclosures M.Z. No disclosures; J.L. No disclosures; M.B. No disclosures. Y.L.: Employment: Bristol Myers Squibb; M.A.P. Consulting or Advisory Role: Bristol Myers Squibb; Cancer Expert Now; Chugai Pharma; Eisai; Erasca, Inc; Intellisphere; Merck; MJH Associates; Nektar; Novartis; Pfizer; WebMD; Research Funding: Array BioPharma (Inst); Bristol Myers Squibb (Inst); Infinity Pharmaceuticals (Inst); Merck (Inst); Novartis (Inst); Rgenix (Inst); K.S.P. Stock ownership in 23and Me, Vincerx, Eyepoint, & Kyverna T.J.H. Employment: Bristol Myers Squibb; M.K.C. Bristol Myers Squibb: Research support (Inst), advisory role/consulting; Medimmune: advisory role/consulting; Immunocore: advisory role/consulting; Merus: family member employee; R.S. No disclosures ### Funding Statement This work was supported in part by the Memorial Sloan Kettering Cancer Center (MSK) NCI Core Grant P30 CA008748 and grant support from the V Foundation (T2021-007, M.K.C and R.S.). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Memorial Sloan Kettering Cancer Center Institutional Review Board gave ethical approval for this work I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All code describing cell phenotyping pipeline, application of DBSCAN for quantification of B cell aggregates, and B cell neighborhood analysis are available on GitHub. Raw mIF data files and de-identified clinical data are available by request to the corresponding author.