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Background There is an urgent need for more robust methods to differentiate immunotherapy responders from non-responders. In this study, by utilizing a novel multiplex imaging (MI)-based panel and analysis pipeline, we found that the spatial distribution and function of immune cells correlate with response in a cohort of immunotherapy-treated melanoma patients.Methods We designed a 28-plex sequential immunofluorescence panel (seqIF™) on the COMET™ platform 1 to target key biomarkers associated with tumor microenvironment composition (TME), immune cell infiltration, and immune checkpoint pathways. Utilizing Nucleai’s deep-learning-based analysis pipeline,2 we profiled 42 pre-treatment biopsies from the SECOMBIT trial3–5 (NCT02631447). We identified 15 cell types, including 10 different immune cell populations, and 10 cell state markers. Following cell typing, cellular neighborhoods were designated as previously described6 and were assigned to tumor or stromal areas. By analyzing 1,941 spatial features within each treatment arm, we identified the top 3 spatial features associated with progression-free survival (PFS), overall survival (OS), and prolonged clinical benefit. To perform the survival analysis, each spatial feature was binarized by proximity to the 0.25 and 0.75 quantiles, and the survival of the resulting groups was compared using the logrank test.Results In Arm A (MAPK inhibitor, MAPKi, until progression followed by immune checkpoint blockade, ICB), several spatial interactions were significantly associated with patient outcomes. The spatial features associated with good MAPKi outcome showed activation of the anti-tumor immune response, including enhanced antigen presentation and PD-L1 positivity within CD8 T-cells and antigen-presenting cells, and ICOS positivity within CD4 T-cells. The high content of stromal cells was associated with a worse outcome.In Arm B (ICB until progression followed by MAPKi), PD-1+ CD8 T-cells presence in the tumor invasive margin and their interaction with PD-L1+ CD4 T-cells were associated with improved PFS and OS. High proportions of TCF1+ CD4 Tregs interactions with macrophages and T-cell interactions with endothelial cells were associated with worse outcome.In Arm C (8 weeks of MAPKi, followed by ICB until progression, followed by MAPKi), PD-L1+ and VISTA+ antigen-presenting cells’ interaction with CD4+ and CD8+ T-cells within the tumor invasive margin was associated with better outcome. In contrast, CD8+ T-cells and CD4+ T-cells interaction with CD163+ macrophages in the outer TME was associated with a worse outcome.Conclusions Our data demonstrates that area-specific immune niches contribute to the success or failure of immunotherapy response, highlighting the importance of spatial biology in predicting outcomes.Acknowledgements The authors thank the patients and families who made this trial possible. Additionally, the authors acknowledge the clinical study teams and CRO who participated in the trial and in particular Paola Schiavo e Mariarita Arenella from CRT (Clinical Research Technology - Salerno). We thank Bristol-Myers Squibb (Princeton, NJ) and Array Biopharma Inc/Pfizer (Boulder, CO) for support. Moreover, the authors thank the participating investigators who did not enroll any patients and thus are not included as authors on the paper, Koelblinger P, Hafner C, Hoeller C (Austria), Weide B (Germany), Larkin J, Lorigan P (UK).References Rivest F, et al. Fully automated sequential immunofluorescence (seqIF) for hyperplexspatial proteomics. Sci Rep. 2023;13(1):16994.Markovits E, et al. A novel deep learning pipeline for cell typing and phenotypic marker quantification in multiplex imaging. bioRxiv. 2022.Ascierto PA, et al. Sequencing of ipilimumab plus nivolumab and encorafeni bPlus binimetinib for untreated BRAF-mutated metastatic melanoma (SECOMBIT): a randomized, three-arm, open-label phase II trial. J Clin Oncol. 2023 Jan 10;41(2):212–221.Ascierto PA, et al. Sequential immunotherapy and targeted therapy for metastatic BRAF V600 mutated melanoma: 4-year survival and biomarkers evaluation from the phase II SECOMBIT trial. Nat Commun. 2024;15(1):146.Ascierto PA, et al. Sequencing of checkpoint of BRAF/MEK inhibitors on brain metastasis in Melanoma. NEJM Evid. 2024;3(10).Schürch, CM, et al. coordinated cellular neighborhoods orchestrate antitumoral immunity at the colorectal cancer invasive front. Cell. 2020;182:1341–1359.e19 (2020).Ethics Approval This study was designed in 2015 as a phase II, open-label randomized trial with no formal comparative test and a single-stage design for each arm. Patients were enrolled at 37 academic medical centers in 9 countries. The trial protocol was approved by the appropriate ethics body at each participating institution and is available in the Supplementary Information file. An independent data monitoring committee oversaw the trial. SECOMBIT is registered at ClinicalTrials.gov ( NCT02631447). The study design and conduct complied with all current regulations regarding the use of human study participants and was conducted in accordance with the criteria set by the Declaration of Helsinki.