{
  "abstract": "Background Recent clinical trials have shown that neoadjuvant immune checkpoint blockade (ICB) improves event-free survival and major pathologic responses in stage III melanoma compared to adjuvant therapy, establishing a new standard of care. 1 2 However, nearly half of patients who undergo neoadjuvant ICB do not achieve adequate response, and recurrence remains common.3 Thus, there is a critical need for validated biomarkers to optimize patient selection, as current clinical and molecular predictors remain suboptimal. Here, we apply a spatial proteomics approach to evaluate the tumor immune microenvironment both prior to and after neoadjuvant therapy for markers of durable response.Methods Tumor samples were collected from 20 patients with stage III melanoma who underwent ICB. Pre-treatment specimens consisted of core biopsies or fine needle aspirates, whereas post-treatment specimens were derived from lymph node dissections. Post-ICB samples were evaluated for standardized pathologic response according to the International Neoadjuvant Melanoma Consortium guidelines. 4 Formalin-fixed paraffin-embedded slides were preprocessed using the PT Module (Epredia) for dewaxing and antigen retrieval. Multiplex immunofluorescence staining was performed on the COMET (Lunaphore) using antibody panels targeting up to 27 phenotypic and functional markers. Pixel-wise autofluorescence correction was applied for each marker using Horizon Viewer software to reduce background subtraction artifacts. Image segmentation, data filtering, and spatial profiling were conducted using a custom Python-based analysis pipeline.Results Analysis of regions of interest enriched for melanoma (HMB45 +) revealed differential immune cell distribution in both pre- and post-treatment samples. In pre-ICB tissue, non- and partial responders exhibited sharper spatial segregation between immune cell-rich and melanoma-dense regions (figure 1A). Notably, CD4+ T cells in these pre-treatment samples were predominantly enriched in the peritumoral stroma rather than infiltrating the tumor-dense regions, a pattern associated with poorer pathologic response. Additionally, a higher CD4+:CD8+ T cell ratio in the pre-ICB samples correlated with reduced pathologic response. In post-treatment tissue, the degree of immune cell infiltration aligned with pathologic response (figure 1B): non-responders showed sparse scattered leukocytes (CD45+), partial responders exhibited patchy immune cell aggregates, and complete responders demonstrated dense immune cell infiltration surrounding residual melanoma. A marked increase in CD8+ T cell density was observed with increasing pathologic response, underscoring the association between cytotoxic T cell infiltration and treatment efficacy.Conclusions Our findings highlight spatial immune features, such as CD4 + localization and CD4+:CD8+ ratio, as potential predictors of response to neoadjuvant ICB in stage III melanoma.References Patel SP, Othus M, Chen Y, Wright GP Jr, Yost KJ, Hyngstrom JR, Hu-Lieskovan S, Lao CD, Fecher LA, Truong TG, Eisenstein JL, Chandra S, Sosman JA, Kendra KL, Wu RC, Devoe CE, Deutsch GB, Hegde A, Khalil M, Mangla A, Reese AM, Ross MI, Poklepovic AS, Phan GQ, Onitilo AA, Yasar DG, Powers BC, Doolittle GC, In GK, Kokot N, Gibney GT, Atkins MB, Shaheen M, Warneke JA, Ikeguchi A, Najera JE, Chmielowski B, Crompton JG, Floyd JD, Hsueh E, Margolin KA, Chow WA, Grossmann KF, Dietrich E, Prieto VG, Lowe MC, Buchbinder EI, Kirkwood JM, Korde L, Moon J, Sharon E, Sondak VK, Ribas A. Neoadjuvant-adjuvant or adjuvant-only pembrolizumab in advanced melanoma. N Engl J Med. 2023 Mar 2;388(9):813-823.Blank CU, Lucas MW, Scolyer RA, van de Wiel BA, Menzies AM, Lopez-Yurda M, Hoeijmakers LL, Saw RPM, Lijnsvelt JM, Maher NG, Pulleman SM, Gonzalez M, Torres Acosta A, van Houdt WJ, Lo SN, Kuijpers AMJ, Spillane A, Klop WMC, Pennington TE, Zuur CL, Shannon KF, Seinstra BA, Rawson RV, Haanen JBAG, Ch’ng S, Naipal KAT, Stretch J, van Thienen JV, Rtshiladze MA, Wilgenhof S, Kapoor R, Meerveld-Eggink A, Grijpink-Ongering LG, van Akkooi ACJ, Reijers ILM, Gyorki DE, Grünhagen DJ, Speetjens FM, Vliek SB, Placzke J, Spain L, Stassen RC, Amini-Adle M, Lebbé C, Faries MB, Robert C, Ascierto PA, van Rijn R, van den Berkmortel FWPJ, Piersma D, van der Westhuizen A, Vreugdenhil G, Aarts MJB, Stevense-den Boer MAM, Atkinson V, Khattak M, Andrews MC, van den Eertwegh AJM, Boers-Sonderen MJ, Hospers GAP, Carlino MS, de Groot JB, Kapiteijn E, Suijkerbuijk KPM, Rutkowski P, Sandhu S, van der Veldt AAM, Long GV. Neoadjuvant nivolumab and ipilimumab in resectable stage III melanoma. N Engl J Med. 2024 Nov 7;391(18):1696-1708.Long GV, Menzies AM, Scolyer RA. Neoadjuvant checkpoint immunotherapy and melanoma: the time is now. J Clin Oncol. 2023 Jun 10;41(17):3236-3248.Tetzlaff MT, Messina JL, Stein JE, Xu X, Amaria RN, Blank CU, van de Wiel BA, Ferguson PM, Rawson RV, Ross MI, Spillane AJ, Gershenwald JE, Saw RPM, van Akkooi ACJ, van Houdt WJ, Mitchell TC, Menzies AM, Long GV, Wargo JA, Davies MA, Prieto VG, Taube JM, Scolyer RA. Pathological assessment of resection specimens after neoadjuvant therapy for metastatic melanoma. Ann Oncol. 2018 Aug 1;29(8):1861-1868.Ethics Approval All human materials were collected and processed according to Stanford University Institutional Review Board (IRB: 65607).Abstract 108 Figure 1Multiplex Immunofluorescence of Pre- and Post-ICB Melanoma Samples. A) Multiplex immunofluorescence images of pre-ICB melanoma samples stained for DAPI (nuclei, blue), HMB45 (melanoma, red), CD45 (leukocyte, green), CD68 (monocyte/macrophage, yellow), CD20 (B cell, grey), CD4 (T cell, cyan), and CD8 (T cell, magenta) demonstrating a pathologic non-responder, partial responder, and complete responder (scale bar = 200 µm). B) Same as A) for post-ICB melanoma samples",
  "authors": [
    {
      "affiliations": [
        "Stanford University School of Medicine, Stanford, CA, USA"
      ],
      "name": "Yichen Zhang"
    },
    {
      "affiliations": [
        "Stanford University School of Medicine, Stanford, CA, USA"
      ],
      "name": "Emma Wagner"
    },
    {
      "affiliations": [
        "Stanford University School of Medicine, Stanford, CA, USA"
      ],
      "name": "Sherry Hsu"
    },
    {
      "affiliations": [
        "Stanford University School of Medicine, Stanford, CA, USA"
      ],
      "name": "Gernot Kaber"
    },
    {
      "affiliations": [
        "Stanford University School of Medicine, Stanford, CA, USA"
      ],
      "name": "Amanda Kirane"
    },
    {
      "affiliations": [
        "Stanford University School of Medicine, Stanford, CA, USA"
      ],
      "name": "Allison Betof Warner"
    }
  ],
  "title": "108 Spatial immune profiling of the melanoma tumor microenvironment under neoadjuvant immunotherapy",
  "uid": "a33294b1-429d-5f53-891b-fb9f1212c113"
}