842 Development of a multiplex test for predicting response to combined immunotherapies in patients with metastatic melanoma (2022)

<h3>Background</h3> In recent years, advanced melanoma treatment has improved dramatically thanks to the advent of immunotherapy. Particularly immune checkpoint inhibitors (ICI), in some patients, have demonstrated to improve long-term outcomes associated with limited toxicity. However, only a small population of patients achieve a durable response to therapy, owing to the lack of clinically validated predictive biomarkers (reviewed in<sup>1</sup>). The availability of improved predictive biomarkers may allow the identification of patients who will most benefit from ICI treatment and those who may be susceptible to immune-related adverse events. This difficulty in obtaining clinically relevant predictive biomarkers underscores the complexity of the immune system and the heterogeneity of the tumor microenvironment. In the present study, we take the first steps towards stratification of advanced melanoma patients who received a combination of ICI. We studied the predictive value of a multi-parameter spatial signature composed of lymphocyte-activation gene-3 (LAG-3), programmed death-ligand 1 (PD-L1) and cluster of differentiation 8 (CD8) in a retrospective cohort of patients with metastatic melanoma treated with a combination of ICI. <h3>Methods</h3> In this retrospective study, from the biobank of the Istituto Nazionale Tumori IRCCS Fondazione G. Pascale, we recovered FFPE skin metastasis samples obtained from 10 melanoma patients with AJCC 8th edition stage IV<sup>2</sup> subsequently treated with combined ICI, enrolled from September 2016 to November 2017. According to the Response Evaluation Criteria in Solid Tumors (RECIST 1.1),<sup>3</sup>4 patients achieved response to the treatment, while 6 patients were non-responders. A single FFPE tumor tissue of each patient was stained using LabSat® platform (Lunaphore Technologies) performing a Tyramide Signal Amplification multiplex immunohistochemistry staining for PD-L1, CD8, and LAG-3 expression, followed by DAPI counterstaining and whole slide fluorescent scanning. Single cell segmentation, phenotyping and quantification were performed in QuPath (0.3.0). The workflow is depicted in figure 1. <h3>Results</h3> Responder patients showed a statistically significant increase in CD8+ single-positive cell frequency compared to non-responders (figure. 2A and 2C). Non-responder patients displayed a statistically significant increase of PD-L1+ single-positive cell frequency, as well as statistically significant increased frequency of double CD8+PD-L1+ positive cells, previously found to be a poor prognostic in multiple cancer types,<sup>4,5</sup> and triple positive cells (figure 2B and 2C). <h3>Conclusions</h3> We show here preliminary evidence of the predictive value of a spatial biomarker signature in patients who underwent combined ICI therapy for advanced melanoma. To further demonstrate clinical relevance, a more detailed analysis using larger retrospective and prospective cohorts is ongoing. <h3>References</h3> Garutti M, Bonin S, Buriolla S, Bertoli E, Pizzichetta MA, Zalaudek I, <i>et al</i>. Find the Flame: Predictive Biomarkers for Immunotherapy in Melanoma. <i>Cancers</i> 2021;<b>13</b>:1819. Gershenwald JE, Scolyer RA. Melanoma Staging: American Joint Committee on Cancer (AJCC) 8th Edition and Beyond. <i>Ann. Surg. Oncol</i>. 2018; 2105–10. Eisenhauer EA, Therasse P, Bogaerts J, <i>et al</i>. New response evaluation criteria in solid tumors: revised RECIST guideline (version 1.1) <i>Eur J Cancer</i>. 2009;<b>45</b>:228–247. Brochez L, Meireson A, Chevolet I, Sundahl N, Ost P, Kruse V. Challenging PD-L1 expressing cytotoxic T cells as a predictor for response to immunotherapy in melanoma. <i>Nat Commun</i> 2018;<b>9</b>:2921. Zhang L, Chen Y, Wang H, Xu Z, Wang Y, Li S, <i>et al</i>. Massive PD-L1 and CD8 double positive TILs characterize an immunosuppressive microenvironment with high mutational burden in lung cancer. <i>J Immunother Cancer</i> 2021;<b>9</b>:e002356. <h3>Ethics Approval</h3> This study was conducted in accordance with the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of the Istituto Nazionale Tumori IRCCS Fondazione "G. Pascale" (Naples, Italy) (protocol code 33/17oss, approved on 10 January 2018).