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INTRODUCTION Despite significant improvements in advanced melanoma therapy, there is still a pressing need for innovative therapies. Here, we optimized a method for testing the drug sensitivity of PDOCs and evaluated whether this preclinical model could be a valid tool for rapidly determining the patient’s tumor response profile to approved and alternative therapies. METHODS PDOCs were generated from melanoma metastases (> 60 specimens) after mechanical or enzymatic dissociation. Tissue fragments ( RESULTS The first experiments were set up to validate PDOCs as predictive preclinical models of patient response to therapy. We tested approved melanoma therapies (i.e., the combination of BRAF and MEK inhibitors, or anti-PD-1 antibodies) on the same melanoma specimens at the IDI and IEO institutes. More than 10 different samples were compared, finding reproducible results between the two Institutions. Furthermore, drug response in PDOCs was comparable to the clinical response of matched patients undergoing the tested therapy, demonstrating PDOCs are reliable predictive tools. Then, we used primary cell lines to screen a pool of ten different targeted agents selected through functional screening of a bioactive library of 512 compounds on patient-derived xenografts, and based on known drug resistance mechanisms in melanoma. The three best-performing compounds were subsequently studied in PDOCs as single agents or in combination with immunotherapy. We found high heterogeneity of drug efficacy in different melanoma samples with no obvious correlation to BRAF or NRAS mutations, metastasis type, or patients’ prior therapies. Of note, in some cases, the combination with an anti-PD-1 inhibitor significantly improved the efficacy of one or more of the three drugs. CONCLUSIONS We optimized and validated PDOCs as personalized preclinical models suitable for assessing the drug sensitivity/resistance profile of individual patient-derived melanomas. By retaining tumor stromal components and heterogeneity, PDOCs could be used to predict patients’ clinical response to currently approved agents, helping oncologists expedite decision-making when several treatment options are possible. Furthermore, the model could be used to evaluate the effectiveness of alternative treatments on tumors resistant to approved therapies, and to explore new drug combinations. Citation Format: Antonella Bresin, Fiorenza Lotti, Lauretta Levati, Pier F. Ferrucci, Francesca Ricci, Francesco Scicchitano, Zorika C. Di Rocco, Giandomenico Russo, Luisa Lanfrancone. Patient-derived organotypic cultures (PDOCs) from melanoma metastases as a precision medicine test to improve patient management. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5500.

Efficacy, safety, together with molecular and immunological biomarkers were studied in a sequential clinical trial of neoadjuvant immunotherapy, surgery and adjuvant immunotherapy in locally advanced or oligometastatic melanoma patients (pts), within an open label, single arm and two sites study. Treatment schedule consisted in four primary cycles of inverted dose ipilimumab 1 mg/kg and nivolumab 3 mg/kg every 3 weeks, followed by radical surgery and adjuvant nivolumab 480 mg every 4 weeks for 6 cycles. Primary objective was pathological complete remission (pCR) rate, according to International Neoadjuvant Melanoma Consortium (INMC) criteria, while secondary objectives were: safety, feasibility and efficacy; QoL; identification of biomarkers of response and resistance; degree of immune activation; longitudinal evaluation of the gut microbiome. From March 2019 to April 2021, 43 pts were enrolled in the trial and, with an intent to treat of 35 pts, 34 completed the primary phase, 31 received surgery and 28 completed the adjuvant phase. Four pts were withdrawn during primary phase for progression (2), toxicity (1) and consent withdrawal (1). Study primary endpoint has been met since 20/31 pts undergoing surgery reached a pCR/near pCR (65%), 4/31 (13%) a pathological partial remission (pPR) and 7/31 (22%) pts a pathological no response. With a median follow-up of 17 months, 33/35 pts are alive. Treatment failure occurred in 9 pts: 2 pts progressed during primary phase and did not undergo surgery; 7 pts progressed during adjuvant (3 pts) or follow-up phase (4 pts). Six out of these 7 pts were classified as pNR at surgery, while the other, classified as pCR, did not receive adjuvant therapy. Both pts in stage IV relapsed. Unfortunately, one pt died for ischemic stroke after 5 months from adjuvant therapy while on CR. Treatment related toxicities were mainly G1-2 and only 6 pts (17%) developed G3-4 adverse events (AE): 3 transaminitis, 1 pneumonitis, 1 myocarditis, 1 CPK increase and 1 dermatomyositis. Translational studies on samples collected before, during therapy and at progression have been performed: whole exome sequencing and gut microbiota dynamics on longitudinal samples showed some relationships with responses and developing resistances. These data, never presented elsewhere previously, are in part new and in part confirmatory of immunological or molecular signatures described by other groups. In conclusion, primary immunotherapy with Ipilimumab/Nivolumab in pts affected by locally advanced/oligometastatic melanoma is able to achieve an elevated pCR/near pCR rate which appears to be predictive of long term relapse free survival. Translational data analyzed longitudinally on each patient can allow for a better selection of pts, giving new insight on the mechanisms of melanoma progression and resistance. Citation Format: Pier Francesco Ferrucci, Bruno Achutti Duso, Luigi Nezi, Luca Mazzarella, Fiorenza Lotti, Sara Gandini, Gianmarco Orsolini, Elisabetta Pennacchioli, Patrizia Gnagnarella, Teresa Manzo, Simone Ribero, Maria Teresa Fierro, Rebecca Senetta, Concetta Riviello, Virginia Caliendo, Pietro Quaglino, Massino Barberis, Giuseppina Bonizzi, Emilia Cocorocchio. Neoadjuvant ipilimumab/nivolumab in locally advanced or oligometastatic melanoma: An open label, single arm, multi-institutional clinical study with molecular and immunological biomarker’s analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT167.