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BACKGROUND: The Rome Trial is a randomized phase II trial (NCT04591431). The aim is to evaluate efficacy and safety of a tailored treatment (TT) compared to standard of care (SoC) in patients with solid tumors. Here we report the preliminary results of the molecular alterations, microsatellite status (MS) and tumor mutational burden (TMB) in metastatic breast cancer (mBC) cohort. METHODS: MBC patients who received at least 1 and no more than 2 systemic treatments were enrolled. Tissue samples were collected within 6 months from the screening. Centralized Next Generation Sequencing (NGS) was performed on both tissue and liquid biopsy. Molecular alterations were evaluated by the Molecular Tumor Board (MTB) using COSMIC, ClinVar, OncoKB and VarSome datasets. Genes with at least 10% frequency of mutation, MS and TMB are reported. RESULTS: From Oct 2020 to June 2022, 980 pts with solid tumors were enrolled. Complete screening mutational data are available for sixty-two pts from the mBC cohort (63% HR+/HER2-, 35% triple negative, 2% HR-/HER2+). NGS was available both on tissue and liquid biopsy in 48 (77%) pts, 14 had only liquid biopsy available due to tissue test failure. 328 genes resulted altered with a median of 7 alteration per pts (0-31). Some pathways were frequently altered: PIK3CA/AKT/MTOR (60%), TP53 (60%), Cell cycle/cycline (35%), FGF/FGFR (26%), BRCA1/2 (17%). The most frequent altered genes were: TP53 (61%), PIK3CA (50%), ESR1 (27%), CCND1 (27%), FGF19 (24%), FGF3 (24%), FGF4 (22%), MYC (22%), FGFR1 (21%), PTEN (21%), EMSY (16%), RB1 (14%), RAD21 (14%), TET2 (13%), BRCA2 (11%), GATA3 (11%), KRAS (10%). No pts with MSI status were reported. Eight (13%) had a high TMB (>10) and the overall median TMB was 5.5 (0-24). Median TMB was similar in tissue and liquid samples (5 and 5.3 mut/mb, p= 0.8). Actionable mutations were detected in 34 pts (54%). Twenty-eight (45%) pts were assigned to a specific TT after the MTB discussion: ipatasertib (16), pemigatinib (5), ipilimumab plus nivolumab (4), lapatinib plus trastuzumab, TDM1 and everolimus (1). MTB requested a germline test for 6 pts: 4 were confirmed (66%; 2 BRCA, 1 PALB2, 1 BRIP1). CONCLUSIONS: The extensive NGS analysis performed in the ROME trial shown that several pathways are commonly mutated in mBC, with target drug potentially available. About 15% of pts had a high TMB but MSI is confirmed as a rare event in breast cancer. Germline mutations have been identified in patients with no prior indication for germline testing. Citation Format: Andrea Botticelli, Simone Scagnoli, Pierfranco Conte, Chiara Cremolini, Paolo Antonio Ascierto, Federico Cappuzzo, Massimo Aglietta, Federica Mazzuca, Ettore Capoluongo, Giovanni Blandino, Umberto Malapelle, Marianna Nuti, Giulia D’Amati, Bruna Cerbelli, Giancarlo Pruneri, Mauro Biffoni, Giuseppe Giannini, Francesco Cognetti, Giuseppe Curigliano, Paolo Marchetti. Mutational landscape of breast cancer patients in ROME trial: preliminary results [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-09.

Background BRAF V600 mutations are frequently found in metastatic melanoma. This mutation constitutively activates the MAPK pathway, which leads to melanoma progression. Patients with BRAF V600-mutant metastatic melanoma typically receive BRAF inhibitors (BRAFi) + MEK inhibitors (MEKi), such as enco + bini, or immune checkpoint inhibitors (CPIs; eg, pembro). However, these have limitations. BRAFi and MEKi may increase BRAF V600-mutant tumor sensitivity to CPIs. Previous studies reported improved progression-free survival (PFS) in patients with advanced BRAF V600-mutant melanoma receiving BRAFi + MEKi + CPI compared with targeted therapy alone. This phase 3 trial will evaluate the efficacy and safety of enco + bini + pembro vs placebo + pembro for unresectable locally advanced or metastatic BRAF V600-mutant melanoma. A safety lead-in (SLI) was built in to determine the recommended phase 3 dose (RP3D) prior to starting phase 3. Trial design STARBOARD (NCT04657991) is a randomized, double-blind, placebo-controlled, phase 3 study evaluating approximately 600 patients with BRAF V600 advanced melanoma. Patients will be stratified by prior systemic adjuvant treatment (CPI, BRAFi/MEKi, or none) and by disease stage (per AJCC 8th edition; IIIB, IIIC, IIID, IV M1a[0], and IV M1b[0] vs IV M1a[1], IV M1b[1], IV M1c[0], IV M1c[1], IV M1d[0], and IV M1d[1]). Patients must have measurable disease (per RECIST 1.1); ECOG performance status of 0 or 1; and adequate bone marrow, hepatic, and renal function. Patients must not have received prior systemic therapy for unresectable or metastatic melanoma; adjuvant treatment with BRAFi/MEKi, anti-PD-1, or anti-CTLA-4 is permitted. Patients cannot have symptomatic brain metastases. Study treatments and end points are shown in Table 1. RP3D was established in May 2022; phase 3 enrollment began in June 2022. Table 1. Study treatments and end points Phase 3 Treatment (21-day cycle) RP3D from SLI: enco + bini + pembro Control: placebo + pembro Primary PFS (RP3D vs control; by BICR) Secondary Key: OS (RP3D vs control) Other: PFS by investigator assessment, objective response rate, duration of response, disease control rate, and time to response, all by BICR and investigator assessment, PFS2, quality of life, safety, pharmacokinetics BICR, blinded independent central review; OS, overall survival. Citation Format: Dirk Schadendorf, Reinhard Dummer, Caroline Robert, Antoni Ribas, Ryan J. Sullivan, Timothy Panella, Meredith McKean, Edgardo S. Santos, Jill Clancy, Anna Polli, Alessandra di Pietro, Paolo A. Ascierto. Randomized phase 3 study (STARBOARD) evaluating encorafenib (enco) + binimetinib (bini) + pembrolizumab (pembro) for first-line treatment of unresectable locally advanced or metastatic BRAF V600-mutant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT069.

This abstract was not presented at the conference. Citation Format: Botticelli A, Cerbelli B, Pisano A, Naso G, Monti M, Ascierto PA, Costarelli L, Magri V, Mauri M, Pignataro MG, Campagna D, Pernazza A, Nuti M, Fortunato L, Della Rocca C, D'Amati G, Marchetti P. Not presented [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-12-08.

Background: In the randomized, two-part, phase 3 COLUMBUS study (NCT01909453), encorafenib (enco) + binimetinib (bini) and enco alone improved 5-year progression-free survival (PFS) and overall survival (OS) vs vemurafenib (vem) in patients (pts) with advanced BRAF V600E/K-mutant melanoma. ctDNA offers a less invasive alternative to tissue biopsy for identifying mutations in pts with advanced melanoma and may have prognostic value. We evaluated whether ctDNA from pts enrolled in COLUMBUS Part 1 correlate with pts’ efficacy outcomes. Methods: In COLUMBUS Part 1, 577 pts were randomized 1:1:1 to enco + bini, enco, or vem. 650 plasma samples were collected at baseline (Cycle 0 or Cycle 1 Day 1; all arms) and during treatment (Cycle 2 Day 1 [C2D1]; enco + bini and enco arms); 426 samples from COLUMBUS Part 1 were successfully analyzed using the GuardantOMNI assay. Analyses were exploratory with no multiplicity adjustments. Results: Survival outcomes in the biomarker cohort were similar to those in the intent-to-treat population. ctDNA was detected in 279 (96%) pts at baseline and 107 (79%) pts at C2D1. 92% concordance was observed between two plasma genomics assays (Inostics and Guardant) and 75% between tumor and plasma with respect to BRAF mutation status. BRAF V600 variant allele frequency (VAF) at baseline was prognostic for PFS and OS (P Conclusions: These exploratory analyses of Part 1 of the COLUMBUS study suggested that treatment with enco + bini significantly reduced BRAF V600 VAF, which was prognostic for PFS and OS at both baseline and C2D1. ctDNA provides a powerful tool for understanding the molecular basis for response and resistance to treatment and may impact future treatment decisions. Citation Format: Reinhard Dummer, Nuzhat Pathan, Zhou Zhu, Caroline Robert, Ana Arance, Jan Willem B. de Groot, Claus Garbe, Helen J. Gogas, Ralf Gutzmer, Ivana Krajsová, Gabriella Liszkay, Carmen Loquai, Mario Mandala, Dirk Schadendorf, Naoya Yamazaki, Michelle Edwards, Jean Cantey-Kiser, Alessandra di Pietro, Shibing Deng, Paolo A. Ascierto, Keith Flaherty. Molecular correlates of clinical benefit from circulating tumor DNA (ctDNA): Analysis of the COLUMBUS study [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 6403.

Background: MAPK pathway dysregulation is a characteristic molecular abnormality of melanoma. NRAS mutations in melanoma lead to constitutive MAPK activation and subsequent MEK and ERK activation. Inhibiting the pathway with a combination of BRAF/CRAF and MEK inhibitors (LXH254 and TMT, respectively) has demonstrated synergistic activity in preclinical models of NRAS-mutant melanoma. Methods: We report data from an open-label, Phase Ib study (NCT02974725), that tested LXH254 + TMT combination in a dose-escalation (ESC) part in pts with KRAS- or BRAF-mutant NSCLC or NRAS-mutant melanoma and in a dose-expansion (EXP) part in NRAS-mutant melanoma pts. In ESC, five dose levels were explored: LXH254 200 mg twice daily (BID) + TMT (1 mg or 0.5 mg) daily (QD); LXH254 400 mg BID + TMT (1 mg or 0.5 mg) QD; LXH254 400 mg BID + TMT 1 mg (QD, 2 wk on/2 wk off). Primary objectives were to evaluate safety and tolerability, and determine the recommended dose for expansion (RDE). Secondary objectives were to characterize pharmacokinetics and pharmacodynamics of LXH254 + TMT, and evaluate antitumor activity. Results: As of Dec 9, 2021, 36 and 30 pts were enrolled in ESC and EXP, respectively; median age was 63.5 and 69 y, respectively. Pts in ESC and EXP received a median of 3 (range: 1-6) and 2 (range: 1-7) prior treatments, respectively, including IO (ESC: 88.9% pts; EXP: 96.7% pts). All pts from ESC and 24 pts from EXP discontinued, mainly due to progressive disease (61.1% and 60%, respectively); 6 pts were ongoing in EXP at data cut-off. In ESC, Grade ≥3 (G≥3) DLTs were reported in 6 pts: dermatitis acneiform (n=2), maculopapular rash (n=2), increased lipase (n=1), and Stevens-Johnson syndrome (n=1). The RDEs were LXH254 200 mg BID + TMT 1 mg QD and LXH254 400 mg BID + TMT 0.5 mg QD. Treatment-related adverse events (TRAEs) were reported in 34 pts (94.4%) in ESC and all pts in EXP. G≥3 TRAEs in ESC and EXP were experienced by 19 (52.8%) and 24 (80%) pts, respectively; the most common being rash or dermatitis acneiform (13.9% each) in ESC and rash (33.3%) in EXP. One fatal TRAE (hypovolemic shock) was reported in the LXH254 400 mg BID + TMT 0.5 mg QD group. The ORR in EXP was 46.7% (7 pts; 95% CI: 21.3-73.4) for LXH254 200 mg BID + TMT 1 mg QD and 13.3% (2 pts; 95% CI: 1.7-40.5) for LXH254 400 mg BID + TMT 0.5 mg QD; overall in EXP, 9 pts (30%) achieved PR and 13 pts (43.3%) reported SD. In EXP the median DOR was 3.8 months (95% CI: 2.6-7.4). Exposures of LXH254 (200 mg or 400 mg BID) + TMT (0.5 mg or 1 mg QD) vs LXH254 single agent were comparable, indicating no apparent LXH254-TMT interactions. Limited paired tumor biopsy data (n=8) showed substantial MAPK pathway inhibition (DUSP6). Conclusions: LXH254 in combination with TMT has shown preliminary antitumor activity in the pretreated, NRAS-mutant melanoma population. A Phase II trial of LXH254 combinations in pts with pretreated BRAF- or NRAS-mutant melanoma is currently ongoing (NCT04417621). Citation Format: Filippo de Braud, Christophe Dooms, Rebecca S. Heist, Celeste Lebbe, Martin Wermke, David Planchard, Dirk Schadendorf, Piotr Rutkowski, Jürgen Wolf, Paolo A. Ascierto, Ignacio Gil-Bazo, Shumei Kato, Maria Wolodarski, Meredith McKean, Eva Muñoz Couselo, Martin Sebastian, Armando Santoro, Vesselina Cooke, Luca Manganelli, Kitty Wan, Anil Gaur, Tanay S. Samant, Jaeyeon Kim, Giordano Caponigro, Xuân-Mai Couillebault, Michele Moschetta, Adil Daud. Phase Ib study of LXH254 + trametinib (TMT) in patients (pts) with NRAS-mutant melanoma [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 CT197.

High tumor mutational burden assessed in tissue biopsies (tTMB-H) or blood (bTMB-H) is associated with clinical efficacy in patients treated with immunotherapies. CheckMate 848 (NCT03668119) is a prospective phase 2 study of nivolumab (NIVO) with or without ipilimumab (IPI) in patients with advanced or metastatic solid tumors that are tTMB-H or bTMB-H (≥ 10 mutations/megabase) who were immunotherapy-naive and refractory to standard local therapies. The primary endpoint was objective response rate (ORR) in patients with tTMB-H or bTMB-H, assessed by FoundationOne® CDx-based and Clinical Trial Assays (Foundation Medicine), respectively. The study was not powered to compare NIVO + IPI vs NIVO. We present the interim and final analyses for the tTMB-H and bTMB-H cohorts, respectively (≥ 12 months follow-up, database lock June 2021). Of 1954 screened patients, 212 were randomized 2:1 to NIVO 240 mg Q2W + IPI 1 mg/kg Q6W or NIVO 480 mg Q4W for ≤ 24 months, and 201 (135 tTMB-H; 147 bTMB-H) were refractory to standard therapies. Of > 40 tumor types, colorectal (10.8%), small-cell lung (7.5%), breast (7.1%), and uterine (7.1%) were the most common. ORR and survival outcomes with NIVO + IPI were improved in patients with tTMB-H. The responses were independent of bTMB-H status in the tTMB-H cohort but improved with tTMB-H status in the bTMB-H cohort (Table). The safety profile of NIVO + IPI was manageable, and clinical outcomes with NIVO were comparable with previous studies. The impact of TMB cutoff, PD-L1 expression, and microsatellite instability were explored. In conclusion, NIVO + IPI demonstrated clinical efficacy with a manageable safety profile in patients with advanced or metastatic solid tumors that are tTMB-H or bTMB-H and refractory to standard therapies, with increased efficacy observed in patients with tTMB-H. NIVO + IPI tTMB-H cohort bTMB-H cohorta Patients, n (%)b,c 68 (32.1) 80 (37.7) Number of prior treatments, median (range) 2 (0–7) 2 (1–9) ORR, n (%)c, 95% CI 24 (35.3), 24.1–47.8 18 (22.5), 13.9–33.2 ORR in patients with bTMB-H by tTMBc: < 10 mut/Mb (n = 31), n (%), 95% CI NA 3 (9.7), 2.0–25.8 ≥ 10 mut/Mb (n = 39), n (%), 95% CI NA 13 (33.3), 19.1–50.2 ≥ 10 to < 16 mut/Mb (n = 18), n (%), 95% CI NA 3 (16.7), 3.6–41.4 ≥ 16 mut/Mb (n = 21), n (%), 95% CI NA 10 (47.6), 25.7–70.2 ORR in patients with tTMB-H by bTMBc: < 10 mut/Mb (n = 20), n (%), 95% CI 7 (35.0), 15.4–59.2 NA ≥ 10 mut/Mb (n = 43), n (%), 95% CI 16 (37.2), 23.0–53.3 NA ≥ 10 to < 16 mut/Mb (n = 12), n (%), 95% CI 3 (25.0), 5.5–57.2 NA ≥ 16 mut/Mb (n = 31), n (%), 95% CI 13 (41.9), 24.5–60.9 NA Percentage of responders (≥ 9 months) (95% CI) 91 (68–98) 88 (61–97) Median PFS, months (95% CI)c 4.1 (2.8–11.3) 2.8 (2.3–3.0) Median OS, months (95% CI)c 14.5 (7.7–NE) 8.5 (5.8–10.5) aThe bTMB cohort was randomized prior to December 20, 2019. bOut of 212 randomized patients; data presented in this table are from patients who were refractory to standard therapies. cMinimum follow-up 12 months. bTMB, blood tumor mutational burden; NA, not applicable; NE, not evaluable; PFS, progression-free survival; OS, overall survival; tTMB, tissue tumor mutational burden. Citation Format: Michael Schenker, Mauricio Burotto, Martin Richardet, Tudor Ciuleanu, Anthony Goncalves, Neeltje Steeghs, Patrick Schöffski, Paolo A. Ascierto, Michele Maio, Iwona Lugowska, Lorena Lupinacci, Alexandra Leary, Jean-Pierre Delord, Julieta Grasselli, David S. Tan, Jennifer E. Friedmann, Jacqueline Vuky, Marina Tschaika, Ruta Slepetis, Georgia D. Kollia, Misena Pacius, Ning Huang, Parul Doshi, Jonathan Baden, Massimo Di Nicola. CheckMate 848: A randomized, open-label, phase 2 study of nivolumab in combination with ipilimumab or nivolumab monotherapy in patients with advanced or metastatic solid tumors of high tumor mutational burden [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 CT022.

Background: MEDI5752 is a monovalent bispecific antibody targeting PD-1 and CTLA-4. The bispecific format is engineered to preferentially inhibit these two checkpoints on activated T cells in tumors, where co-expression is particularly abundant. This may maximize tumor-specific response and reduce the peripheral toxicity that is common when targeting these checkpoints independently. Here we present dose-escalation data from a phase I, open-label, multicenter study in advanced solid tumors (NCT03530397). Methods: Eligible patients (pts) were ≥18 years old (ECOG PS 0-1) with tumors not amenable to standard therapy. Pts were treated at 10 dose levels from 2.25-2500 mg IV Q3W until progression or unacceptable toxicity. Primary objectives were safety and identification of maximum tolerated dose (MTD). Secondary objectives included preliminary antitumor activity by RECIST v1.1, PK and immunogenicity. Exploratory objectives included evaluation of pharmacodynamic (PD) biomarkers. Results: At data cutoff (Sept 30, 2021), 86 pts (median age, 60.5 years; male, 75.6%; Asian, 57.0%) were enrolled. The most common tumor types were RCC (22.1%), NSCLC (16.3%), and head and neck cancer (8.1%); 95.2% had received prior systemic therapy and 90.7% were immunotherapy-naïve. MEDI5752 showed dose-dependent PK and sustained peripheral PD-1 receptor occupancy (>90%) at doses >225 mg. Dose-dependent increases in peripheral T cell proliferation (Ki67+) and activation (ICOS+) plateaued at doses ≥500 mg and demonstrated CTLA-4 specific blockade in the range of tremelimumab 6-10 mg/kg. At doses ≥500 mg, MEDI5752 significantly expanded new and existing T-cell clones, likely broadening the tumor antigen-driven T-cell response. Across all doses, objective responses were seen in 19.8% of pts (n=17, 1 complete and 16 partial [PR]). Median duration of response was 17.5 months. Molecular response (defined as ≥50% reduction in ctDNA) occurred in 36.5%. Doses ≥1500 mg (n=53) were poorly tolerated with Grade (G) 3/4 treatment-related AEs (TRAEs) in 50.9%; discontinuations due to TRAEs in 45.3%, and death in 1 (at 2000 mg). However, doses Conclusions: MEDI5752 showed encouraging antitumor activity with durable clinical benefit. Doses Citation Format: Ben Tran, Mark Voskoboynik, Sang-We Kim, Charlotte Lemech, Enric Carcereny, Sun Young Rha, Myung-Ju Ahn, Enriqueta Felip, Ki H. Lee, Eduardo C. Alvarez, James Chih-Hsin Yang, Paolo A. Ascierto, Mariano P. Pulla, Dan Freeman, Xuyang Song, Shelby D. Gainer, Patrick Mitchell, Ikbel Achour, Deepa S. Subramaniam, Seock-Ah Im. MEDI5752, a novel PD-1/CTLA-4 bispecific checkpoint inhibitor for advanced solid tumors: First-in-human study [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 CT016.

Background: NIVO has demonstrated significant benefit over IPI as adjuvant treatment in pts with resected stage IIIB–C or stage IV melanoma (AJCC v7). Combination NIVO + IPI has shown numerically longer survival than NIVO alone in pts with metastatic melanoma. CheckMate 915 evaluated adjuvant NIVO + IPI 1 mg/kg Q6W vs NIVO. Methods: Pts aged ≥ 12 y with completely resected stage IIIB–D or stage IV melanoma (AJCC v8) were stratified by tumor PD-L1 expression and stage and treated with NIVO 240 mg Q2W + IPI 1 mg/kg Q6W (NIVO + IPI1) or placebo-controlled NIVO 480 mg Q4W alone for ≤ 1 y (NIVO + IPI were to be discontinued together). Dual endpoints were recurrence-free survival (RFS) in the PD-L1 < 1% and intent-to-treat populations. Distant metastasis-free survival (DMFS) in pts with stage III disease was an exploratory endpoint. Results: Of 920 pts randomized to NIVO + IPI1 and 924 to NIVO alone, most had stage IIIB (31% vs 31%) or IIIC (53% vs 52%) disease and had undergone complete lymph node dissection (64% vs 64%). Pts treated with NIVO + IPI1 vs NIVO alone had a shorter median duration of therapy (7.6 vs 11.1 mo) and therefore, a lower median cumulative NIVO dose (3840 vs 6240 mg). At a minimum follow-up of 24 mo, RFS and DMFS did not differ between treatment groups (table). Grade 3/4 treatment-related adverse events (TRAEs) occurred in 33% of pts treated with NIVO+IPI1 and 13% with NIVO alone; any-grade TRAEs led to discontinuation of therapy in 32% vs 10% of pts, respectively. There were 4 treatment-related deaths (all with NIVO + IPI1). Conclusions: The NIVO + IPI1 regimen did not result in RFS or DMFS improvement vs NIVO in stage IIIB–D/IV resectable melanoma; safety profiles were consistent with previous studies. NIVO 480 mg Q4W outcomes in CheckMate 915 were similar to previous NIVO results and reinforce NIVO as an adjuvant standard of care in a study population that included pts with and without complete lymphadenectomy. NIVO 240 mg Q2W + IPI 1 mg/kg Q6W (ITT)NIVO 480 mg Q4W (ITT)NIVO 240 mg Q2W + IPI 1 mg/kg Q6W (tumor PD-L1 < 1%)NIVO 480 mg Q4W (tumor PD-L1 < 1%)RFS24-mo rate64.6% (95% CI, 61.3-67.7)63.2% (95% CI, 59.9-66.4)53.6% (95% CI, 48.0-58.8)52.4% (95% CI, 46.8-57.7)Median, mo (events/pts)Not reached (327/920)Not reached (347/924)33.2 (159/349)25.3 (166/351)HR, NIVO + IPI1 vs NIVO0.92 (97.295% CI, 0.77-1.09); P = 0.2690.91 (95% CI, 0.73-1.14)DMFS in pts with stage III disease24-mo rate75.4% (95% CI, 72.1-78.4)77.4% (95% CI, 74.1-80.3)67.9% (95% CI, 61.9-73.1)68.4% (95% CI, 62.5-73.7)Median, mo (events/pts)Not reached (195/797)Not reached (194/798)Not reached (92/305)Not reached (96/307)HR, NIVO + IPI1 vs NIVO1.01 (95% CI, 0.83-1.23)0.94 (95% CI, 0.70-1.25) Citation Format: Georgina V. Long, Dirk Schadendorf, Michele Del Vecchio, James Larkin, Victoria Atkinson, Michael Schenker, Jacopo Pigozzo, Helen J. Gogas, Stéphane Dalle, Nicolas Meyer, Paolo A. Ascierto, Shahneen Sandhu, Thomas Eigentler, Ralf Gutzmer, Jessica C. Hassel, Caroline Robert, Matteo Carlino, Anna Maria Di Giacomo, Marcus O. Butler, Eva Muñoz-Couselo, Michael P. Brown, Piotr Rutkowski, Andrew Haydon, Jean-Jacques Grob, Jacob Schachter, Paola Queirolo, Alexander Menzies, Sandra Re, Tuba O. Bas, Veerle de Pril, Daniel Tenney, Hao Tang, Jeffrey S. Weber. Adjuvant therapy with nivolumab (NIVO) combined with ipilimumab (IPI) vs NIVO alone in patients (pts) with resected stage IIIB-D/IV melanoma (CheckMate 915) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT004.

Skin toxicity after anti-PD1 treatment in melanoma patients is the most common type of immune related adverse effect (irAE) and has been associated with improved overall response rate and survival. Nonetheless, not many mechanistic biomarkers have been identified so far, that could be associated with low-grade skin toxicity and good response rates. In this study we have addressed this question by analyzing tumor tissue samples from late-stage melanoma patients with first-line anti-PD1 treatment. Samples obtained prior to treatment were submitted for an unbiased deep proteomic analysis using mass spectrometry (LC-MS) and a targeted transcriptomic analysis. Using the unbiased analysis as a discovery platform we were able to define a potential biomarker panel associated not only with improved response but also low-grade skin toxicity. Unbiased quantification of proteins in tumor tissues was done using data-independent acquisition (DIA) LC-MS technology. Proteins from tissue samples were denatured, digested, and analyzed on a mass spectrometer. A deep spectral library was generated, and proteins were quantified using Spectronaut software (Biognosys). In addition, from the same tumor tissue RNA was extracted and subjected to transcriptomic analysis with NanoString nCounter using the PanCancer IO 360 panel. Subsequent data analysis was done using a sPLS-DA using combined factor of skin toxicity and response. Unbiased analysis of 22 baseline tumor tissue samples from late-stage melanoma patients treated in first line with anti-PD1 resulted in identification and quantification of more than 8000 proteins. Progression free survival analysis showed difference between patients with reported low-grade skin toxicity against all others. Therefore, for sPLS-DA both factors, presence/absence of skin toxicity and response status, were used (non-responders with low-grade skin toxicity were not present in this cohort). Complete separation of subjects was achieved with a panel of 21 proteins. This panel was used for hierarchical clustering and was able to fully restore all three groups of patients. Among all proteins identified in the proteomic panel, melanoma-associated antigen C1 (MAGEC1) has been also assessed in the targeted transcriptomic analysis and represents strikingly similar results. MAGEC1 is also found as a strong predictor in the Human Protein Atlas project. Interestingly, MAGE protein family are tumor-specific antigens that can be recognized by autologous cytolytic T lymphocytes and could serve as a novel ICI target or predictive biomarker. In this study we confirm prior observations of a survival benefit related to irAEs after treatment with PD-1 blockade in late-stage melanoma patients. We also demonstrate the power of deep proteomic profiling and transcriptomic analysis in molecular biomarker selection associated to response and irAEs which further benefit patient survival. Citation Format: Jakob Vowinckel, Domenico Mallardo, Kamil Sklodowski, Martin Soste, Mariaelena Capone, Gabriele Madonna, Vito Vanella, Sarah Warren, Kristina Beeler, Paolo A. Ascierto. Response and skin toxicity related protein signature in late stage melanoma patients after anti-PD-1 treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1623.

Background: Approved systemic treatments for advanced melanoma include immune checkpoint inhibitor therapy (CIT) and targeted therapy with BRAF plus MEK inhibitors for BRAFV600E/K mutant melanoma. Response rates with CITs are typically lower than those observed with targeted therapy, but CIT responses are more durable. Preclinical and clinical data suggest a potential for synergy between CIT and BRAF plus MEK inhibitors. We therefore evaluated whether combining CIT with targeted therapy could improve efficacy vs targeted therapy alone. Methods: Treatment-naive patients with unresectable stage IIIc/IV melanoma (AJCC 7th ed), measurable disease by RECIST 1.1, and BRAFV600 mutations in their tumors were randomized to the anti-programmed death-ligand 1 antibody A + C + V or placebo (Pbo) + C + V. A or Pbo were given on days 1 and 15 of each 28-day cycle. Treatment was continued until disease progression or unacceptable toxicity. The primary outcome was investigator-assessed progression-free survival (PFS). Results: 514 patients were enrolled (A + C + V = 256; Pbo + C + V = 258) and followed for a median of 18.9 months. Investigator-assessed PFS was significantly prolonged with A + C + V vs Pbo + C + V (15.1 vs 10.6 months, respectively; hazard ratio: 0.78; 95% confidence interval: 0.63-0.97; P=0.025), an effect seen in all prognostic subgroups. While objective response rates were similar in the A + C + V and Pbo + C + V groups, median duration of response was prolonged with A + C + V (21.0 months) vs Pbo + C + V (12.6 months). Overall survival data were not mature at the time of analysis. Common treatment-related adverse events (AEs; >30%) in the A + C + V and Pbo + C + V groups were blood creatinine phosphokinase (CPK) increase (51.3% vs 44.8%), diarrhea (42.2% vs 46.6%), rash (40.9% in both arms), arthralgia (39.1% vs 28.1%), pyrexia (38.7% vs 26.0%), alanine aminotransferase (ALT) increase (33.9% vs 22.8%), and lipase increase (32.2% vs 27.4%). Common treatment-related grade 3/4 AEs (>10%) that occurred in the A + C + V and Pbo + C + V groups were lipase increase (20.4% vs 20.6%), blood CPK increase (20.0% vs 14.9%), ALT increase (13.0% vs 8.9%), and maculopapular rash (12.6% vs 9.6%). The incidence of treatment-related serious AEs was similar between the A + C + V (33.5%) and Pbo + C + V (28.8%) groups. 12.6% of patients in the A + C + V group and 15.7% in the Pbo + C + V group stopped all treatment because of AEs. The safety profile of the A + C + V regimen was generally consistent with the known profiles of the individual components. Conclusion: Combination therapy with A + C + V was tolerable and manageable, produced durable responses, and significantly increased PFS vs Pbo + C + V. Thus, A + C + V represents a viable treatment option for BRAFV600 mutation-positive advanced melanoma. ClinicalTrials.gov ID: NCT02908672 Citation Format: Grant A. McArthur, Daniil Stroyakovskiy, Helen Gogas, Caroline Robert, Karl Lewis, Svetlana Protsenko, Rodrigo Pereira, Thomas Eigentler, Piotr Rutkowski, Lev Demidov, Georgy Moiseevich Manikhas, Yibing Yan, K. C. Huang, Anne Uyei, Virgina McNally, Ralf Gutzmer, Paolo Ascierto. Evaluation of atezolizumab (A), cobimetinib (C), and vemurafenib (V) in previously untreated patients with BRAFV600 mutation-positive advanced melanoma: Primary results from the phase 3 IMspire150 trial [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT012.

Background and Aims: Hepatocellular carcinoma (HCC) is the third leading cause of death from cancer globally with an extremely variable 5-year survival rate. Immunotherapy strategies for HCC may represent a key therapeutic tool to improve clinical outcome in HCC patients. HepaVac-101 (EudraCT Number: 2015-003389-10; NCT03203005), is a single-arm, first-in-man Phase I/II clinical trial evaluating a therapeutic cancer vaccine in patients affected by HCC. It is a highly innovative, novel approach based on a multi-peptide vaccine (IMA970A) combined with the TLR7/8/RIG I agonist CV8102 as an adjuvant. Method: The IMA970A off-the-shelf vaccine includes 5 HLA-A*24 and 7 HLA-A*02 as well as 4 HLA-DR restricted peptides identified and selected from native human HCC tumor tissue by applying the XPRESIDENT® discovery platform. CV8102 is a novel ribonucleic acid (RNA) based immunostimulatory agent inducing a balanced Th1/Th2 immune response.HLA-A*02 and/or A*24-positive patients with very early, early and intermediate stage HCCs have been enrolled to be treated with 9 intradermal vaccinations consisting of IMA970A plus CV8102 following a single pre-vaccination infusion of low-dose cyclophosphamide acting as an immunomodulator. Patients have completed standard of care treatments at time of enrolment and received study drugs without concomitant anti-tumor therapy with no evidence of disease reactivation. The primary endpoints of the HepaVac-101 clinical trial are safety, tolerability, and antigen specific T cell response. Secondary/exploratory endpoints are additional immunological parameters in blood, infiltrating T-lymphocytes in tumor tissue, biomarkers in blood and tissue, disease-free survival/progression-free survival and overall survival. Results: Patients were enrolled in 6 centers located in 5 European countries i.e. Italy (Naples and Negrar/Varese), Germany (Tübingen), UK (Birmingham), Spain (Pamplona) and Belgium (Antwerp). The end of trial (EOT) was reached on last December 20, 2019. 82 HCC patients have been screened for suitable HLA haplotypes, 22 patients were put on study treatment (i.e. received at least the pre-treatment with cyclophosphamide). The vaccination showed a good safety profile with no major SAE nor AESI reported during the protocol. Immunogenicity data are currently evaluated and will be available at time of the meeting. Conclusion: The HEPAVAC-101 clinical trial is the achievement of the HEPAVAC Consortium supported by the European Commission's 7th framework program with contract No. 602893 (www.hepavac.eu). Citation Format: Luigi Buonaguro, Andrea Mayer, Markus Loeffler, Sarah Missel, Roberto Accolla, Yuk Ting Ma, Tanguy Chaumette, Regina Heidenreich, Alfred Königsrainer, Joerg Ludwig, Diego Alcoba, Heiko Schuster, Cecile Gouttenfangeas, Greta Forlani, Maria Tagliamonte, Paolo A. Ascierto, Antonio Avallone, Marco Borrelli, Hans-Georg Rammensee, Bruno Sangro, Mercedes Iñarrairaegui, Sven Francque, Luisa Vonghia, Stefania Gori, Tony Weinschenk, Carsten Reinhardt, Ulrike Gnad-Vogt, Harpreet Singh. Hepavac-101 first-in-man clinical trial of a multi-peptide-based vaccine for hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-094.

In relation to the spread of severe acute respiratory syndrome coronavirus 2 (SARS CoV2), oncologists need to face some difficult situations. First of all, safety considerations about cancer patients during this pandemia. To keep safe both patients and healthy workers is the most important rule. In our daily practice we adopted a specific policy about the conduct of our clinical managements of melanoma patients, in order to minimise the risk of any potential exposure. We are prioritizing patients, according to the kind of treatment and the stage of disease. In order to avoid any gathering condition which could increase the risk of infection for patients, we are optimizing access about adjuvant treatment, considering of high priority the higher risk patients, privileging longer infusion schedule of treatment for immunotherapy and/or selecting patients with BRAF mutation for starting target therapy. Metastatic patients have the highest priority, and nothing changed in their daily management except for preferring, when possible, the longer schedule of treatment in order to reduce the access in the hospital. The enrolment in clinical trials is on hold and the patients still in treatment inside clinical study are managed in the respect of GCPs as well as possible. Some additional issues are side effects in patients on treatment with target therapy and immunotherapy. About target therapy (especially dabrafenib and trametinib), patients who develop fever higher than 37.5 without resolving after discontinuation, should perform COVID test before to restart the treatment. Patients on treatment with immunotherapy who show pneumonitis at CT scan (even without fever), should perform COVID test before start steroids. Immuno-oncologists have an important experience in the management of immuno-related adverse events. The hyperactivation of immune system due to the immunotherapy strategies can develop some conditions which need of immuno-suppressive drugs to reduce the harmful immune reaction. Since the acute respiratory stress syndrome COVID-19 related seems to occur from an excess of cytokine production, we focused our attention on the cytokines storm which probably lead to ARDS by COVID-19 and how to prevent or treat it. We know very well the Cytokine Release Syndrome (CRS), one of the most prominent and well described toxicity from chimeric antigen receptor T cell therapy (CAR-T), as well as from some bispecific antibodies. In particular, we know the key role played by IL-6 in the pathogenesis of these kind of hyperinflammation syndromes. Considering elevated serum concentration of IL-6 and CPR in patients admitted in ICUs department, we started to use monoclonal antibodies against IL6, above all tocilizumab. In Italy we started on 19th of March a phase II study (NCT04317092) which enrolled 330 patients in 24 hours, with the ability of tocilizumab to reduce the one-month mortality rate as main endpoint. Results should be reported at the end of April. Other drugs active in reducing the CRS are at moment in clinical trial or expanded access program like JAK inhibitors, complement inhibitors, toll like receptor inhibitors and others. Citation Format: Paolo A. Ascierto. Experience in using oncology drugs in patients with COVID-19 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT405.

Background Immune checkpoint inhibitors (ICI) have greatly improved the treatment options for patients with advanced stage melanoma, with improved clinical responses and overall survival compared to standard systemic therapies. However, a large percentage of melanoma patients do not respond to ICIs, highlighting the need for a greater understanding of the tumor environment and host immune response. Here, we apply unbiased discovery proteomics, based on label-free data independent acquisition (DIA) mass spectrometry, to deeply characterize global tumor proteomes to identify proteins and pathways that are associated with pre-treatment response to anti-PD-1 immunotherapy. Methods Unbiased, data-independent acquisition (DIA) mass spectrometry was used to analyze formalin fixed paraffin imbedded (FFPE) tumor tissue samples from subjects with Stage III-IV melanoma which were resected prior to initiation of first-line anti-PD-1 ICI therapy. The selected samples represent two distinct clinical subgroups; those who received clinical benefit, with a stable disease or better (SD, PR and CR, n = 13), and those with no clinical benefit (PD, n = 9). Samples were prepared for mass spectrometry using standard procedures. All samples were analyzed using 4-hour gradients on a LC-MS/MS setup operated in DIA mode. Data was extracted using Spectronaut (Biognosys) with a sample specific spectral library which was combined with a large human tissue resource library. Statistical analysis was conducted to identify proteins that are up- or down-regulated with respect to benefit group. Pathway analysis was also conducted to highlight dysregulated biological functions and pathways. Results In analysis with 2-hour gradients, >7,500 proteins were quantified across all samples. Univariate statistical testing between groups identified 254 proteins are dysregulated (120 up- and 134 down-regulated) in subjects who received clinical benefit, of which a subset of 25 proteins was identified that describe the variance between the two sample groups. When annotated to their sub-cellular location, all up-regulated species are identified as mitochondrial proteins, indicating an enhanced metabolic environment in the responder subgroup. Additionally, GM2A and PLEKHA5 were strong diagnostic predictors of responder status. This updated analysis, conducted with a deeper level of characterization, will focus on additional metabolic pathways as well as known proteins associated with metabolic resistance (e.g. CD39 and CD73) to more fully characterize the tumor metabolic environment. Conclusions Global profiling of the tumor proteome provides a unique characterization of melanoma tumor biology. A pathway level analysis shows increased metabolic processes may underly some of the differences in benefit related to ICI therapy. Citation Format: Nicholas Dupuis, Jakob Vowinckel, Domenico Mallardo, Mariaelena Capone, Madonna Gabriele, Antonio Sorrentino, Vito Vanella, Daniel Heinzmann, Paolo Ascierto. Proteomic profiling of FFPE tumors samples from melanoma subjects treated with anti-PD-1 immunotherapy identifies proteins associated with response to treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5529.

Background: Significant advancements, including development of immune checkpoint inhibitors and targeted therapies, have transformed outcomes in patients (pts) with unresectable or metastatic melanoma. However, pts who do not respond or who progress while receiving these regimens have limited options. Spartalizumab (PDR001) is a high-affinity, humanized monoclonal antibody blocking the programmed cell death 1 (PD-1) receptor. This study is evaluating combinations of spartalizumab with novel compounds to restore antitumor T-cell activity in pts with melanoma progressing after prior PD-1 blockade therapy. Methods: This randomized, open-label, 2-part, multicenter, open-platform, Phase II study (NCT03484923; PLATforM) will evaluate the safety and efficacy of spartalizumab combination treatment in pts with unresectable or metastatic melanoma progressing after prior anti-PD-1/L1 therapy and, if the tumor harbors a BRAF V600 mutation, a BRAF inhibitor. The primary endpoint is objective response rate per RECIST v1.1, with duration of response and assessment of paired tumor biopsies for biomarkers of antitumor T-cell activity as part of the secondary endpoints. Part 1, “selection”, will begin with 3 combination arms: (1) spartalizumab + LAG525 (LAG-3 antibody), (2) spartalizumab + capmatinib (c-MET inhibitor), and (3) spartalizumab + canakinumab (IL-1β antagonist). The PLATforM study will use an adaptive design during the selection part that allows for dropping arms for futility, adding new arms, and selecting 1 or multiple arms for further expansion. Bayesian methodology will be used with specific probability criteria for futility and efficacy assessments at each interim analysis. Pts (≈ 60-85) will be stratified by baseline lactate dehydrogenase level and randomized equally to all open arms during the selection part. Part 2, “expansion”, will further investigate the efficacy and safety of treatment combination(s) selected during part 1. The sample size for part 2 will be adaptive and based on predictive power calculations considering the results from part 1. Previously presented at ESMO 2018, FPN 1304TiP, Weber et al. Reused with permission. Citation Format: Caroline Robert, Reinhard Dummer, Ana Arance, Antoni Ribas, Jeffrey Weber, Georgina V. Long, John Haanen, Paul Nathan, Paolo A. Ascierto, Eduard Gasal, Anthony D'Amelio, Severine Bettinger, Aislyn D. Boran, Dirk Schadendorf. A randomized, open-label, open-platform, Phase II study evaluating the efficacy and safety of novel spartalizumab (PDR001) combinations in previously treated unresectable or metastatic melanoma (PLATforM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT120.

Background: Increased tumor mutational burden (TMB) and inflammation are associated with improved clinical outcomes to immuno-oncology (I-O) therapy in many tumor types. Genomic correlates of response to nivolumab (N) vs dacarbazine (D) (CheckMate [CM] 066; NCT01721772) and N+ipilimumab (I) combination therapy or N vs I (CM 067; NCT01844505) were evaluated for association of TMB and an inflammatory gene signature with clinical outcomes. Methods: In pretreatment tumor samples from eligible patients (pts), TMB was analyzed by whole-exome sequencing using median number of total missense mutations to define high vs low TMB (TMB-H/TMB-L). Associations of TMB with progression-free survival (PFS) and overall survival (OS) were evaluated per protocol-defined exploratory analyses using 4-year follow-up data for both trials. PFS and OS associations with an inflammatory signature were assessed by RNAseq in CM 067 samples only and grouped into tertiles for analysis. HR and 95% CI were calculated using Cox modeling and P values were calculated using the Wald test. Results: In CM 066, TMB was evaluable in 122 of 411 pts. PFS and OS were significantly longer in pts treated with N, but not with D, for TMB-H vs TMB-L tumors. The benefit of PFS and OS for N vs D was greater in pts with TMB-H than TMB-L (Table). In CM 067, 583 of 937 pts were evaluable for TMB and 299 were evaluable using an inflammatory signature. PFS and OS were longer in each treatment (tx) arm in pts with TMB-H vs TMB-L (Table). Across tx arms, a numerical benefit was observed for PFS and OS in pts with high vs low inflammatory signature (Table). TMB-H and a high inflammatory signature score were independently associated with clinical outcomes to I-O. Conclusions: High TMB or high inflammatory signature was associated with benefit to I-O. TMB status did not differentiate between N+I and N, and higher inflammatory status increased the likelihood of benefit for N+I and N, suggesting further evaluation of these biomarkers to characterize the response to I-O regimens in melanoma. Association of TMB (CM 066) or TMB and inflammatory signature (CM 067) with PFS and OSCategorical comparisonClinical measureHR (95% CI; P value)CM 066NDPatients with evaluable TMB, n (high, low)53 (23, 30)69 (38, 31)TMB-H vs TMB-LPFSOS0.33 (0.16-0.69; 0.0031)0.43 (0.2-0.91; 0.027)0.67 (0.39-1.10; 0.13)0.66 (0.39-1.10; 0.13)N vs D in TMB-HPFSOS0.28 (0.14-0.59; 0.00083)0.35 (0.17-0.73; 0.0046)N vs D in TMB-LPFSOS0.57 (0.33-0.99; 0.047)0.55 (0.31-0.98; 0.044)Categorical comparisonClinical measureHR (95% CI; P value)CM 067N+INIPatients with evaluable TMB, n (high, low)197 (94, 103)192 (95, 97)194 (101, 93)TMB-H vs TMB-LPFSOS0.56 (0.39-0.81; 0.002)0.53 (0.34-0.81; 0.0032)0.45 (0.31-0.65; 0.00002)0.48 (0.31-0.72; 0.00046)0.64 (0.47-0.87; 0.005)0.59 (0.42-0.83; 0.0022)N+I vs N in TMB-HPFSOS0.95 (0.63-1.40; 0.81)0.97 (0.60-1.60; 0.88)-N+I vs N in TMB-LPFSOS0.77 (0.56-1.10; 0.12)0.87 (0.61-1.30; 0.46)-Patients with evaluable inflammatory signature, n (low, med, high)93 (33, 32, 28)106 (39, 31, 36)100 (28, 36, 36)Inflammatory signature, high vs lowPFSOS0.38 (0.18-0.80; 0.01)0.53 (0.24-1.18; 0.12)0.60 (0.34-1.00; 0.069)0.35 (0.18-0.69; 0.0025)0.48 (0.27-0.85; 0.012)0.52 (0.27-1.00; 0.049) Citation Format: F. Stephen Hodi, Jedd D. Wolchok, Dirk Schadendorf, James Larkin, Max Qian, Abdel Saci, Tina C. Young, Sujaya Srinivasan, Han Chang, Megan Wind-Rotolo, Jasmine I. Rizzo, Donald G. Jackson, Paolo A. Ascierto. Genomic analyses and immunotherapy in advanced melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT037.

Introduction: The phase 3 COMBI-i study (NCT02967692) is evaluating spartalizumab (S; PDR001) in combination with dabrafenib + trametinib (D+T) in treatment-naive patients (pts) with BRAF V600–mutant metastatic melanoma. COMBI-i comprises 3 parts: (1) safety run-in; (2) biomarker part; and (3) double-blind, randomized, placebo-controlled part. In part 1, 9/9 pts had confirmed responses (Dummer et al. ASCO-SITC 2018). The primary objective for part 2 is to evaluate changes in PD-L1 levels and CD8+ cells after treatment with S+D+T. Here, we report first efficacy, safety, and biomarker data for part 2. Methods: In the biomarker cohort, tissue samples were collected at baseline (BL) and, if visible lesions were present, after 2-3 wk, after 8-12 wk, and at disease progression. CD8 and PD-L1 (using DAKO 28-8 assay) were examined by IHC. Any remaining tumor material was analyzed for additional biomarkers (eg, LAG3, TIM3, FOXP3, CD163). Peripheral blood mononuclear cells and plasma samples were collected at several time points for flow cytometry, ctDNA analysis, and cytokine profiling. Results: At the data cutoff (27 Oct 2017) 23 pts received S 400 mg Q4W + D 150 mg BID + T 2 mg QD. 13 pts had completed 8 wk of treatment and median follow-up was 2.0 mo (range, 0.1-3.5). At study entry, median age was 62 y. 20 pts (87%) had stage IV disease and 3 had unresectable stage III disease; 8/9 pts with stage IV M1c had elevated LDH. Adverse events (AEs) of any grade occurred in 91% of pts; 22% (n=5) were grade 3/4. No AEs led to discontinuation. Any-grade AEs occurring in ≥ 15% of pts included pyrexia (n=14, 61%), arthralgia, fatigue, rash, and vomiting (n=4 each, 17%). The only grade 3/4 event occurring in > 1 pt was pyrexia (n=2, 8.7%), which was managed with dose interruption. All 7 pts evaluable for response at first post-BL assessment at wk 12 had an unconfirmed partial response; no pts had progressive disease. In preliminary analyses of matched paired BL and on-treatment tumor samples (n=9), 4/9 tumors had no intratumoral CD8+ cells and 5/9 had very few intratumoral CD8+ cells at BL. After 2-3 wk of S+D+T treatment, 8/9 pts had an increase in intratumoral CD8+ cells; most increases were substantial. CD8+/PD-L1 and other tissue-specific and circulating biomarkers will be presented in a larger set of samples. Conclusions: Although follow-up was limited and pt numbers were small, initial data support use of the anti–PD-1 antibody S with D+T in pts with BRAF V600–mutant unresectable or metastatic melanoma. The manageable safety profile and promising efficacy are consistent with part 1 findings from COMBI-i. Initial biomarker data suggest that S+D+T has a beneficial effect on the tumor immune microenvironment, particularly on enhancing intratumoral CD8+ T-cell infiltration. Citation Format: Reinhard Dummer, Dirk Schadendorf, Paul Nathan, Hussein Tawbi, Caroline Robert, Paolo A. Ascierto, Antoni Ribas, Celeste Lebbé, Mario Mandala, Naoya Yamazaki, Erika Richtig, Wilson H. Miller, Eduard Gasal, Mathilde Kaper, Jan C. Brase, Bijoyesh Mookerjee, Georgina V. Long. The anti-PD-1 antibody spartalizumab (PDR001) in combination with dabrafenib and trametinib in previously untreated patients with advanced BRAF V600-mutant melanoma: First efficacy, safety, and biomarker findings from the part 2 biomarker cohort of COMBi-i [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT182.

Introduction: The successful deployment of checkpoint inhibitors in cancer immunotherapy relies on the responsiveness of an individual's immune system for relief of that particular blockade in the cancer immunity cycle. As most patients fail to respond to immunotherapy, there is a need for biomarkers that can predict clinical benefit of the therapeutic by identifying the patient population most likely to respond. Gene expression signatures characterizing basal immune state within tumor have shown utility in retrospective analysis of clinical trials. This study utilizes tumor and PBMCs from patients with metastatic melanoma receiving either anti-CTLA-4 or anti-PD-1/PD-L1 to characterize local and peripheral patterns of gene expression associated with clinical benefit of therapy. The goal of this study is to evaluate existing gene signatures and develop novel signatures that predict response to checkpoint inhibitors in melanoma. Methods: Pretreatment biopsies from metastatic lesions of melanoma stage IV patients treated with ipilimumab (anti-CTLA4) or pembrolizumab (anti-PD-L1) were retrieved from the Department of Pathology archives. Cohorts were assembled of 30 patients receiving each drug, equally stratified between responders and nonresponders. RNA expression in tumor biopsies will be profiled with a pilot version of the NanoString® IO360 gene expression panel. In parallel, pre- and post-treatment PBMC from independent cohorts receiving either ipilimumab (anti-CTLA4) or pembrolizumab (anti-PD-1) were collected and profiled with the NanoString PanCancer Immune Panel (gene expression) and Immune Profiling Panel (protein expression). Results: Patterns of gene expression in the tumor biopsies and gene and protein expression in the PBMCs will be assessed for correlation to clinical outcome (PFS, OS, ORR). Specifically, the Tumor Inflammation Signature described by Ayers et al. (1), an investigational 18-gene signature of suppressed adaptive immune response that enriches for clinical response to pembrolizumab, will be assessed in these cohorts. Other patterns of gene and protein expression that correlate with response to immunotherapy or lack thereof will also be evaluated. Conclusion: Correlating patterns of gene expression from tumor with clinical response can lead to the development of biomarkers to better select patients who will respond to immunotherapy. It can also lead to identifying immune evasion. Gene and protein profiling from a readily accessed sample such as PBMC may give insights into early on-treatment signatures of efficacy. Utilization of a clinical grade platform such as the NanoString nCounter® may speed the development of diagnostic assays that can be used to predict and monitor patient response to immunotherapy. Reference: 1. Ayers et al., J Clin Invest 2017;127:2930. Citation Format: Mariaelena Capone, Gabriele Madonna, Paolo Ascierto, Patrick Danaher, SuFey Ong, Sarah Warren, Joseph M. Beechem, Alessandra Cesano. Prognostic gene signature use in checkpoint inhibitor monotherapy for melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 558.

Background: The checkpoint inhibitors ipilimumab and nivolumab are approved by FDA as adjuvant therapy for patients with resected high-risk stage III melanoma. We conducted the phase 3 double-blind EORTC 1325/KEYNOTE-054 trial to evaluate pembrolizumab vs placebo in patients with resected high-risk stage III melanoma. Methods: Eligible patients included those ≥18 years of age with complete resection of cutaneous melanoma metastatic to lymph node(s), classified as stage IIIA (excluding lymph node metastasis ≤1 mm), IIIB or IIIC (excluding those with in-transit metastasis). A total of 1019 patients were randomized (stratification by stage and region) to pembrolizumab at a flat dose of 200 mg (N=514) or placebo (N=505) every 3 weeks for a total of 18 doses (~1 year) or until disease recurrence or unacceptable toxicity. In the absence of brain metastases, patients with recurrence were eligible for cross-over or re-challenge (those who received pembrolizumab and recurred more than 6 months after completing 1 year of therapy). The 2 co-primary endpoints were recurrence-free survival (RFS) in the intention-to-treat overall population and in patients with PD-L1-positive tumors. A total of 409 RFS events were needed to provide 92% power to detect a hazard ratio (HR) for recurrence or death of 0.70 (1-sided alpha=1.4%). The interim analysis was performed at 1-sided alpha=0.8% (O'Brien-Fleming alpha error spending function) in the overall patient population, and at 2.5% in the PD-L1 positive population. Results: Overall, 15%/46.5%/37.5% of patients had stage IIIA/IIIB/IIIC. At 1.25 yr median follow-up, pembrolizumab (135 events) compared with placebo (216 events) significantly prolonged RFS in the overall population (12-month RFS rate: 75.4% vs 61.0%; HR 0.57, 98.4% CI 0.43-0.74; P Conclusions: As adjuvant therapy for resected high-risk stage III melanoma pembrolizumab 200 mg every 3 weeks, for up to 1 year, resulted in significantly prolonged RFS with a favorable benefit-risk profile. Citation Format: Alexander M. Eggermont, Christian U. Blank, Mario Mandala, Georgina V. Long, Victoria Atkinson, Stéphane Dalle, Andrew Haydon, Mikhail Lichinitser, Adnan Khattak, Matteo S. Carlino, Shahneen Sandhu, James Larkin, Susana Puig, Paolo A. Ascierto, Piotr Rutkowski, Dirk Schadendorf, Rutger Koornstra, Leonel Hernandez-Aya, Michele Maio, Alfonsus J. van den Eertwegh, Jean-Jacques Grob, Ralf Gutzmer, Rahima Jamal, Veronica Rivas, Nageatte Ibrahim, Sandrine Marreaud, Sven Janssen, Alexander van Akkooi, Stefan Suciu, Caroline Robert. Pembrolizumab versus placebo after complete resection of high-risk stage III melanoma: Efficacy and safety results from the EORTC 1325-MG/Keynote 054 double-blinded phase III trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT001.

Background and aim: FKBP51 is an immunophilin encoded by FKBP5 gene on chromosome 6. It is a protein resident in lymphocytes and involved in immune response. Recently, our group demonstrated that the inhibitory checkpoint PD-L1/PD1 promoted the alternative splicing of FKBP5 gene, resulting in increased expression of its variant 4, in PBMC of melanoma patients. The aim of this study was to address whether such a molecular signature could help in identifying an immune profile associated with increased probability of immunotherapy response. Experimental Design: The splicing FKBP51 isoform or FKBP51s was measured in peripheral blood T lymphocytes subsets (CD3/CD4, CD3/CD8, CD25 and PD-L1) and CD14 monocytes from a cohort of 118 patients and 77 age- and sex-matched healthy controls, by flow cytometry. Blood samples were collected before patients underwent ipilimumab treatment. Furthermore, in 64 out of 118 patients, expression of FKBP51s was also assessed in regulatory T cells. Results: Physiologically, each PBMC subset analyzed contained a fraction of an FKBP51spos component, which resulted expanded in melanoma patients. We also measured an increase in CD3/CD8 and PD-L1 lymphocytes in patients. CD4 T lymphocytes showed the FKBP51sneg fraction significantly impaired, which might reflect the condition of impaired T cell help. Treg count was increased, in accordance with previous studies. The count of FKBP51sposTregs defined a subgroup of nonresponder patients to ipilimumab, by 92.6%. A 2D hierarchical partitioning of data from FKBP51s-immunophenotype (heatmap) revealed 3 main clusters: C1 (33 pts, 51,5%), C2 (14 pts, 22%) and C3 (17 pts, 26,5%). FKBP51spos Treg subset appeared globally increased in all clusters. In C1, values of effector T cells and monocytes were not different from normal donors. C2 showed a significant increase of FKBP51spos PD-L1 monocytes and a significant probability of not responding (Chi-square=5.46; p=0.019). This finding is consistent with the hypothesis that response to ipilimumab is prevented by accessory cells exerting a negative immune regulatory control. C3 showed a significant increase of FKBP51s in overall lymphocyte subsets analyzed. Patients in this cluster showed reduced overall survival. This finding suggests that a prevalence of activation (CD25) and co-inhibitory (PD-L1) markers together with the expansion of FKBP51spos effector T cells might reflect a condition of chronic lymphocyte stimulation in some advanced melanoma patients, contributing to T cell-exhaustion. Conclusions: FKBP51s-based immunophenotype of melanoma patients revealed several profiles virtually related to a negative immune regulatory control and highlights an impairment of a Treg subset endowed with increased suppressive potential. Such FKBP51sTreg subset is likely to be associated with immunotherapy response (Chi-square=9.916, p=0.002) . Citation Format: Simona Romano, Ester Simeone, Anna D'Angelillo, Paolo D'Arrigo, Mario Capasso, Vito Alessandro Lasorsa, Martina Tufano, Anna Rea, Michele Russo, Nicola Zambrano, Paolo Antonio Ascierto, Maria Fiammetta Romano. An FKBP5-based immunophenotype for assessment of the immunosuppression status and possible prediction of immunotherapy response in melanoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2624. doi:10.1158/1538-7445.AM2017-2624

Introduction: Malignant melanoma is the most aggressive form of skin cancer presenting in approximately 50% of cases activating mutations in the BRAF oncogene. For these patients new therapeutic options are represented by the combination of BRAF inhibitors and MEK-inhibitors. However, a major limitation is the occurrence of resistance related in the majority of cases to mutations causing either reactivation of the MAPK/ERK pathway or of the PI3K/PTEN/AKT pathway. This complex scenario is worsened by the absence, in a significant proportion of cases, of new mutations. This suggests the involvement also of epigenetic or post-transcriptional changes at the basis of resistance. We have focused our attention over the past years to adaptive changes occurring in melanoma cells upon exposure to BRAF and/or MEK inhibitors in order to identify new therapeutic targets. Here we hypothesize that miRNAs deregulation upon cell exposure to kinase inhibitors may contribute to the development of drug resistance. Experimental procedures: The involvement of specific miRNAs in the establishment of drug resistance in melanoma was investigated through the online software miRò while target predictions were performed with TargetScan and microrna.org. Impact on melanoma cell growth, migration and establishment of drug resistance was assessed through enforced-transient miR overexpression. Tumor biopsies were obtained from patients before therapy with kinase inhibitors or after relapse and used for RNA extraction. Results: We identified by bioinformatics analysis a novel miRNA, miR-579-3p, relevant for BRAF mutated melanoma progression and found that it targets both BRAF, the relevant oncoprotein in these melanomas, and MDM2. miR-579-3p expression levels are high in nevi, heavily decline in stage III-IV melanomas and further decrease in melanomacells selected in vitro for resistance to BRAF and/or MEK inhibitors. Through in vitro studies we obtained evidence that mir-579-3p is able to inhibit melanoma cell growth and migration and to induce apoptosis alone or in combination with BRAF and/or MEKi. Moreover we found that miR-579-3p overexpression impairs the establishment of resistance to BRAFi in human melanoma cells. Finally we found this miR to be down-regulated in tumor samples obtained from patients who developed resistance to target therapies and its expression to be inversely correlated to the expression levels of its target genes, BRAF and MDM2. Conclusions: Our results strongly demonstrate that miR-579-3p is a novel oncosuppressor miRNA, controlling melanoma progression and development of drug resistance and which could be used as a new therapeutic target for intervention. Citation Format: Luigi Fattore, Mario Acunzo, Giulia Romano, Alessandro Laganà, Debora Malpicci, Maria Elena Pisanu, Antonio Maria Grimaldi, Franco Fulciniti, Carlo Maria Croce, Rita Mancini, Paolo Antonio Ascierto, Gennaro Ciliberto. miR-579-3p is a novel master regulator of melanoma progression and drug resistance in metastatic melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1070.

Background. BRAFV600E mediated-MAPK pathway activation is associated in melanoma cells with IFNAR1 down-regulation. IFNAR1 regulates melanoma cell sensitivity to IFNα, a cytokine currently used for the adjuvant treatment of melanoma patients. These findings in conjunction with limited therapeutic efficacy of BRAF-I prompted us to examine whether the efficacy of IFNα therapy of melanoma harboring BRAFV600E can be increased by its combination with BRAF-I. Methods. BRAF/NRAS genotype, ERK activation, IFNAR1 and HLA class I antigen expression were tested in 60 primary melanoma tumors of treatment naïve patients. The effect of BRAF-I on IFNAR1 expression was assessed in 3 melanoma cell lines and in 4 tumor biopsies of BRAFV600E metastatic melanomas. The anti-proliferative, pro-apoptotic and immunomodulatory activity of BRAF-I and IFNα combination was tested both in vitro and in vivo utilizing 3 melanoma cell lines, HLA class I antigen-MA derived peptide complex-specific T cells and immunodeficient mice (5 mice /group for mice survival and 10 mice/group for inhibition of tumor growth). All statistical tests were two-sided. Differences were considered statistically significant when the P value was Citation Format: Francesco Sabbatino, Yangyang Wang, Giosuè Scognamiglio, Elvira Favoino, Steven A. Feldman, Vincenzo Villani, Keith T. Flaherty, Sjoerd Nota, Diana Giannarelli, Ester Simeone, Anna M. Anniciello, Giuseppe Palmieri, Stefano Pepe, Gerardo Botti, Paolo A. Ascierto, Cristina R. Ferrone, Soldano Ferrone. Anti-tumor activity of a BRAF inhibitor and IFNα combination in BRAF mutant melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4017.

Melanoma tumors often harbor mutations in the mitogen-activated protein kinase-signaling pathway family members BRAF or NRAS. NRAS mutations, observed in about 20% of patients with melanoma, are associated with higher tumor proliferation and poorer prognosis. A large retrospective analysis (n = 677) demonstrated that NRAS mutations are independently predictive of poor survival in patients with cutaneous melanoma (Jakob et al., 2011). There are no approved targeted therapies for melanoma patients with NRAS-mutant tumors; treatments are currently limited to chemotherapy and/or immunotherapy. Binimetinib (MEK162), a potent and selective inhibitor of MEK1/2, has demonstrated promising phase 2 clinical activity in this patient subset. Here we describe the “NRAS mElanoma and MEK inhibitOr” (NEMO) trial, an ongoing 2-arm, open-label, 2:1 randomized phase 3 study designed to compare the efficacy of binimetinib vs dacarbazine in patients with metastatic NRAS-mutant melanoma (NCT01763164). Eligible patients must have advanced unresectable or metastatic cutaneous melanoma or melanoma of unknown primary origin with a documented NRAS Q61 mutation (confirmed by central laboratory molecular screening) that was previously untreated or has progressed on or after any number of immunotherapy regimens. Patients are stratified by stage, Eastern Cooperative Oncology Group performance status, and prior immunotherapy. The primary endpoint of the study is progression-free survival, and secondary endpoints include overall survival, overall response, time to response, duration of response, disease control rate, safety, and quality of life. Binimetinib is administered orally at 45 mg twice daily and dacarbazine is dosed intravenously at 1000 mg/m2 once every 3 weeks. This phase 3 trial is designed to enroll 393 patients and is currently recruiting patients at more than 150 centers across North America, South America, Europe, Asia-Pacific, and Africa. The study design, including eligibility criteria, methodology, and endpoints of the phase 3 NEMO trial will be presented. Citation Format: Georgina Long, Reinhard Dummer, Keith Flaherty, Dirk Schadendorf, Petr Arenberger, Lev Demidov, Anna Maria Di Giacomo, Mario Mandalà, Giovanni Rangoni, Pascal Wolter, Naoya Yamazaki, James Ford, Ernesto Wasserman, Marine Weill, Paolo Ascierto. NEMO: A phase 3 trial of binimetinib (MEK162) versus dacarbazine in patients with advanced NRAS-mutant melanoma who are untreated or have progressed on or after immunotherapy. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B16.

Background: Mutations of NRAS and BRAF genes have been identified with high frequency in nevi, cutaneous melanomas, and melanoma metastases. Prevalence of such mutations during the disease progression phases and among the different types of metastasis still remains inconclusive. Methods: A total of 275 tumour tissues from 116 melanoma patients were screened for mutations; among them, paired samples of microdissected primary melanomas (N=92) and synchronous or asynchronous metastases (N=156) from same patients were included. Tissue samples underwent mutation analysis by automated DNA sequencing. Secondary lesions were from: lymph nodes (LM; N=77), skin (SM; N=36), visceral (VM; N=23) and brain (BM; N= 44) sites. The full coding sequences and splice junctions of p16CDKN2A (exons 1, 2, and 3) and NRAS (exons 2 and 3) genes as well as the entire sequence at the exon 15 of the BRAF gene were screened for mutations by direct sequencing on automated fluorescence-cycle sequencer (ABIPRISM 3130, Applied Biosystems, Foster City, CA). Results: Overall, mutations were identified in 56/95 (59%) primary melanomas [43% BRAF - 16% NRAS], 49/77 (64%) lymph nodes [48% BRAF - 16% NRAS], 22/36 (61%) subcutaneous metastases [53% BRAF - 8% NRAS], 13/23 (57%) visceral metastases [43% BRAF - 13% NRAS], and 31/44 (70%) brain metastases [48% BRAF - 23% NRAS]. Overall, a slight and not significant increase in mutation frequency after progression from primary melanoma was observed in our series: 115/180 (64%) mutated metastases [48% BRAF - 16% NRAS]. Considering the paired samples from the same patients, LM (92% consistency) and VM (96% consistency) presented a highly similar prevalence and distribution of BRAF/NRAS mutations in comparison with primary melanomas, whereas a discontinuous pattern of mutations was detected in BM (80% consistency) or, mostly, SM (75% consistency). Occurrence of distinct mutation distribution between primary melanomas and correspondent metastases suggests that independent subclones have been generated in a limited fraction of patients. The rate of mutations in p16CDKN2A gene was found to steadily increase moving from primary melanomas (5/69; 7%) to melanoma metastases (21/144; 15%), with the highest prevalence of p16CDKN2A alterations (18/29; 62%) in melanoma cell lines Conclusions: Our results provide further clues about the impact of NRAS and BRAF mutations among the different stages of melanoma progression. Moreover, we confirmed that p16CDKN2A mutation rates are increasing during disease progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3677. doi:1538-7445.AM2012-3677