Abstract Background: Our phase Ib NIBIT-M4 study firstly reported that the hypomethylating agent guadecitabine (G), a prodrug of decitabine (D), followed by ipilimumab (I) is safe, with clinical and tumor-immunomodulatory activity in metastatic melanoma (MM) pts (CCR 2019; Nat Commun 2023). Thus, we designed the NIBIT-ML1 trial investigating the efficacy of G plus I+nivolumab (I+N) in PD-1/PDL-1 resistant MM and NSCLC pts. The Stage I MM Cohort results are reported. Methods: The NIBIT-ML1 is a multicenter, run-in, phase II randomized, non-comparative study, in Stage III/IV MM (Cohort A) or NSCLC (Cohort B) pts progressing on PD-1/PDL-1 therapy. An amendment replaced G with ASTX727 (oral D combined with cedazuridine). After a safety Run-in (6 pts/Cohort), 36 MM pts were randomized (1:1) to ASTX727 plus I+N (Arm A) or to I+N (Arm B) in the Stage I. Immune(i)-ORR and safety, iDCR and PFS were primary and secondary endpoints, respectively; exploratory analysis integrated RNA-seq and DNA methylation profiling, and multiplex immunofluorescence (mIF) for CD3, CD4, CD8, CD20, CD163 of tumor biopsies at week (W)0 and at W12. Results: 36 Stage III (3)/IV (33) MM pts (22 male; median age 62y), were randomized in Arm A or B, in Stage I. As of December 15, 2025, at a median follow-up of 19 months (IQR: 11-20), the iORR was 33% (3 CR, 3 PR) (95% CI: 13.3-59.0) and 17% (1 CR, 2 PR) (95% CI: 3.6-41.4) in Arm A and B, respectively; both Arms met the primary endpoint. The iDCR and the median PFS were 56% (95% CI: 30.7-78.5) and 9.4 (CI 95%: 5.0-13.8) months in Arm A and were 39% (95% CI: 17.3-64.2) and 5.8 (95% CI: 5.0-6.6) months in Arm B. The number of hypermethylated probes at W0 was higher in tumor biopsies from pts with iDCR (R) compared to NR,in Arm A+Run-in (A+R). Comparative analysis of differentially methylated probes identified 35,319 probes hypermethylated specifically in R from Arm A+R at screening; among those, 43 were hypomethylated by treatment only in R, and were associated with genes involved viral mimicry and antitumor immunity. Integrated DNA methylation and transcriptomic tumor analyses revealed an epigenetic reactivation driven by treatment-induced promoter hypomethylation of 166 immune-related genes in R from Arm A+R but not in Arm B. No significant difference was observed at W0 between Arm A+R and Arm B in intra-tumoral T-cell infiltration; CD8+and CD3+ enriched on-therapy in over 50% of R from Arm A+R. Conclusions: ASTX727 plus I+N induces clinically meaningful objective responses that correlate with epigenetic immune reprogramming in PD-1 refractory MM pts. Baseline tumor methylation profiling may identify MM pts who will benefit from the addition of a DNA hypomethylating agent to ICI therapy. Citation Format: Anna Maria Di Giacomo, Alessia Covre, Maria Fortunata Lofiego, Francesca Pia Caruso, Maura Colucci, Vincenzo D'Alonzo, Raffaella Grifoni, Roberta Depenni, Laura Solmonese, Francesco Marzani, Emma Bello, Antonio De Falco, Monica Valente, Ramiz Rana, Eleonora Carbonari, Giovanni Amato, Elena Manenti, Ilenia Vizzari, Sandra Coral, Harold Keer, Aram Oganesian, Danna Chan, Roberta Mortarini, Maresa Altomonte, Diana Giannarelli, Andrea Anichini, Teresa Maria Rosaria Noviello, Michele Ceccarelli, Michele Maio. Epigenetic immune reprogramming overcomes PD-1 resistance in metastatic melanoma patients: the phase II NIBIT-ML1 study [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr LB-C004.

Abstract Brain metastases affect up to 30% of patients with metastatic cancer and are a major cause for morbidity and mortality. Treatment approaches include neurosurgery, various approaches to radiotherapy and systemic pharmacotherapy. Encouraging response rates have been observed in patients with melanoma and non-small cell lung cancer (NSCLC) with asymptomatic or oligo-symptomatic brain metastases treated with novel systemic therapies, including immune checkpoint inhibitors and targeted therapy, challenging the need for immediate radiosurgery. Eligible patients for STRIKE must have newly diagnosed and untreated asymptomatic or oligo-symptomatic brain metastases from melanoma or NSCLC, with an indication for systemic therapy. The treatment regimen consists of standard systemic treatment with (Arm A) or without (Arm B) stereotactic radiosurgery. Systemic therapy follows the current standard of care according to the primary tumor. Primary endpoint is CNS-specific progression-free survival (PFS), locally assessed according to RANO criteria. Secondary endpoints include CNS-specific PFS per tumour cohort, objective CNS response rate, duration of CNS response, pattern of CNS-specific progression, extra-CNS progression, incidence of radionecrosis and pseudoprogression, overall survival, neurocognitive function, quality of life and functional independence, and toxicity. We assume that the addition of radiosurgery to systemic treatment will increase median CNS-specific PFS by 62% from a median of 4 and 8 months for melanoma and NSCLC, respectively, corresponding to an overall hazard ratio of 0.62 for time to CNS failure. According to the log-rank test, at a 5% one-sided significance level, 143 events provide 88% power for a sample size of 180 patients. The USZ-STRIKE trial is an academic study sponsored by ETOP IBCSG Partners Foundation, with substantial funding from the USZ Foundation. The trial is being conducted in 15 centres in Switzerland, Italy, the Netherlands, Spain and the United Kingdom. Current accrual is 57 patients. he study is registered on ClinicalTrials.gov: NCT05522660.

BACKGROUND:Melanoma is the most aggressive skin cancer, with a 50% five-year mortality in metastatic or unresectable cases. Non-invasive biomarkers are crucial for guiding treatment. MicroRNAs (miRNAs), especially those encapsulated in extracellular vesicles (EVs), are stable in plasma and hold promise as biomarkers. METHODS:This study analyzed EV-associated miRNAs (EV-miRNAs) from 50 blood samples of 18 stage III-IV melanoma patients treated with anti-CTLA-4 immunotherapy and a DNA hypomethylating agent. Samples were collected at baseline, week 4, and week 12. Patients were classified as responders (R) or non-responders (NR). RESULTS:A baseline signature of four EV-miRNAs predicted primary resistance. Treatment altered 15 EV-miRNAs at week 4 and 51 at week 12; nine were consistently modulated. At week 12, 27 EV-miRNAs differed between NR and R, with miR-1203 and miR-566-3p up-regulated in NR, linked to resistance and poor survival. CONCLUSIONS:These results highlight EV-miRNAs as non-invasive biomarkers for predicting and monitoring therapy response.

Abstract Background: The multicenter phase III NIBIT-M2 study, sponsored by the NIBIT Foundation, showed a 41% 7-y overall survival (OS) of melanoma patients (pts) with asymptomatic brain metastases (BM) treated with ipilimumab (I) plus nivolumab (N) (I+N) (Di Giacomo AM, CCR 2021; EJC 2024). In spite of the significant therapeutic efficacy of I+N in this patient population, no biomarkers predictive of response have been identified yet, also due to the limited accessibility of BM. Here, we report the final analysis of the NIBIT-M2 study with the 10-y survival and its correlation with cell free (cf) DNA profiles. Methods: The NIBIT-M2 study recruited melanoma pts with active, untreated, asymptomatic BM from 9 Italian Centers, randomized (1:1:1) to receive fotemustine (F) (Arm A), I+F (Arm B), or I+N (Arm C). Primary endpoint was OS; among secondary was intracranial progression-free survival (iPFS). Exploratory analyses were conducted on cfDNA plasma samples collected at baseline and week (W) 12 on therapy. Tumor fraction (TF) was estimated from low pass WGS using IchorCNA. Tumor-specific methylation Score (T-meth Score) was computed as the ratio between the coverage over methylated regions analyzed by cf-methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq) and melanoma-specific methylated regions previously identified in the TCGA melanoma cohort. Results: From Jan 2013 to Sept 2018, 80 pts were enrolled and 76 were treated with F (23), I+F (26), or I+N (27). As of December 1, 2025, with a median follow-up of 125 months (mo), median OS was 8.5 (95% CI: 4.8-12.2), 8.2 (95% CI: 2.1-14.3) and 29.2 (95% CI: 0-73.5) mo for Arm A, B, and C, respectively. The 10-y OS rate was 13.0% (95% CI: 0-26.7) in Arm A, 7.7% (95% CI: 0-17.9) in Arm B, and 31.2% (95% CI: 13.0-49.4) in Arm C. The 10-y melanoma specific survival was 13.0% (95% CI: 0-26.7), 7.7% (95% CI: 0-17.9), and 35.1% (95% CI: 16.3-53.9) in Arm A, B and C, respectively. The 10-year iPFS rate was 4.3% (95% CI: 0-12.7), 7.7% (95% CI: 0-17.9), and 20% (95% CI: 3.9-35.7) in Arm A, B and C, respectively. Patients were stratified at baseline according to the median values of TF (n=57; median 0.022) and of T-meth Score (n=53; median 0.096): a significantly higher median OS was observed in pts with TF (22.3 vs 8.2 mo; p=0.033) and T-meth Score (26.3 vs 7.9 mo; p=0.002) below their median values. Of note, pts with low TF and T-meth Score were enriched at baseline in Arm C. Additionally, a decrease in TF (n=29) and T-meth Score (n=24) was observed at W12 in pts with an OS above the median (26.3 mo for TF and 24.0 mo for T-meth Score). Conclusions: The 10-y results of the NIBIT-M2 study, with the longest follow-up available to date in melanoma pts with asymptomatic BM, continue to show persistent long-term therapeutic efficacy of I+N. Plasma-derived TF and T-meth Score may predict long-term survival of melanoma pts with asymptomatic BM treated with I+N. Citation Format: Anna Maria Di Giacomo, Vanna Chiarion-Sileni, Michele Del Vecchio, Pier Francesco Ferrucci, Michele Guida, Pietro Quaglino, Massimo Guidoboni, Paolo Marchetti, Vincenzo D'Alonzo, Maura Colucci, Giovanni Amato, Roberto Camerini, Maria Fortunata Lofiego, Monica Valente, Valentina Croce, Emma Bello, Maresa Altomonte, Mario Mandalà, Diana Giannarelli, Piera Grisolia, Antonio De Falco, Michele Ceccarelli, Alessia Covre, Michele Maio. Ten-year survival and cell-free DNA methylation profiling of melanoma patients with asymptomatic brain metastases treated with nivolumab plus ipilimumab: The multicenter phase III NIBIT-M2 trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr CT008.

2008 Background: The NIBIT Foundation-sponsored phase III NIBIT-M2 study showed a 41% 7-y OS of melanoma patients (pts) with asymptomatic brain metastases (BM) treated with ipilimumab (I) plus nivolumab (N) (I+N) ( Di Giacomo AM, CCR 2021 and EJC 2024 ). Despite the significant efficacy of I+N therapy in this pts population, no biomarkers predictive of response have been identified yet also due to the accessibility of BM. We here report the 10-y survival and its correlation with cell-free (cf)DNA analyses on serial plasma samples collected from pts enrolled in the NIBIT-M2 study. Methods: The NIBIT-M2 study recruited melanoma pts with active, untreated, asymptomatic BM from 9 Italian Centers, randomized (1:1:1) to receive fotemustine (F) (Arm A), I+F (Arm B), or I+N (Arm C). Primary endpoint was OS. Exploratory analyses were conducted on cfDNA plasma samples collected at baseline and week (W) 12 on therapy. Tumor fraction (TF) was estimated from low pass WGS using IchorCNA. Tumor-specific methylation Score (T-meth Score) was computed as the ratio between the coverage over methylated regions analyzed by cf-methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq) and melanoma-specific methylated regions previously identified in the TCGA melanoma cohort. Results: From Jan 2013 to Sept 2018, 80 pts were enrolled: 76 received F (23), I+F (26), or I+N (27). As of December 1, 2025, with a median follow-up of 125 months (mo), median OS was 8.5 (95% CI: 4.8-12.2), 8.2 (95% CI: 2.1-14.3) and 29.2 (95% CI: 0-73.5) mo for Arm A, B, and C, respectively. The 10-y OS rate was 13.0% (95% CI: 0-26.7) in Arm A, 7.7% (95% CI: 0-17.9) in Arm B, and 31.2% (95% CI: 13.0-49.4) in Arm C. The 10-y melanoma specific survival was 13.0% (95% CI: 0-26.7), 7.7% (95% CI: 0-17.9), and 35.1% (95% CI: 16.3-53.9) in Arm A, B and C, respectively. Patients were stratified at baseline according to the median values of TF (n=57; median 0.022) and of T-meth Score (n=53; median 0.096): a significantly higher median OS was observed in pts with TF (22.3 vs 8.2 mo; p=0.033) and T-meth Score (26.3 vs 7.9 mo; p=0.002) below their median values. Of note, low TF and T-meth Score were enriched at baseline in pts from Arm C. Additionally, a decrease in TF (n=29) and T-meth Score (n=24) was observed at W12 in pts with an OS above the median (26.3 mo for TF and 24.0 mo for T-meth Score). Conclusions: The 10-y results of the NIBIT-M2 study, with the longest follow-up available to date in melanoma pts with asymptomatic BM treated with I+N, continue to show persistent long-term therapeutic efficacy of the combination. Plasma-derived TF and T-meth Score may predict long-term survival of melanoma pts with asymptomatic BM treated with I+N. Clinical trial information: NCT02460068 .

Anti-programmed cell death-(ligand) 1 (anti-PD-[L]1) agents are approved for advanced and early-stage cancers. While they may offer clinical and economic benefits in the neoadjuvant and/or adjuvant setting, their population-level impact in Italy has not been thoroughly evaluated. This study aims to estimate health and productivity outcomes of introducing anti‑PD‑(L)1 agents for neoadjuvant and/or adjuvant therapy in early‑stage cancers in Italy (melanoma Stage IIB/C, melanoma Stage III, renal cell carcinoma, triple‑negative breast cancer and resectable non‑small‑cell lung cancer) over a 10-year horizon. We developed a model synthesising outputs from five indication-specific Markov models comparing two worlds: one without anti-PD-(L)1 agents use in the neoadjuvant and/or adjuvant settings versus one with their use. Italian-specific population and incidence inputs were used, with clinical and quality-of-life data from individual trials, from a societal perspective with 3

Abstract BACKGROUND: Immune checkpoint inhibitors (ICIs), including anti-PD-1 antibodies, reverse tumor-induced immune suppression and promote effective anti-tumor T-cell responses. They have drastically improved outcome in advanced melanoma therapy, yielding durable improvements in progression-free survival (PFS) and overall survival (OS). Although adding anti-CTLA-4 or anti-LAG-3 enhances efficacy beyond anti-PD-1 alone, these approaches do not directly target dominant myeloid-mediated resistance mechanisms within the tumor microenvironment, and new agents targeting this axis are needed. EIK1001, a TLR7/8 dual-agonist, activates myeloid and plasmacytoid dendritic cells to stimulate innate inflammation and adaptive immunity. Across multiple studies, EIK1001 has been well tolerated and has demonstrated clinical activity both as monotherapy and in combination with ICIs. METHODS: TeLuRide-006 (NCT#06697301) is a global, multicenter, randomized, double-blind, adaptive Phase 2/3 trial of EIK1001 or placebo, in combination with pembrolizumab (pembro), as first-line therapy for participants (pts) with advanced cutaneous melanoma. Approximately 740 pts will be randomized, including a dose-optimization (DO) stage in which pts are randomized 1:1:1 to two EIK1001 dose levels or placebo, each administered with pembro, followed by Phase 2/3 expansion evaluating the selected EIK1001 dose versus placebo, with pembro. Interim analyses will guide progression from DO to Phase 2 and subsequently to Phase 3. EIK1001 or placebo is administered IV QW until the end of Week 27 then Q3W; while pembro is administered IV Q3W. Pts are stratified by prior anti-PD-1 adjuvant therapy, LDH level, and BRAF mutational status. Key eligibility criteria include patients ≥12 years (with country-specific variations), life expectancy ≥3 months, unresectable Stage III or Stage IV melanoma, known BRAF V600 status, at least one RECIST v1.1-measurable lesion, and no prior or current pneumonitis/interstitial lung disease. Primary objectives are to assess the efficacy and safety of two EIK1001 doses with pembro in DO, and to compare PFS (RECIST v1.1 by blinded independent central review [BICR]) and OS between the selected EIK1001 dose and placebo in combination with pembro. Secondary objectives include safety and tolerability of either EIK1001 dose with pembro, objective response rate and duration of response per RECIST v1.1 by BICR. Exploratory objectives include time to response, EIK1001 exposure-response relationships, and health-related quality of life. This study enrolled (randomized) its first participant on 5/21/2025 and is activated at 89 sites in 22 countries at the time of abstract submission. Citation Format: Diwakar Davar, Bernardo Rapoport, Jan C. Simon, Oliver Bechter, Stephane Dalle, Yin Wu, Daniel Brungs, Anna Maria Di Giacomo, Hanna Eriksson, Peter Mohr, Rossanna Pezo, Egle Ramelyte, Gareth Rivalland, Toshifumi Hoki, Geetha Krishna Kaza, Yayan Zhang, Etah Kurland, Alexander Eggermont. Adaptive phase 2/3 study of EIK1001, a TLR7/8 dual agonist, in combination with pembrolizumab, as first-line therapy in participants with advanced melanoma (Teluride-006) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr CT224.

Melanoma plasticity drives immune evasion and therapy resistance through dynamic cell-state transitions beyond genetic alterations. Although epigenetic remodeling is central to this process, its impact under therapeutic pressure remains unclear. We profiled longitudinal biopsies from patients with melanoma treated in the phase 1b NIBIT-M4 epi-immunotherapy trial [NCT02608437, DNA (cytosine-5)-methyltransferase 1 inhibitor plus anti-CTLA-4] using single-cell multiome and spatial transcriptomics. Seven malignant meta-programs were identified, including a rare Wnt/β-Catenin melanocytic state and a dedifferentiated neural crest-like state enriched in nonresponders. Spatial analyses showed that homotypic clustering stabilizes resistant programs, with neural crest-like cells forming compact niches. Responders displayed enrichment of antigen presentation/interferon program and coordinated T and B cell expansion, whereas nonresponders retained stable neural crest-like clusters. Epigenetic therapy reactivated transposable elements, priming innate immunity and enhancing immunogenicity. Nuclear factor of activated T cells, cytoplasmic 2 (NFATC2) emerged as a master regulator of neural crest-like states and resistance; its perturbation promoted differentiation and immunogenicity. These findings define mechanisms of resistance and nominate β-Catenin and NFATC2 as therapeutic vulnerabilities.

Abstract Background: In the NIBIT Foundation-sponsored phase Ib NIBIT-M4 study, we firstly reported that the hypomethylating agent (HMA) guadecitabine (G), a prodrug of decitabine (D), followed by ipilimumab (I), was safe and had promising clinical and tumor-immunomodulatory activity in metastatic melanoma (MM) patients (pts) (CCR 2019; Nature Commun 2023). Thus, we designed the NIBIT-ML1 trial to investigate the efficacy of G plus I+nivolumab (I+N) in MM or non-small cell lung cancer (NSCLC) pts, progressing to PD-1/PDL-1 inhibitors. Clinical results, integrated analyses of transcriptomic and methylome profiles, and immune contextures of serial tumor biopsies are being investigated in the MM Cohort. Methods: The NIBIT-ML1 is a multicenter, run-in, phase II randomized, non-comparative study, in Stage III/IV MM (Cohort A) or NSCLC (Cohort B) pts progressing on PD-1/PDL-1 therapy. An amendment replaced G with ASTX727 (oral D combined with cedazuridine). After a safety Run-in (6 pts/Cohort), 36 MM pts were randomized (1:1) to ASTX727 plus I+N (Arm A) or to I+N (Arm B) in the Stage I. Immune(i)-ORR and safety, iDCR and PFS were primary and secondary endpoints, respectively; exploratory analysis integrated RNA-seq and DNA methylation profiling, and multiplex immunofluorescence (mIF) for CD3, CD4, CD8, CD20, CD163 of tumor biopsies at week (W)0 and W12. Results: Run-in phase: 6 Stage IV MM pts (2 male; median age 71y) received G (2 pts) or ASTX727 (4 pts) plus I+N. No DLT occurred. One CR, 2 PR, 1 SD, and 2 PD were observed. Stage I: 36 Stage III (3)/IV (33) MM pts (22 male; median age 62y), were randomized to Arm A or B. As of December 15, 2025, at a median follow-up of 19 months (IQR: 11-20), the iORR was 33% (3 CR, 3 PR) (95% CI: 13.3-59.0) and 17% (1 CR, 2 PR) (95% CI: 3.6-41.4) in Arm A and B, respectively; both Arms met the primary endpoint. The iDCR and the median PFS were 56% (95% CI: 30.7-78.5) and 9.4 (CI 95%: 5.0-13.8) months in Arm A and were 39% (95% CI: 17.3-64.2) and 5.8 (95% CI: 5.0-6.6) months in Arm B. In Arm A+Run-in (A/R) the number of hypermethylated probes at W0 was higher in tumor biopsies from pts with iDCR (R) compared to non R. Comparative analysis of differentially methylated probes identified 35,319 probes hypermethylated specifically in R from Arm A/R at screening; among those, 43 were hypomethylated by treatment only in R, and were associated with genes involved in viral mimicry and antitumor immunity. Integrated DNA methylation and transcriptomic tumor analyses revealed an epigenetic reactivation driven by treatment-induced promoter hypomethylation of 166 immune-related genes in R from Arm A/R but not in Arm B. No significant difference in intra-tumoral T-cell infiltration was observed at W0 between Arm A/R and Arm B; CD8⁺and CD3⁺ enriched on-therapy in over 50% of R from Arm A/R. Conclusions: ASTX727 plus I+N induces clinically meaningful objective responses that correlate with epigenetic immune reprogramming in PD-1 refractory MM pts. Baseline tumor methylation profiling may identify MM pts who will benefit from the addition of a DNA hypomethylating agent to ICI therapy. Citation Format: Anna Maria Di Giacomo, Alessia Covre, Maria Fortunata Lofiego, Francesca Pia Caruso, Maura Colucci, Vincenzo D'Alonzo, Raffaella Grifoni, Roberta Depenni, Laura Solmonese, Francesco Marzani, Emma Bello, Antonio De Falco, Monica Valente, Ramiz Rana, Elena Carbonari, Giovanni Amato, Elena Manenti, Ilenia Vizzari, Sandra Coral, Harold Keer, Aram Oganesian, Danna Chan, Roberta Mortarini, Maresa Altomonte, Diana Giannarelli, Andrea Anichini, Teresa Noviello, Michele Ceccarelli, Michele Maio. Epigenetic immune reprogramming overcomes PD-1 resistance in metastatic melanoma patients: The phase II NIBIT-ML1 study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr CT236.

Pleural mesothelioma (PM) has a poor prognosis and standard therapy with immune checkpoint inhibitors (ICIs) CTLA-4 and PD-1 is still clinically unsatisfying. No predictive biomarkers of ICI efficacy in PM are available yet. In the retrospective multicenter NIBIT-EPI-MESO study, multi-omics analysis of pre-ICI therapy tumor lesions from 91 patients with PM treated in earlier clinical trials or in daily practice identified four PM subsets with progressively increasing global DNA methylation profiles-demethylated, LOW, intermediate and CpG island methylator phenotype (CIMP). These methylation subsets predicted response and survival to ICI therapy. The LOW subset was enriched in responder patients, who had the longest median overall survival and the highest 3-year overall survival rate, and showed a T cell- and B cell-rich immune microenvironment. Conversely, the CIMP subtype was enriched in nonresponder patients with the shortest median overall survival and overall survival, along with a depleted immune microenvironment. A methylation-based probabilistic decision-making classification tool to predict the outcome of ICI treatment in patients with PM was developed.

TPS9601 Background: Immune checkpoint inhibitors (ICIs), including anti-PD-1 antibodies, reverse tumor induced immune suppression and promote effective anti-tumor T-cell responses. They have drastically improved outcome in advanced melanoma therapy, yielding durable improvements in progression-free survival (PFS) and overall survival (OS). Although adding anti-CTLA-4 or anti-LAG-3 enhances efficacy beyond anti-PD-1 alone, these approaches do not directly target dominant myeloid-mediated resistance mechanisms within the tumor microenvironment, and new agents targeting this axis are needed. EIK1001, a TLR7/8 dual-agonist, activates myeloid and plasmacytoid dendritic cells to stimulate innate inflammation and adaptive immunity. Across multiple studies, EIK1001 has been well tolerated and has demonstrated clinical activity both as monotherapy and in combination with ICIs. Methods: TeLuRide-006 (NCT#06697301) is a global, multicenter, randomized, double-blind, adaptive Phase 2/3 trial of EIK1001 or placebo, in combination with pembrolizumab (pembro), as first-line therapy for participants (pts) with advanced cutaneous melanoma. Approximately 740 pts will be randomized, including a dose-optimization (DO) stage in which pts are randomized 1:1:1 to two EIK1001 dose levels or placebo, each administered with pembro, followed by Phase 2/3 expansion evaluating the selected EIK1001 dose versus placebo, with pembro. Interim analyses will guide progression from DO to Phase 2 and subsequently to Phase 3. EIK1001 or placebo is administered IV QW until the end of Week 27 then Q3W; while pembro is administered IV Q3W. Pts are stratified by prior anti-PD-1 adjuvant therapy, LDH level, and BRAF mutational status. Key eligibility criteria include patients ≥12 years (with country-specific variations), life expectancy ≥3 months, unresectable Stage III or Stage IV melanoma, known BRAF V600 status, at least one RECIST v1.1–measurable lesion, and no prior or current pneumonitis/interstitial lung disease. Primary objectives are to assess the efficacy and safety of two EIK1001 doses with pembro in DO, and to compare PFS (RECIST v1.1 by blinded independent central review [BICR]) and OS between the selected EIK1001 dose and placebo in combination with pembro. Secondary objectives include safety and tolerability of either EIK1001 dose with pembro, objective response rate and duration of response per RECIST v1.1 by BICR. Exploratory objectives include time to response, EIK1001 exposure-response relationships, and health-related quality of life. This study enrolled (randomized) its first participant on 5/21/2025 and is activated at 91 sites in 22 countries at the time of abstract submission.

Background Nivolumab plus ipilimumab (COMBO) is the standard treatment for asymptomatic melanoma brain metastases (MBM), but current guidelines do not provide specific recommendations for treatment discontinuation in responding patients. This study aimed to evaluate outcomes after COMBO discontinuation within 24 months and the role of continuing treatment beyond 24 months. Methods Patients with MBM treated with COMBO who discontinued treatment within 24 months for reasons other than disease progression or continued beyond this time point were retrieved. Overall survival (OS), objective response, progression-free survival (PFS) and toxicities were analyzed. Results 465 patients were included: 392 discontinued COMBO within 24 months, while 73 continued beyond 24 months. Treatment was discontinued due to complete response (CR, n=47), partial response (PR, n=45), stable disease (SD, n=12), toxicity after SD (n=59), toxicity after CR (n=99), or toxicity after PR (n=130). At multivariable analysis, the line of treatment (>first vs first: HR 2.65 (1.62-4.32)), the immune-related adverse events (irrespective of anti-tumor necrosis factor-alpha) (HR 0.18 (0.07-0.42)); COMBO discontinuation after CR (HR 0.15 (0.05-0.40)), or PR (HR 0.08 (0.03-0.26)), as well as stopping due to toxicity after CR (HR 0.14 (0.07-0.27)) or PR (HR 0.51 (0.32-0.82)), were associated with OS. Notably, at a median follow-up of 51 months (IQR 31-70), patients with CR/PR who discontinued COMBO within 24 months had PFS and OS comparable to those who continued treatment beyond this time point. 4-year OS exceeded 83% in patients discontinuing COMBO after CR, PR, or toxicity following CR, compared with 66.4% in those discontinuing due to toxicity after PR; median PFS was not reached in the former groups but was 18.6 months in the toxicity after PR group. Conclusion Discontinuation of COMBO within 24 months appears safe in patients with CR and in selected cases of PR, with no survival disadvantage versus prolonged therapy.

9543 Background: Pts with advanced cMEL R/R to PD-(L)1–based therapy, including PD-(L)1+CTLA-4, have few treatment options. BOT (Fc-enhanced anti–CTLA-4) augments T-cell priming, depletes Tregs, and activates antigen-presenting cells to overcome immune checkpoint inhibitor (ICI) resistance. BOT ± BAL (anti–PD-1) has shown activity in ICI-R/R and cold tumors. Methods: The open-label, global phase 2 C-800-23 trial (NCT05529316) enrolled pts with cMEL (stage III unresectable or IV) R/R to prior anti–PD-(L)1 ± CTLA-4. Part 1: randomized 1:1 to BOT 50 mg or 150 mg every 3 weeks (Q3W; up to 4 doses). Part 2: BOT 75 mg Q3W (up to 4 doses) plus BAL 450 mg Q3W (up to 2 years). Endpoints included confirmed objective response rate (ORR; primary; RECIST 1.1), progression-free survival (PFS), duration of response (DOR), overall survival (OS), and safety. Clinical benefit rate (CBR) was complete or partial response or stable disease for ≥24 weeks. Results: As of Dec 13, 2025, 138 pts received BOT monotherapy (median follow-up, 12.3 mo; range, 0.2–35+) and 36 pts received BOT+BAL (median follow-up, 13.8 mo; range, 2–26+). Overall, 51% had stage M1c or M1d disease, 29% had BRAF mutant disease, and 39% had LDH >ULN. In pts R/R to anti–PD-(L)1, 72% (60/83 with pre-trial data available) had primary resistance (best overall response of progressive disease [PD] or PD ≤6 mo from most recent course of respective ICI type). In pts R/R to anti–PD-(L)1+CTLA-4, 69% (61/88) had primary PD-(L)1 resistance and 69% (60/87) had primary CTLA-4 resistance. With BOT+BAL, ORR was 22% (95% CI, 10–39), CBR was 33% (95% CI, 19–51), median DOR was not reached (NR; 95% CI, 4.17–NR), 12-mo PFS was 29% (95% CI, 15–44), and median OS was 16.6 mo (95% CI, 9.1–NR; 24-mo OS, 46%; 95% CI, 29–61). ORR with BOT+BAL was numerically higher in PD-(L)1+CTLA-4 R/R pts (29%, 4/14; 95% CI, 8–58; 3/4 responders had primary ICI resistance) vs PD-(L)1–only R/R pts (18%, 4/22; 95% CI, 5–40; all responders had primary ICI resistance). With BOT alone, ORR was 6% (95% CI, 3–11), CBR was 14% (95% CI, 9–21), 12-mo PFS was 7% (95% CI, 3–13), and median OS was 12.9 mo (95% CI, 9.7–16.2; 24-mo OS, 28%; 95% CI, 20–36). Responses were irrespective of Fcγ receptor genotype. Treatment-related adverse events occurred in 81% (grade ≥3, 30%) with BOT alone and 94% (grade ≥3, 36%) with BOT+BAL; most common was diarrhea (BOT 33%; BOT+BAL 42%). One possibly treatment-related death was reported with BOT monotherapy (immune-mediated enterocolitis). No new safety signals occurred. Conclusions: In this ICI-R/R cMEL population enriched with primary ICI-resistant pts, BOT+BAL showed durable responses, encouraging survival, and a stronger signal in pts who had prior conventional anti–CTLA-4. BOT monotherapy also showed activity. This supports a differentiated mechanism for BOT and continued development of BOT+BAL, including for pts with dual ICI-R/R cMEL. Clinical trial information: NCT05529316 .

8052 Background: Pleural mesothelioma (PM) is an aggressive malignancy with a poor prognosis. The clinicalefficacy of standard therapy with immune checkpoint inhibitors (ICI) is limited and heterogeneous acrossPM subtypes. Tumor-intrinsic characteristics (i.e., inflammatory phenotype, molecular features, DNAmethylation) may influence immune responsiveness, but predictive biomarkers of ICI therapy efficacy inPM are still lacking. Methods: NIBIT-EPI-MESO is a retrospective, multicenter study, sponsored by theNIBIT Foundation, evaluating biological correlates of clinical outcomes in PM patients (pts) treated withICI (i.e., anti-CTLA-4 plus anti-PD-1, anti-CTLA-4 plus anti-PD-L1, or anti-CTLA-4 monotherapy). Pre-ICI therapy FFPE tumor samples were analyzed by RRBS methylation (n = 83 pts) and RNA-seq (n = 82pts), with methylation subtypes defined by consensus clustering of the top 1% most variable CpGs.Tumor microenvironment (TME) was characterized by multiplex immunofluorescence analysis of CD4,CD8, CD20, CD68, CD163 (n = 35 pts). Integrated multi-omics analyses were used to associate tumorbiology with clinical outcome of PM pts. Results: Unsupervised methylation profiling identified four PMsubsets with increasingly global DNA methylation levels: demethylated (DEM), LOW, intermediate (INT),and CpG island methylator phenotype (CIMP). Methylation subtypes were significantly associated withresponse to ICI, with LOW/DEM enriched among responder (R) pts and INT/CIMP in non-R pts (p =0.002); no association of response to ICI was found with PM histotype (p = 0.33). The LOW subsetexhibited the longest median overall survival (mOS) and the highest 3-year OS rate, expressed genesinvolved in pathways associated with innate and adaptive immune responses, and showed an “inflamed”TME (i.e., CD8+ T cells, CD20+ B cells). Conversely, the CIMP subtype had the shortest mOS and OSrate, was characterized by genes enriched in developmental, morphogenetic and cell cycle-relatedprocesses, along with a “desert” TME. Functional characterization of the identified methylation classes ofPM was validated in the MESOMICS dataset. Accordingly, a PM methylation subtype classifier wasdeveloped to predict response to ICI therapy. Conclusions: Tumor DNA methylation defines biologicallyand clinically distinct immune phenotypes in PM and robustly predicts clinical response and long-termsurvival in ICI-treated PM patients, regardless of tumor histology.

BACKGROUND:Cutaneous squamous cell carcinoma (cSCC) represents the second most common form of non-melanoma skin malignancy, and, when not amenable to curative surgery or radiotherapy, it is a life-threatening disease. The anti-PD-1 monoclonal antibody cemiplimab has transformed the outcome of advanced or metastatic cSCC, with response rates approaching 50% and sustained benefit beyond three years in clinical trials. Cemiplimab is now the first-line standard of care treatment for advanced disease. METHODS:This retrospective observational study included consecutive adult patients with locally advanced (lac) or metastatic (m) cSCC who received cemiplimab (350 mg every three weeks) at the Center for Immuno-Oncology, University Hospital of Siena, Italy, either through an Expanded Access Program or routine clinical practice. Clinical outcome and treatment related adverse events (TRAEs) are reported. RESULTS:Between December 2019 and December 2023, 27 patients (24 male; median age 82 years [range 41-90]) diagnosed with lacSCC (n = 20 [74.0%]) or mcSCC (n = 7 [25.9%]) were treated with cemiplimab as first line therapy and were followed until June 2024. Head and neck were the primary tumor location for 88.8% of patients, followed by trunk (7.4%) and lower extremities (3.7%). All patients had comorbidities, including six patients (22.2%) with hematologic malignancies. With a median follow-up of 31 months (data cut-off June 2024), the ORR was 66.6% (complete response 22.2%) and the disease control rate (DCR) 77.7%. Median progression-free survival (mPFS) and overall survival (mOS) were not reached, while 2-year PFS and OS rates were 65.2% and 71%, respectively. Treatment was well-tolerated, with three (11.1%) patients experiencing grade ≥3 TRAEs, and three patients discontinuing treatment due to TRAEs. CONCLUSIONS:Our real-world experience confirms the high rate of durable objective responses, good tolerability and long treatment duration of cemiplimab in elderly and frail cSCC patients as well.

LBA2512 Background: In the NIBIT Foundation-sponsored phase Ib NIBIT-M4 study, we firstly showed that the hypomethylating agent (HMA) guadecitabine (guade), a prodrug of decitabine (D), followed by ipilimumab (I) was safe with promising clinical and immunologic activity in cutaneous metastatic melanoma (MM) patients (pts) (Di Giacomo, Clin Cancer Res 2019; Noviello, Nat Commun 2023 ). Thus, we further explored the activity of HMA combined with checkpoint inhibitors in the NIBIT-ML1 trial in which we investigated the efficacy of guade plus I+nivolumab (I+N) in PD-1/PDL-1-resistant MM and NSCLC pts. The primary analysis and the correlation between tumor methylome and immune contextures with the MM Cohort clinical outcome will be presented. Methods: The NIBIT Foundation NIBIT-ML1 is a multicenter, run-in, phase II randomized, non-comparative study (Simon two stages optimal design), in unresectable Stage III/IV MM (Cohort A) and NSCLC (Cohort B) pts progressing to PD-1/PDL-1 as last treatment. A Monitoring Committee reviewed safety data throughout the study. A trial amendment replaced guade with ASTX727, an oral fixed-dose combination of D with cedazuridine. Following a safety run-in of 6 pts, 36 eligible MM pts were randomized (1:1) to ASTX727 plus I+N or to I+N. Primary objective was immune(i)-ORR, according to a centralized radiologic assessment, defined as the proportion of pts with an iBOR of confirmed iCR/iPR. Secondary were: safety, DCR and PFS. Tumor methylation and immune contextures of serial tumor biopsies at baseline (W0) and on-treatment (W12 and/or W19) were investigated by EPIC Array and RNAseq. Results: Run-in phase: 6 Stage IV MM pts received guade (2 pts) or ASTX727 (4 pts) plus I+N. No DLT occurred. Three PR, 2 SD, and 1 PD were observed. Stage I: 36 Stage III (3)/IV (33) MM pts, received ASTX727 plus I+N (ARM A) or I+N (ARM B). ORR was 33% (2 CR, 4 PR) and 17% (3 PR) in ARM A and B; DCR was 56% in ARM A and 39% in ARM B. Both ARMs met the Stage I Simon design. The 1-year PFS rate was 43% and 11% for ARM A and B. With no overlapping toxicities, 1 DLT (G5 macrophage activation syndrome) was reported in ARM A. G3/4 TRAEs were 72% and 50% in ARM A and B, respectively, and G3/4 irAEs were 39% in ARM A and 44% in ARM B. A time-dependent reduction in tumor methylation levels in run-in and ARM A pts was observed, with more hypomethylated probes in on-treatment vs baseline tumors. Integrative methylation and transcriptomic analyses showed a promotion of immune regulatory genes, T and B cell activation in on-treatment tumors of ARM A pts. Enrichment of immune pathways was found in the Run-in and in ARM A responder pts. Conclusions: Treatment with ASTX727 plus I+N is feasible and has meaningful clinical and immunologic activity in PD-1 refractory MM pts. Clinical trial information: NCT04250246 .

Lung neuroendocrine tumors (NETs) are well-differentiated neuroendocrine neoplasms of lung origin, including typical and atypical carcinoids (ACs). Therapeutic options for this rare disease are limited in daily clinical practice. Immune-checkpoint inhibitors (ICIs) are under clinical investigation. Here, we report a systematic reappraisal about ICIs results in lung NETs. We reviewed articles on observational or interventional studies that reported efficacy data of ICIs in lung NETs. Case reports and studies with insufficient data were excluded from the analysis. We searched the electronic databases Medline, Embase, Web of Science, and Cochrane Library up to May 2024. Two investigators independently screened the identified records and assessed studies quality. We summarized the results descriptively and in a meta-analysis of ORR according to the type of intervention. The search retrieved 1344 records. After selection, we included 11 studies in the meta-analysis of ORR, with a total of 128 adult patients with lung NET (25% ACs) that were progressing after at least one line of systemic therapy, including treatment with somatostatin analogs. Ten studies were Phase II, and 1 study was phase Ib. The summary ORR was 14.7% (95% CI, 5.8-32.2), 44.4% (27.2-63.1) for ACs. Subgroup analysis by intervention types showed a trend for lower ORR of lung NETs treated with ICI monotherapy (ORR: 2.7%; 0.0-63.7) compared with combinations (p-value: 0.056). The combination of temozolomide plus nivolumab showed the highest ORR (66.7%; 33.3-88.9). The median OS (reported in 2 studies) was not reached. Safety was consistent with historical data of ICIs. Our work suggests that ICIs are a promising treatment for patients with lung NETs, especially ACs, and warrant further investigation in more focused studies.