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.

LBA9517 Background: PIVOTAL (NCT02938299) is an open-label, randomized, multicenter, phase 3 trial evaluating daromun as a neoadjuvant intralesional (IT) therapy for resectable, locally advanced stage III melanoma. The trial enrolled 256 efficacy-evaluable patients (pts) in the EU and met its primary endpoint, demonstrating a statistically significant improvement in recurrence-free survival (RFS; HR = 0.59; p = 0.005) for daromun versus upfront surgery, at a median follow-up (FU) of 21 months from randomization (Kähler et al, Ann Onc 2025, 36, 1166). Methods: Pts with skin and/or lymph node metastatic melanoma amenable to complete surgical resection were randomized (1:1) to receive 4 weekly IT injections of daromun (13 Mio IU of L19IL2 and 400 μg of L19TNF) followed by surgery or upfront surgery alone. Prior surgery, radiotherapy (RT) and/or systemic therapies (ST) were permitted, as well as any approved adjuvant treatment post-surgery.The primary endpoint was RFS, the secondary endpoints included DMFS, OS and safety. An event-free survival (EFS) sensitivity analysis was conducted, considering progression to unresectable melanoma before surgery, recurrence of the disease, or death due to melanoma or treatment as events. Results: An updated primary outcome analysis at a median FU of over 36 months from randomization (database cut-off Nov. 29, 2025) confirmed a clinically relevant improvement in RFS (HR = 0.60; p = 0.003) for daromun versus upfront surgery. The EFS sensitivity analysis carried out in the overall population was consistent with the RFS results (HR = 0.66; 95% CI = 0.47-0.92).The PIVOTAL trial included two clinically distinct subgroups: pts with newly diagnosed disease (n = 32; 12%) and pts with recurrence(s) after surgery and/or systemic (neo)adjuvant treatment (n = 224; 88%). Among the recurrent cohort, 86 pts (38.3%) had received and failed previous ST, while 138 pts (61.6%) had only received surgery/RT. Sensitivity EFS analyses were also performed in i) the recurrent cohort (HR = 0.59; 95% CI = 0.41-0.85), ii) the subgroup of pts in the recurrent cohort who had only received previous surgeries and/or RT (HR = 0.55; 95% CI = 0.34-0.89), and iii) the pts subgroup in the recurrent cohort who received prior ST (HR = 0.63; 95% CI = 0.36-1.08 ). The rate and type of treatment-related adverse events at a longer FU were consistent with previous reports, and no new safety risks were identified. Conclusions: With longer FU the highly significant reduction in the risk of recurrence or death with neoadjuvant daromun in pts with locally advanced stage III melanoma is confirmed. The sensitivity EFS analyses provide consistent, confirmatory evidence of daromun’s efficacy across the entire trial population and, more importantly, in the subgroup of pts with recurrent disease, regardless of any prior ST. No new safety signals were reported. Clinical trial information: NCT02938299 .

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 .

Daromun (L19IL2/L19TNF) was investigated as a neoadjuvant, intralesional therapy for patients with fully resectable stage III melanoma in the phase III PIVOTAL trial (ClinicalTrials.gov identifier: NCT02938299). The trial enrolled 256 patients in the European Union and met its primary end point, demonstrating a statistically significant improvement in recurrence-free survival (RFS; hazard ratio, 0.59; P = .005) for daromun followed by surgery versus up-front surgery, at a median follow-up (FU) of 21 months from random assignment. PIVOTAL included two clinically distinct subgroups, namely, patients with de novo diagnosed metastatic disease (n = 34; 13%) and patients with recurrence(s) after surgery with or without radiotherapy and/or adjuvant systemic therapies (n = 222; 87%). Here, we present an updated analysis of the primary and secondary end points, including safety data, at a median FU of 36.8 months from random assignment (database cutoff: November 28, 2025), alongside new sensitivity analyses of event-free survival (EFS). The updated analysis confirms the clinically and statistically meaningful improvements in RFS and distant metastasis-free survival recorded in the neoadjuvant daromun versus control arm. The EFS post hoc analysis, conducted in both the overall population and the recurrent patient subgroups (with or without prior systemic therapies), provides consistency and robustness to the benefit of neoadjuvant daromun observed for the primary efficacy end point. No new safety signals of concern were recorded.

BACKGROUND:Fulzerasib, a KRASG12C inhibitor, has shown clinical activity in previously treated non-small-cell lung cancer (NSCLC). Clinical studies indicate that combining a KRASG12C inhibitor with an anti-EGFR antibody is effective in colorectal cancer; however, its benefit in NSCLC remains to be explored. METHODS:This single-arm, phase 1b/2 trial enrolled patients at 30 medical centres in Spain, Italy, and Greece. Eligible patients had KRASG12C-mutated NSCLC with no previous systemic anti-tumour therapy for advanced or metastatic disease and were aged 18 years and older; had at least one measurable lesion according to the Response Evaluation Criteria in Solid Tumours (RECIST) version 1.1; had an Eastern Cooperative Oncology Group performance status of 0 or 1; and had a life expectancy of 3 months or more. Enrolled patients received 600 mg oral fulzerasib twice daily plus 500 mg/m2 intravenous cetuximab every 2 weeks. The primary endpoint was investigator-assessed objective response rate per RECIST 1.1 assessed in the full response analysis set. The study was registered with ClinicalTrials.gov (NCT05756153) and is complete. FINDINGS:Between April 23, 2023, and April 15, 2024, a total of 60 patients were screened, and 47 patients (78%) received treatment. The median age was 68 years (IQR 59-72); 25 patients (53%) were male and 22 (47%) were female. 45 (95%) patients were current or former smokers. As of Jan 14, 2025, the median follow-up time and treatment duration were 12·8 months (90% CI 11·6-14·6) and 10·1 months (IQR 6·3-13·0) months, respectively. The confirmed objective response rate was 69% (90% CI 56-80). Treatment-related adverse events (TRAEs) occurred in 41 (87%) of the 47 patients (grade 3 in seven [15%]). No grade 4 or 5 TRAEs were observed. TRAEs led to drug discontinuation in three (6%) patients, with only cetuximab being discontinued. INTERPRETATION:In treatment-naive KRASG12C-mutated NSCLC, fulzerasib plus cetuximab showed encouraging activity with a favourable safety profile. The combination is currently being planned for investigation in a phase 3 study. FUNDING:Zhejiang GenFleet Therapeutics.

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.

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.

The COMBI-I trial (ClinicalTrials.gov identifier: NCT02967692) evaluating spartalizumab plus dabrafenib and trametinib (sparta-DabTram, n = 267) versus placebo plus dabrafenib and trametinib (placebo-DabTram, n = 265) for BRAF V600-mutant unresectable or metastatic melanoma failed to reach its primary end point of progression-free survival at 24 months. This final analysis reports overall survival (OS) during at least 5 years of extended follow-up. At the end of the trial (August 21, 2024), the median duration of follow-up was 76.9 months (range, 73.7-83.3 months). The median OS was 61.5 months (95% CI, 41.6 to not evaluable) for the sparta-DabTram arm and 41.6 months (95% CI, 30.6 to 56.9) for the placebo-DabTram arm (hazard ratio, 0.760 [95% CI, 0.598 to 0.966]). The safety findings were consistent with the known safety profile for sparta-DabTram. The most common treatment-related adverse event (TRAE) was pyrexia (65.9% v 46.2%, respectively, in the two study arms). Grade ≥3 TRAEs were reported in 57.3% and 36.7% of patients in the two arms, respectively. The combination of sparta-DabTram appears to improve OS compared with dabrafenib and trametinib alone in patients with BRAF V600-mutant metastatic melanoma.

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 .

After its practice-changing success, immune checkpoint inhibitors (ICI) have possibly reached a treatment plateau and novel drug candidates are currently being investigated to overcome resistance to ICI including patients who never responded to ICI. With some of the recent failures of novel checkpoint inhibitors, there has been a shift of focus to other modalities such as antibody drug conjugates and radiopharmaceuticals. And skepticism towards novel agents in immunotherapy, including cell therapies, has yet to be replaced by renewed optimism. The combination of ICI with anti-angiogenic agents appeared as a promising new approach to avoid or revert resistance to ICI. Following these encouraging observations, bispecific agents have followed suit and are emerging as possible advancements. Targeting other immune cells, such as macrophages and epigenetic modulation hold additional promise to overcome resistance. Finally, the recent progress in vaccine development may provide a novel approach to selectively activate the global immune system and re-direct its components to reject tumors. Overall, it appears that the full potential of immunomodulators has not yet been fully achieved. Thus, improving the understanding of cancer biology, the interaction with the immune system and identifying patient subsets who might benefit from resetting their immune system continue to be opportunities for oncology drug development.

Background:The immune-balancing role of thymosin alpha 1 (Tα1) is well-recognized in contexts of immune dysregulation. Within the anti-tumor context, Tα1 demonstrated to act as an immune-enhancer, with potential roles in immunotherapy-based treatments. However, Tα1 immunomodulatory potential on tumor cells is poorly understood. Additionally, Tα1 pleiotropic effects on immune cells require in-depth investigations to unravel its specific impact on different immune cell populations. Thus, we first aimed to investigate whether Tα1 treatments influenced the transcriptional immune profile of various cancer cell lines. Alongside, CD4+ T, CD8+ T, B, and natural killer cells from healthy donors (HDs) were treated individually with Tα1, to assess its direct effects on each immune cell population. Methods:Cutaneous melanoma, glioblastoma, and pleural mesothelioma cell lines and HD immune cell subsets were treated with Tα1 for 48 hours. Total RNA was subsequently isolated, and gene expression profiles were analyzed by the nCounter® SPRINT Profiler. Genes with a log2ratio ≥0.58 and ≤-0.58 in Tα1-treated vs untreated cells were defined as differentially expressed (DEGs) and subsequently evaluated for the enrichment of Gene Ontology terms to identify biological processes potentially affected by Tα1 in tumor and immune cells. Results:Tα1 minimally changed cancer cell DEGs and immune-related biological processes, suggesting a comprehensive lack of transcriptional immunomodulatory potential on the tumor counterpart. Conversely, Tα1 exhibited to directly affect the proliferation and/or transcription processes of each studied immune cell subset, with the greatest transcriptional impact observed for activated CD8+ T cells, crucial players in anti-tumor immunity. Conclusion:Our findings question the tumor immunomodulatory properties of Tα1, simultaneously underscoring the importance of further investigating Tα1 influence on specific immune cell subsets in the periphery or within the tumor microenvironment of cancer patients. This would contribute to understand Tα1 potential in immunotherapy-based combination strategies, within the anti-tumor setting.