Anna Maria Di Giacomo

Phosphatidylinositol 3-kinase delta (PI3Kδ) promotes tumour cell growth directly or indirectly by activating immune-suppressive cells. In contrast to previous PI3Kδ inhibitors, roginolisib is a selective, non-ATP competitive inhibitor, which locks PI3Kδ into an inactive state and causes an allosteric modulation. In a First-in-Human dose study, continuous daily dosing of roginolisib was investigated in patients with solid and haematologic malignancies. In Part A, 24 patients received escalating doses of roginolisib. In Part B, 20 mostly pre-treated metastatic uveal melanoma (mUM) patients received a dose associated with continuous PI3Kδ inhibition of >90%. The primary endpoint was safety, and secondary endpoints included pharmacokinetic (PK) and pharmacodynamic (PD) profile, clinical anti-tumour responses assessments. Exploratory endpoints included immunophenotyping, proteomics, genomics. No Dose Limiting Toxicity or drug related toxicities requiring dose modifications were observed ( ≥ Grade 3 toxicities: 3/44; 6.8%). In mUM, median Overall Survival (mOS) was 20.8 months (data-cut off December 2023) and 18.4 months (data-cut-off February 2026). mOS in mUM patients with Stable Disease at Week 16 ( = Cycle 5) was 28.5 months, while patients who progressed at Week 16 had a median OS of 12.3 months. Roginolisib was safe, had a predictable PK profile, T regulatory cell abundance was decreased while activated CD8⁺ T cells increased together with soluble plasma IL-15. ClinicalTrials.gov registration: NCT04328844.

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.

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. 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). 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. These results highlight EV-miRNAs as non-invasive biomarkers for predicting and monitoring therapy response.

BackgroundPure ground-glass opacities (pGGOs) represent a growing diagnostic challenge in the era of lung cancer screening. Although often perceived as indolent, a subset of these lesions may harbor biologically aggressive features such as PD-L1 expression or EGFR mutations. Identifying non-invasive predictors of these molecular markers could enhance risk stratification and guide clinical management.MethodsThis multicenter retrospective study analyzed invasive pGGOs from the Italian GORDON database. Only nodules with preoperative high-resolution CT, histologically confirmed invasive adenocarcinoma, and available molecular profiling for EGFR mutations and/or PD-L1 expression were included. Qualitative and quantitative CT features, including attenuation values, Difference CT value, and Relative Attenuation, were assessed.ResultsPD-L1 expression was found in 29.5% of 132 analyzed nodules and was significantly associated with lower unenhanced attenuation (–451 HU vs. –328 HU, p = 0.01), lower Difference CT value (p = 0.02), and lower Relative Attenuation (p = 0.01). Arterial vascular supply was less common in PD-L1+ nodules (p = 0.02). ROC analysis of attenuation parameters showed moderate predictive performance. EGFR mutations were identified in 38.8% of 129 nodules, significantly more frequent in women and younger patients. No radiologic predictors were significantly associated with EGFR status, except for a lower prevalence of bubble-like lucency in EGFR-mutant nodules (p = 0.01).ConclusionQuantitative CT features, particularly attenuation metrics, may aid in identifying PD-L1–expressing pGGOs, even in the absence of morphological aggressiveness. In contrast, conventional imaging lacks predictive value for EGFR mutations, highlighting the need for further molecular integration.

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.

DATA SUPPLEMENT Adjuvant Nivolumab Versus Ipilimumab in Resected Stage III/IV Melanoma: 5-Year Efficacy and Biomarker Results From CheckMate 238

Melanoma plasticity drives immune evasion and therapy resistance through dynamic cell-state transitions beyond genetic alterations. Epigenetic remodeling critically influences such processes, yet its role in reshaping the tumor ecosystem under therapeutic pressure remains unresolved. Here, we profiled longitudinal biopsies from melanoma patients treated in the phase Ib NIBIT-M4 epi-immunotherapy clinical trial ( NCT02608437 ), testing the combination of a DNMT1 inhibitor with anti-CTLA4 using single-cell multiome and high-resolution spatial transcriptomics. Integrated analyses resolved seven malignant meta-programs, including a rare Wnt/β-catenin–driven melanocytic state and a de-differentiated neural crest–like state enriched in non-responders. Spatial modeling revealed that homotypic clustering stabilizes resistant programs, with neural crest–like cells forming compact, centrally localized niches, whereas Wnt/β-catenin subpopulations displayed a bimodal architecture, either cohesive clusters sustained by adhesion or dispersed, transcriptionally plastic cells. Responders exhibited progressive enrichment of an antigen presentation/interferon program and coordinated remodeling of the tumor microenvironment with T and B cell expansion, whereas tumors from non-responder patients maintained stable composition of neural crest–like clusters. Epigenetic therapy reactivated transposable elements, providing both regulatory signals that prime innate immunity within microenvironment and generating antigens that drive immunoediting and immunogenicity of Antigen presentation/interferon cell states in responders. Finally, NFATC2 emerged as a master regulator of neural crests–like transcriptional phenotypes and promoter of resistance to therapeutic interventions in melanoma patients. NFATC2 perturbation was able to shift tumor cells towards more differentiated and immunogenic states. These findings reveal how epigenetic-based immunotherapy reshapes melanoma ecosystems, provide mechanistic insights into how multiple transcriptional programs promote tumor plasticity and resistance to both combinatorial therapies and immune checkpoint blockade, identify spatial clustering as a principle stabilizing resistant niches, and highlight β-catenin and NFATC2 as actionable vulnerabilities to overcome resistance.

Background: The immune-balancing role of thymosin alpha 1 (Tα 1) is well-recognized in contexts of immune dysregulation. Within the anti-tumor context, Tα 1 demonstrated to act as an immune-enhancer, with potential roles in immunotherapy-based treatments. However, Tα 1 immunomodulatory potential on tumor cells is poorly understood. Additionally, Tα 1 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 Tα 1 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 Tα 1, 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 Tα 1 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 Tα 1-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 Tα 1 in tumor and immune cells. Results: Tα 1 minimally changed cancer cell DEGs and immune-related biological processes, suggesting a comprehensive lack of transcriptional immunomodulatory potential on the tumor counterpart. Conversely, Tα 1 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 Tα 1, simultaneously underscoring the importance of further investigating Tα 1 influence on specific immune cell subsets in the periphery or within the tumor microenvironment of cancer patients. This would contribute to understand Tα 1 potential in immunotherapy-based combination strategies, within the anti-tumor setting.