Identifying high-risk features and improving follow-up strategies in thin melanoma: a retrospective cohort study Antonella Vecchiato, Claudia Cozzolino, Paolo Del Fiore, Fortunato Cassalia, PierPaola Vallese, Chiara Trevisiol, Marco Rastrelli, Valentina Salizzato, Luisa Piccin, Irene Russo, Alessandra Buja, Jacopo Pigozzo, Rocco Cappellesso, Mauro Alaibac, Simone Mocellin Clinical and Experimental Dermatology, 2026 Background Thin melanomas (TMs) are typically associated with favourable outcomes, but a subset of patients experience recurrence and worse survival. Prognostic factors for recurrence in TM remain inadequately characterized. Objectives To identify clinical and histopathological factors associated with recurrence and explore implications for follow-up strategies in TM. Methods A retrospective cohort study included 308 patients with TM (Breslow thickness 0.1–1 mm) treated at two medical institutions between 1998 and 2017. Patients were stratified into groups with recurrence (n = 53) and no recurrence (n = 255). Clinical and histopathological variables were analysed, and survival outcomes were assessed. Results Recurrence (vs. no recurrence) was associated with nodular histology (13% vs. 3.1%, P = 0.02), presence of ulceration (17% vs. 2.7%, P < 0.001) and higher mitotic rate (mean 2.1 vs. 0.6 mitoses mm−2, P < 0.001). Ultrathin melanoma (Breslow ≤ 0.5 mm) was protective against recurrence (hazard ratio 0.08, P < 0.001). Recurrence (vs. no recurrence) reduced 10-year overall survival (62% vs. 95.7%, P < 0.001) and melanoma-specific survival (64% vs. 100%, P < 0.001). Conclusions Prognostic factors such as ulceration, mitotic rate and histological subtype should guide individualized follow-up strategies. Ultrathin melanomas may require less intensive monitoring, while melanomas > 0.5 mm could benefit from extended follow-up beyond current guidelines. Tailoring surveillance based on recurrence risk could improve outcomes and quality of care for patients with TM.
A model workflow for microfluidic enrichment and genetic analysis of circulating melanoma cells Cristina Poggiana, Alessandro Francesco Piazza, Cristina Catoni, Ilaria Gallingani, Luisa Piccin, Stefania Pellegrini, Vittorio Aneloni, Valentina Salizzato, Jacopo Pigozzo, Alessio Fabozzi, Antonella Facchinetti, Chiara Menin, Paolo del Fiore, Simone Mocellin, Vanna Chiarion-Sileni, Antonio Rosato, Maria Chiara Scaini Scientific Reports, 2025 Circulating melanoma cells (CMCs) are responsible for the hematogenous spread of melanoma and, ultimately, metastasis. However, their study has been limited by the low abundance in patient blood and the heterogeneous expression of surface markers. The FDA-approved CellSearch platform enriches CD146-positive CMCs, whose number correlates with progression-free survival and overall survival. However, a single marker may not be sufficient to identify them all. The Parsortix system allows enrichment of CMCs based on their size and deformability, keeping them viable and suitable for downstream molecular analyses. In this study, we tested the strengths, weaknesses and potential convergences of both platforms to integrate the counting of CMCs with a protocol for their genetic analysis. Samples run on Parsortix were labeled with a customized melanoma antibody cocktail, which efficiently labeled and distinguished CMCs from endothelial cells/leukocytes. The capture rate of CellSearch and Parsortix was comparable for cell lines, but Parsortix had a higher capture rate in real-life samples. Moreover, double enrichment with both CellSearch and Parsortix succeeded in removing most of the leukocyte contamination, resulting in an almost pure CMC sample suitable for genetic analysis. In this regard, a proof-of-concept analysis of CMCs from a paradigmatic case of a metastatic uveal melanoma patient led to the identification of multiple genetic alterations. In particular, the GNAQ p.Q209L was identified as homozygous, while a deletion in BAP1 exon 9 was found hemizygous. Moreover, an isochromosome 8 and a homozygous deletion of the CDKN2A gene were detected. In conclusion, we have optimized an approach to successfully enrich and retrieve viable CMCs from metastatic melanoma patients. Moreover, this study provides proof-of-principle for the feasibility of a marker-agnostic CMC enrichment followed by CMC phenotypic identification and genetic analysis.Kindly check and confirm the processed contributed equally is correctly identify We confirm.
Molecular Basis of BRAF Inhibitor Resistance in Melanoma: A Systematic Review Ilaria Cosci, Valentina Salizzato, Paolo Del Fiore, Jacopo Pigozzo, Valentina Guarneri, Simone Mocellin, Alberto Ferlin, Sara Mathlouthi, Luisa Piccin, Mariangela Garofalo Pharmaceuticals, 2025 Background: Melanoma, the deadliest human skin cancer, frequently harbors activating BRAF mutations, with V600E being the most prevalent. These alterations drive constitutive activation of the MAPK pathway, promoting uncontrolled cell proliferation, survival, and dissemination. The advent of BRAFi and MEKi has significantly improved outcomes in BRAF V600-mutant melanoma. However, therapeutic resistance remains a major clinical barrier. Methods: This review integrates recent findings from preclinical and clinical studies to delineate resistance mechanisms to BRAF-targeted therapy. It categorizes resistance into primary (intrinsic), adaptive, and acquired forms, and analyzes their molecular underpinnings, including genetic and epigenetic alterations, pathway reactivation, and microenvironmental interactions. Results: Primary resistance is linked to pre-existing genetic and epigenetic changes that activate alternative signaling pathways, such as PI3K-AKT. Adaptive and acquired resistance includes secondary BRAF mutations, pathway redundancy, phenotype switching, and immune and stromal interactions. High-throughput sequencing has revealed novel mutations, including NRAS, NF1, and PTEN alterations, that contribute to resistance. Discussion: Understanding the multifaceted nature of resistance is critical to improving outcomes in advanced melanoma. This review highlights emerging strategies to overcome resistance, including combinatorial therapies, metabolic targeting, and biomarker-driven approaches, aiming to inform future therapeutic development and precision oncology strategies.
Critical role of protein kinase CK2 in chronic myeloid leukemia cells harboring the T315I BCR::ABL1 mutation Camila Paz Quezada Meza, Valentina Salizzato, Elisabetta Calistri, Marco Basso, Manuela Zavatti, Sandra Marmiroli, Mauro Salvi, Bing Z. Carter, Arianna Donella-Deana, Christian Borgo, Maria Ruzzene International Journal of Biological Macromolecules, 2025 Chronic myeloid leukemia (CML) is characterized by the fusion protein BCR::ABL1, a constitutively active tyrosine kinase. The frontline treatment, represented by tyrosine kinase inhibitors (TKIs), has dramatically improved the clinical outcomes of patients. However, TKI resistance through various mechanisms has been reported. In particular, the BCR::ABL11 T315I mutation is associated with resistance to first- and second-generation TKIs and poor survival outcomes. For patients harboring this mutation, treatments with third generation TKIs are indicated, which are however accompanied by adverse events. Protein kinase CK2 is implicated in several human diseases. Although its role in CML has already been proven, its essentialness in T315I-mediated TKI resistance has yet to be investigated. Here we show that CK2 contributes to the aberrantly high signaling pathways in T315I-cells, and that its pharmacological or genetic targeting diminishes those signals, induces apoptosis, and reduces the proliferation and clonogenic potential of T315I-cells. The effects of CK2 inhibition are also observed in the presence of bone marrow stromal cells and under hypoxic conditions, and, remarkably, in patient-derived cells. Moreover, CK2 inhibition or genetic ablation of the CK2α catalytic subunit sensitizes T315I-cells towards TKIs. Collectively, our results suggest the potential benefit of inhibiting CK2 in CML characterized by T315I-dependent resistance.
Real-world multicentre observational study of effectiveness and toxicity in patients with advanced cutaneous squamous cell carcinoma treated with cemiplimab L. Faoro, V. Salizzato, M. Coppola, D. Carpanese, A. Fabozzi, L. Tondulli, R. Marconcini, F. Spagnolo, P. Del Bianco, V. Chiarion‐Sileni, L. Piccin Journal of the European Academy of Dermatology and Venereology, 2025 The annual incidence of cutaneous squamous cell carcinoma (CSCC) is increasing worldwide, as a consequence of life expectancy and sun damage exposure increase, with 5% cases evolving into not resectable locally advanced (aCSCC) and metastatic diseases.1, 2 Cemiplimab has led to a breakthrough in the treatment of aCSCC resulting in up to 50% overall response rate and long-lasting responses.3-6 Real-world evidence may close the gap between clinical trials and real practice: affected subjects are often underrepresented in clinical trials due to their frailty, advanced age, immunosuppression, comorbidities and polypharmacy. Our retrospective, observational, multicentre study enrolled 90 patients, treated with cemiplimab 350 mg intravenously Q3W between May 2020 and July 2022, in four Italian centres. The median age was 82 years (Q1, Q3 = 78, 87), 70.0% ECOG-PS 1–2, males accounted for 77.8% of the treated population and autoimmune disorders were present in 7.8% of patients. 61.1% was diagnosed with locally advanced while 38.9% with metastatic CSCC; head and neck (H&N) was the most frequent primary tumour site (80.1%), followed by limbs (12.2%), trunk (4.4%) and genitalia (3.3%). The median number of administrations was 9 and the median follow-up was 16.28 months (IQR: 11.41–20.66). Objective response rate (ORR) and disease control rate (DCR) were 47.8% and 73.4%, respectively. The median time to achieve ORR was 4.8 months (IQR: 3.4–7.6) and 3.8 months (IQR: 2.1–6.1) for DCR. Median duration of response (mDoR) among patients with disease control was 12.1 months (95% CI: 4.7-NR), and 75.3% of responding patients maintained the response at 12 months. Noteworthy, DoR was lower compared to the EMPOWER-CSCC-1 trial,6 likely due to differences in the duration of exposure to cemiplimab and number of administered doses. Table 1 summarizes patients' characteristics according to response. The 12 months OS and PFS of whole cohort were 56.6% (95% CI: 45.2, 66.5) and 47.9% (95% CI: 36.5, 58.4), respectively (Figure 1a). Interestingly, patients with H&N primary tumour exhibited better OS (17.34 months), which halved to 8.62 months for patients with tumour located in the limbs (Figure 1b). Noteworthy, poor ECOG-PS (Figure 1c) or the presence of concomitant autoimmune disorders (Figure 1d), even not requiring immunosuppressive agents, are related to worse prognosis, pointing out the necessity to carefully tailor the treatment in these patients. Our data confirmed the safety profile of cemiplimab, as treatment-related adverse events (TRAEs) occurred in 51.1% of patients, but none of Grade 4 or 5. Recorded TRAEs were as follows: 24.4% cutaneous (pruritus and rash), 16.7% fatigue, 11.1% endocrine/metabolic (thyroid and hypophysis dysfunction and diabetes), 7.8% gastrointestinal (diarrhoea and vomiting), 7.8% arthralgia and 32.2% others (hepatic, renal, hematologic, pancreatic, cardiologic, pulmonary or general events). Interestingly, the presence of any toxicity was associated with a significantly longer mOS (NR, 95% CI: 11.64-NR) as compared to no TRAE (mOS: 8.62 months, 95% CI: 3.09–17.3, p = 0.003) (Figure 1e), and for subjects with skin AEs there was a better outcome (OS: NR months, 95% CI: 11.64-NR vs. 12.2 months, 95% CI: 7.63-NR) (Figure 1f). These data suggest a potential correlation between treatment response and the occurrence of TRAEs, above all in presence of mild/moderate toxicities and when steroid immunosuppressive therapy is not required. Our work confirmed the effectiveness and the acceptable safety profile of cemiplimab in aCSCC patients, in agreement with both clinical trials3-6 and real-world studies.7, 8 Further research, aimed at the identification of clinical and laboratory predictive factors to treatment, could be useful to tailor and personalize the therapy in this heterogeneous and frail class of patients. AF received speaker's fees from BMS, MSD, Novartis, SUN Pharma. VCS received speaker's fees from Pierre Fabre and Immunocore; received support for attending meetings and travel from Pierre Fabre and Sanofi. LP received speaker's fees from Novartis, MSD, BMS. FS received speaker's fees from BMS, MSD, Novartis, Pierre Fabre, Sanofi, Merck, Sun Pharma; had advisory roles for Pierre Fabre, MSD, Novartis and Sun Pharma; received support for attending meetings and travel from Pierre Fabre and MSD. RM received speaker's fees from BMS, MSD, Novartis, SUN Pharma, Sanofi, Ipsen, Pierre Fabre, advisory roles for Pierre Fabre, Immuncore, MSD, Novartis, BMS, Ipsen; received support for attending meetings and travel from Pierre Fabre, Merck Sharp & Dohme, Novartis, Ipsen. LT received support for attending meetings and travel from Sanofi, Merck Serono and Roche. All the other authors (LF, VS, MC, DC, PDB) declare that they have no competing interests. The data that support the findings of this study are available from the corresponding author upon reasonable request.
Immunohistochemical and molecular profiling of uveal melanoma: clinicopathological correlations from an Italian cohort Francesco Fortarezza, Giovanni Zarrilli, Giada Munari, Valentina Angerilli, Mariangela Balistreri, Luisa Piccin, Mariangela Balistreri, Valentina Salizzato, Jacopo Pigozzo, Giulia Midena, Raffaele Parrozzani, Valentina Guarneri, Edoardo Midena, Marta Sbaraglia, Matteo Fassan, Angelo Paolo Dei Tos Pathologica, 2025 Objective. Uveal melanoma (UM) is the most common primary intraocular malignancy in adults, characterized by distinct histopathological and molecular features and often associated with poor prognosis due to its high metastatic potential. While histology, BAP1 status, and chromosomal changes are established prognostic markers, integration of morphological, immunophenotypic, and molecular data is evolving. Methods. We retrospectively analyzed 84 UM cases from a single institution using an integrated approach combining histological classification, immunohistochemical profiling, and targeted next-generation sequencing with a 63-gene panel. Tissue microarrays were used for immunophenotyping, and mutation data were stratified by prognostic outcomes. Results. Most tumors were localized to the choroid and predominantly exhibited spindle-cell morphology. Mutations in GNAQ or GNA11 were identified in 83% of sequenced cases. Loss of BAP1 expression correlated with epithelioid histology and denser T-cell infiltration yet lacked PD-L1 expression. Aberrant p53 staining was more frequent in spindle-cell tumors, though TP53 mutations were rare, suggesting functional inactivation through other mechanisms. Notably, mutations typically associated with cutaneous melanomas (e.g., BRAF, KIT, CDKN2A) were also detected, particularly in a single iris melanoma, suggesting site-specific molecular convergence. Additional recurrent alterations were found in NOTCH1, PTEN, PIK3CA, and KDR, implicating the mTOR and VEGF signaling pathways. A high mutational burden, along with mutations in genes such as H3F3A, IDH2, JAK3, and ESR1, was more frequent in tumors with poorer prognosis, supporting their potential role in disease aggressiveness. Conclusions. This study highlights the heterogeneous molecular landscape of UM and underscores the importance of integrating histopathological and molecular data for improved prognostic stratification. The identification of potential therapeutic targets and atypical mutations typically associated with other melanoma subtypes suggests avenues for future research and tailored therapeutic strategies.
Sex-related differences in serum biomarker levels predict the activity and efficacy of immune checkpoint inhibitors in advanced melanoma and non-small cell lung cancer patients Giulia Pasello, Aline S. C. Fabricio, Paola Del Bianco, Valentina Salizzato, Adolfo Favaretto, Luisa Piccin, Fable Zustovich, Alessio Fabozzi, Costanza De Rossi, Jacopo Pigozzo, Mattia De Nuzzo, Elia Cappelletto, Laura Bonanno, Dario Palleschi, Gian Luca De Salvo, Valentina Guarneri, Massimo Gion, Vanna Chiarion-Sileni Journal of Translational Medicine, 2024 Background Immune Checkpoint Inhibitors (ICIs) lead to durable response and a significant increase in long-term survival in patients with advanced malignant melanoma (MM) and Non-Small Cell Lung Cancer (NSCLC). The identification of serum cytokines that can predict their activity and efficacy, and their sex interaction, could improve treatment personalization. Methods In this prospective study, we enrolled immunotherapy-naïve patients affected by advanced MM and NSCLC treated with ICIs. The primary endpoint was to dissect the potential sex correlations between serum cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, GM-CSF, MCP-1, TNF-ɑ, IP-10, VEGF, sPD-L1) and the objective response rate (ORR). Secondly, we analyzed biomarker changes during treatment related to ORR, disease control rate (DCR), progression free survival (PFS) and overall survival (OS). Blood samples, collected at baseline and during treatment until disease progression (PD) or up to 2 years, were analyzed using Luminex xMAP or ELLA technologies. Results Serum samples from 161 patients (98 males/63 females; 92 MM/69 NSCLC) were analyzed for treatment response. At baseline, IL-6 was significantly lower in females (F) versus males (M); lower levels of IL-4 in F and of IL-6 in both sexes significantly correlated with a better ORR, while higher IL-4 and TNF-ɑ values were predictive of a lower ORR in F versus M. One hundred and sixty-five patients were evaluable for survival analysis: at multiple Cox regression, an increased risk of PD was observed in F with higher baseline values of IL-4, sPD-L1 and IL-10, while higher IL-6 was a negative predictor in males. In males, higher levels of GM-CSF predict a longer survival, whereas higher IL-1β predicts a shorter survival. Regardless of sex, high baseline IL-8 values were associated with an increased risk of both PD and death, and high IL-6 levels only with shorter OS. Conclusions Serum IL-1β, IL-4, IL-6, IL-10, GM-CSF, TNF-ɑ, and sPD-L1 had a significant sex-related predictive impact on ORR, PFS and OS in melanoma and NSCLC patients treated with ICIs. These results will potentially pave the way for new ICI combinations, designed according to baseline and early changes of these cytokines and stratified by sex.
Cytokine and soluble programmed death-ligand 1 levels in serum and plasma of cancer patients treated with immunotherapy: Preanalytical and analytical considerations Elia Cappelletto, Laura Tiozzo Fasiolo, Valentina Salizzato, Luisa Piccin, Alessio Fabozzi, Anna Contato, Paola Del Bianco, Giulia Pasello, Vanna Chiarion-Sileni, Massimo Gion, Aline S. C. Fabricio International Journal of Biological Markers, 2024 Aim To evaluate cytokine and soluble programmed death ligand-1 (sPD-L1) levels in the serum and plasma of cancer patients treated with immunotherapy, and to test different assays. Methods Three Luminex xMAP assays and two ELLA microfluidic cartridges were used to screen 28 immune-related biomarkers in 38 paired serum and citrate-theophylline-adenosine-dipyridamole (CTAD) plasma samples collected from 10 advanced melanoma or non-small cell lung cancer (NSCLC) patients at different time points during immunotherapy. Results Twenty-three of 28 biomarkers were detected both in serum and plasma by at least one of the assays, including IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, GM-CSF, IFN-γ, TNF-α, VEGF, IP-10, MCP-1, eotaxin, fractalkine, G-CSF, IFN-α, IL-1RA, IL-13, IL-17A, MIP-1β and sPD-L1. Conversely, FGF-2 and IL-1α were not detected in both matrices; GRO-α factor and EGF were detected only in serum and MIP-1α only in plasma. sPD-L1, MCP-1, IFN-γ, IL-8, MIP-1β and VEGF were, respectively, 1.15-, 1.44-, 1.83-, 2.43-, 2.82-, 6.72-fold higher in serum, whereas IL-10, IL-4, IL-2 and IL-5 were 1.05-, 1.19-, 1.92- and 2.17-fold higher, respectively, in plasma. IP-10 levels were higher in plasma but, as well as for VEGF, the bias serum versus plasma varied depending on the assay used (IP-10: −5.7% to −145%; VEGF: 115% to 165%). No significant differences were found for the remaining nine analyzed cytokines. Conclusion The cytokine and sPD-L1 levels may differ between serum and plasma samples collected from cancer patients treated with immunotherapy, and the results obtained can be influenced by the different characteristics of the tested assays. The standardization of pre-analytical and analytical procedures is therefore needed for the future implementation of these circulating biomarkers in clinical practice.
Protein kinase CK2 subunits exert specific and coordinated functions in skeletal muscle differentiation and fusogenic activity Valentina Salizzato, Sofia Zanin, Christian Borgo, Elisa Lidron, Mauro Salvi, Rosario Rizzuto, Giorgia Pallafacchina, Arianna Donella-Deana FASEB Journal, 2019 Casein kinase 2 (CK2) is a tetrameric protein kinase composed of 2 catalytic (α and α′) and 2 regulatory β subunits. Our study provides the first molecular and cellular characterization of the different CK2 subunits, highlighting their individual roles in skeletal muscle specification and differentiation. Analysis of C2C12 cell knockout for each CK2 subunit reveals that: 1) CK2β is mandatory for the expression of the muscle master regulator myogenic differentiation 1 in proliferating myoblasts, thus controlling both myogenic commitment and subsequent muscle‐specific gene expression and myotube formation; 2) CK2α is involved in the activation of the muscle‐specific gene program; and 3) CK2α′ activity regulates myoblast fusion by mediating plasma membrane translocation of fusogenic proteins essential for membrane coalescence, like myomixer. Accordingly, CK2α′ overexpression in C2C12 cells and in mouse regenerating muscle is sufficient to increase myofiber size and myonuclei content via enhanced satellite cell fusion. Consistent with these results, pharmacological inhibition of CK2 activity substantially blocks the expression of myogenic markers and muscle cell fusion both in vitro in C2C12 and primary myoblasts and in vivo in mouse regenerating muscle and zebrafish development. Overall, our work describes the specific and coordinated functions of CK2 subunits in orchestrating muscle differentiation and fusogenic activity, highlighting CK2 relevance in the physiopathology of skeletal muscle tissue.—Salizzato, V., Zanin, S., Borgo, C., Lidron, E., Salvi, M., Rizzuto, R., Pallafacchina, G., Donella‐Deana, A. Protein kinase CK2 subunits exert specific and coordinated functions in skeletal muscle differentiation and fusogenic activity. FASEB J. 33, 10648–10667 (2019). www.fasebj.org
