Stefano Landi

Pleural mesothelioma (PM) often presents loss of BAP1 and MTAP genes. Digital PCR (dPCR) can accurately measure gene copy numbers (CNs). We hypothesized that cell‐free DNA (cfDNA) extracted from the supernatant of pleural fluids (SPFs) could allow an efficient detection of these genetic events. BAP1/MTAP CN was measured with dPCR in SPFs and in solid tumor biopsies (STBs) of 37 PM patients. Results were compared with immunohistochemistry (IHC) carried out in STBs. IHC alone detected BAP1/MTAP‐losses in 32/37 (86.5%). However, the sensitivity improved to 34/37 (91.9%) when low CN of BAP1/MTAP was included. Among 20 patients presenting SPFs, dPCR alone detected a low CN in 18 samples (sensitivity = 90%). Finally, this two‐gene signature was applied on SPFs in a prospective series of six consecutive patients presenting an undefined diagnosis (negative for PM) and two showed reduced CN. After 7 months of follow‐up, these subjects were further re‐evaluated for the persistence/worsening of the symptoms and were diagnosed for PM. We highlighted dPCR's potential for detecting subtle BAP1/MTAP losses in SPFs of PM patients. This appears as a complementary approach for a more accurate diagnosis in specific contexts, when high‐risk patients receive negative results after IHC analysis.

Monoclonal gammopathy of undetermined significance (MGUS) is a necessary precursor condition to multiple myeloma (MM). Given the role of autophagy in modulating MM risk, we investigated whether genetic variation in autophagy‐related genes influences susceptibility to MGUS. We analyzed the association of 34,042 common autophagy‐related single nucleotide polymorphisms (SNPs) with MGUS across six independent cohorts, five from Europe and one from North America, comprising 2317 MGUS cases and 282,358 controls. We also assessed their impact on immune parameters, including absolute counts of 91 blood‐derived immune cell subsets and 103 circulating immunological proteins. Meta‐analysis revealed a genome‐wide significant association between the ULK4 _rs6599175C allele and increased MGUS risk ( p = 3.35 × 10 ⁻⁸ ). Carriers of this allele showed reduced counts of memory B cell subsets (IgM+CD38+CD27+ and IgD+IgM+CD27+; p = .0038 and p = .0056, respectively) and natural effector B cells (CD24+CD38+IgD+IgM+ cells; p = .0060). Although these associations were not statistically significant after multiple testing correction, they suggest a role of ULK4 in early B‐cell differentiation. Additionally, the CDKN2A _rs2811710 variant showed a suggestive association with MGUS risk ( p = 2.17 × 10 ⁻⁴ ), affecting transcription factor binding involved in B‐cell proliferation and differentiation, although it lacked association with immune markers. In conclusion, we confirm a genome‐wide significant association of the ULK4 locus and MGUS risk, supporting its role in early B‐cell differentiation, and identify CDKN2A as a candidate susceptibility locus warranting further investigation.

Mesothelin (MSLN) is among the most studied cancer-related antigens, and it is extensively studied as a therapeutic target for the treatment of various malignancies, including pleural mesothelioma, pancreatic ductal adenocarcinoma, and ovarian cancer. However, despite the development of many MSLN-targeting strategies, such as antibody–drug conjugates (ADC), bispecific antibodies, and CAR-T cells, clinical responses have remained limited, underscoring the need for a deeper understanding of MSLN biology. Over the past decades, many studies have highlighted a link between MSLN and cancer progression and its association with specific features within the tumor microenvironment (TME). More recently, mechanistic evidence has emerged showing the involvement of MSLN in the establishment of key malignant features, such as the epithelial-to-mesenchymal transition (EMT) and matrix metalloproteinase 7-mediated remodeling of the extracellular matrix (ECM). Furthermore, these studies also show a direct role for MSLN in the immunosuppressive polarization of the TME through the interaction with CD206 macrophage receptors (leading to an M2-like polarization) and by promoting the transition of mesothelial cells into specific cancer-associated fibroblasts (CAFs). This review synthesizes current evidence on MSLN transcriptional regulation and its functional implications in invasion, metastasis, and immune evasion. We also summarize ongoing therapeutic strategies targeting MSLN and discuss how TME-driven resistance mechanisms are shaping the next generation of MSLN-directed therapies. By integrating molecular insights with translational perspectives, this work provides a comprehensive overview of MSLN biology and its emerging therapeutic relevance in cancer.

We investigated the influence of 55 583 autophagy-related single-nucleotide polymorphisms (SNPs) on chronic lymphocytic leukemia (CLL) risk across 4 independent populations comprising 5472 CLL cases and 726 465 controls. We also examined their impact on overall survival (OS), time to first treatment (TTFT), autophagy flux, and immune responses. A meta-analysis of the 4 populations identified, to our knowledge, for the first time, significant associations between CDKN2A (rs3731204) and BCL2 (rs4940571, rs12457371, and rs1026825) SNPs and CLL risk, with CDKN2A showing the strongest association (P = 1.57 × 10−12). We also validated previously reported associations for FAS, BCL2, and BAK1 SNPs with CLL risk (P = 4.73 × 10−21 to 3.39 × 10−9). The CDKN2Ars3731204 and FASrs1926194 SNPs associated with increased CDKN2A and ACTA2 messenger RNA expression levels in the whole blood and/or lymphocytes (P = 5.1 × 10−7, P = 1.58 × 10−21, and P = 7.8 × 10−41), although no significant effect on autophagy flux was observed. However, associations were found between CDKN2A, BCL2, and FAS SNPs and various T-cell subsets, cytokine production, and circulating concentrations of interferon gamma, tumor necrosis factor–related apoptosis-inducing ligand, CD40, chemokine ligand 20, and interleukin-2 receptor subunit β proteins (P ≤ .005). No significant association was detected between autophagy variants and OS or TTFT, suggesting that these variants drive disease initiation rather than progression. In conclusion, this study identified 4 novel associations for CLL and provided insights into the biological pathways that influence CLL development.

In our previous drug screening, cephalomannine, fludarabine, and crenolanib were among the most active compounds against permanent cell lines of Pleural Mesothelioma (PM). Here, firstly we assessed the efficacy of these compounds on PM-xenograft NOD-SCID mice and evaluated the overall survival (OS) as major endpoint. Then, we evaluated in vitro the underlying mechanisms involving CPM (at 0.1 µM) as anticancer agent on REN (derived from PM) and MeT-5A, a mesothelial cell line often used as a non-malignant model of mesothelial cells. We measured the oxygen consumption rate (OCR), the intracellular reactive oxygen species (ROS) production and the protein level of BAX and Bcl2, as proxy for apoptosis. Moreover, we measured IL-18 levels, as marker of the inflammasome pathway activation. While no effects were found for fludarabine (40, 100, or 200 mg/kg body weight) and crenolanib (15 mg/kg body weight), for cephalomannine (CPM, 2 mg/kg body weight) we observed a prolonged median OS in the treated group (75 days) vs vehicle control group (55 days; P = 0.0021). We found an increase in ROS production and a reduction in OCR in the REN cell line, compared to MeT-5A. We also noticed an activation of apoptosis, but not of inflammation. These results confirm the effectiveness of CPM on PM, suggesting novel targets and pathways for investigation.

High-grade serous carcinoma (HGSC) of the ovary is characterized by two major histological patterns: a classic papillary/micropapillary architecture and a solid pseudo-endometrioid transitional (SET) variant. We investigated whether the distinct morphologic subtypes are underpinned by transcriptomic differences in the tumor microenvironment (TME). We profiled 21 HGSC tumors (7 SET, 14 classic) using a 770-gene NanoString PanCancer Progression panel. Differential expression analysis revealed ~20 genes with significantly different expression (>4-fold, adjusted p < 0.01) between SET and classic tumors. Unsupervised clustering partially separated SET and classic tumors, suggesting that global gene expression patterns correlate with histologic subtype. SET tumors exhibited upregulation of cell-cycle and epithelial genes (e.g., PTTG1, TRAIL, HER3) and downregulation of genes involved in epithelial–mesenchymal transition (EMT), extracellular matrix (ECM) organization, and angiogenesis (e.g., TWIST2, FGF2, decorin) relative to classic tumors. Notably, PTTG1 and TRAIL were upregulated ~6–9-fold in SET tumors, whereas TWIST2 was ~7-fold downregulated, consistent with reduced EMT in SET tumors. Pathway analysis indicated that SET tumors appear to have an immune-active, stroma-poor microenvironment, in line with an “immunoreactive” phenotype, whereas classic tumors showed a mesenchymal, stroma-rich profile. These molecular distinctions could have diagnostic utility and may inform therapeutic stratification, with key dysregulated genes (e.g., HER3, TRAIL, FGF2) representing potential prognostic or predictive biomarkers. For example, high HER3 expression in SET tumors might predict sensitivity to ERBB3/PI3K inhibitors, whereas stromal factors (e.g., FGF2) enriched in classic HGSC could be targeted with microenvironment-modulating therapies. These preliminary findings require validation before translation into pathology practice via immunohistochemical (IHC) assays (e.g., for HER3 or TRAIL), potentially enabling improved classification and personalized treatment of HGSC. We report effect sizes as log₂ fold change with 95% confidence intervals and emphasize FDR-adjusted q-values. Given the small sample size and the absence of outcome data (OS/PFS/PFI), results are preliminary and hypothesis-generating. Orthogonal protein-level validation and replication in larger, independent cohorts are required before any translational inference.

High-grade serous carcinoma (HGSC) of the ovary is characterized by two major histological patterns: a classic papillary/micropapillary architecture and a solid pseudo-endometrioid transitional (SET) variant. We investigated whether the distinct morphological subtypes are underpinned by transcriptomic differences in the tumor microenvironment (TME). We profiled 21 HGSC tumors (7 SET, 14 classic) using a 770-gene NanoString PanCancer Progression panel. Differential expression analysis revealed ~20 genes with significantly different expression (>4-fold, adjusted p < 0.01) between SET and classic tu-mors. Unsupervised clustering partially separated SET and classic tumors, suggesting that global gene expression patterns correlate with histologic subtype. SET tumors exhibited upregulation of cell-cycle and epithelial genes (e.g., PTTG1, TRAIL, HER3) and down-regulation of genes involved in epithelial–mesenchymal transition (EMT), extracellular matrix (ECM) organization, and angiogenesis (e.g., TWIST2, FGF2, decorin) relative to classic tumors. Notably, PTTG1 and TRAIL were upregulated ~6–9-fold in SET tumors, whereas TWIST2 was ~7-fold downregulated, consistent with reduced EMT in SET tumors. Pathway analysis indicated that SET tumors appear to have an immune-active, stroma-poor microenvironment, potentially consistent with an "immunoreactive" phenotype, whereas classic tumors showed a mesenchymal, stroma-rich profile. These molecular distinctions could have diagnostic utility and may inform therapeutic stratification, with key dysregulated genes (e.g., HER3, TRAIL, FGF2) representing potential prognostic or predictive biomarkers. For example, high HER3 expression in SET tumors might predict sensitivity to ERBB3/PI3K inhibitors, whereas stromal factors (e.g., FGF2) enriched in classic HGSC could be targeted with microenvironment-modulating therapies. These preliminary findings require validation before translation into pathology practice via immunohistochemical (IHC) assays (e.g., for HER3 or TRAIL), potentially enabling improved classification and personalized treatment of HGSC.

Differentiated thyroid cancer (DTC) is a prevalent malignancy with increasing global incidence, yet its genetic susceptibility remains poorly understood. Although previous genome-wide association studies (GWAS) have identified several susceptibility loci, the genetic, transcriptomic, and proteomic factors influencing DTC risk remain unclear. We conducted a large-scale GWAS of 7,681 cases and 963,550 controls of European ancestry. Transcriptome-wide association studies (TWAS) used the joint tissue imputation across thyroid, pituitary, blood, and hypothalamus tissues. Proteome-wide association studies (PWAS) integrated brain and plasma proteomic data to identify proteins influencing DTC risk. Mendelian randomization (MR) and Bayesian colocalization were conducted to infer causality. GWAS identified 18 novel loci associated with DTC risk, four of which were previously suggested and are now confirmed. TWAS identified 29 significant genes, including five genes ( LRRC34 , NRG1 , HEMGN , PTCSC3 , and SMAD3) located within known loci and three novel genes ( SAMD4A, RAD51-AS1, and MPHOSPH6 ) validated as causal through MR and Bayesian colocalization. PWAS identified seven significant proteins, with three ( MTHFR , KDELC2 , and SAMD4A ) confirmed as causal, further highlighting 15q15.1 as a novel risk locus consistently emerging across all omics layers. This integrated multi-omics approach reveals novel genetic and molecular mechanisms underlying DTC, linking genomic variation to gene expression and protein abundance.

Background: Excess adipose tissue has been associated with the increased prevalence and aggressiveness of many human cancers. While its role in differentiated thyroid cancer is well established, in medullary thyroid cancer (MTC), data are conflicting. We aimed to evaluate the impact of excess adipose tissue on MTC prevalence, aggressiveness at diagnosis, and outcome in a large series of patients. Methods: We evaluated 529 patients with sporadic MTC from a prospectively maintained database. Weight and height were measured in all patients at the time of surgery, and body mass index (BMI) was calculated. Therefore, patients were classified according to BMI categories suggested by the WHO for Caucasian patients. Data about somatic mutations were available in 254/529 patients (48.0%). Results: The prevalence of subjects with obesity was higher than that in the Italian general population (20.2 vs. 12.0%). Patients with obesity presented smaller tumors, lower T and N stage, and lower AJCC 8th edition stage, as well as lower preoperative calcitonin values compared to under/normal and overweight ones. The prevalence of somatic RET and RAS mutations did not differ significantly across the WHO BMI categories. At the end of follow-up, structural disease was less common in patients with obesity (15.4%) compared to under/normal (25.6%, p < 0.05) and overweight (24.1%, p = 0.079). Further surgeries for local recurrence were less common in patients with obesity (3.8%) compared to overweight ones (11.0%, p < 0.05). Survival was superimposable in the different categories of BMI. Conclusions: Among patients with sporadic MTC, there is a higher prevalence of patients with obesity compared to the general population. A higher BMI was associated with less aggressive clinical presentation and better clinical outcomes.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers with patients having unresectable or metastatic disease at diagnosis, with poor prognosis and very short survival. Given that genetic variation within autophagy‐related genes influences autophagic flux and susceptibility to solid cancers, we decided to investigate whether 55,583 single nucleotide polymorphisms (SNPs) within 234 autophagy‐related genes could influence the risk of developing PDAC in three large independent cohorts of European ancestry including 12,754 PDAC cases and 324,926 controls. The meta‐analysis of these populations identified, for the first time, the association of the BID_rs9604789 variant with an increased risk of developing the disease (OR_Meta = 1.31, p = 9.67 × 10⁻⁶). We also confirmed the association of TP63_rs1515496 and TP63_rs35389543 variants with PDAC risk (OR = 0.89, p = 6.27 × 10⁻⁸ and OR = 1.16, p = 2.74 × 10⁻⁵). Although it is known that BID induces autophagy and TP63 promotes cell growth, cell motility and invasion, we also found that carriers of the TP63_rs1515496G allele had increased numbers of FOXP3+ Helios+ T regulatory cells and CD45RA+ T regulatory cells (p = 7.67 × 10⁻⁴ and p = 1.56 × 10⁻³), but also decreased levels of CD4+ T regulatory cells (p = 7.86 × 10⁻⁴). These results were in agreement with research suggesting that the TP63_rs1515496 variant alters binding sites for FOXA1 and CTCF, which are transcription factors involved in modulating specific subsets of regulatory T cells. In conclusion, this study identifies BID as new susceptibility locus for PDAC and confirms previous studies suggesting that the TP63 gene is involved in the development of PDAC. This study also suggests new pathogenic mechanisms of the TP63 locus in PDAC.