Biogem

Abstract Background: Pleural mesothelioma (PM) is an aggressive malignancy with a poor prognosis. The clinical efficacy of standard therapy with immune checkpoint inhibitors (ICI) is limited and heterogeneous across PM subtypes. Tumor-intrinsic characteristics (i.e., inflammatory phenotype, molecular features, DNA methylation) may influence immune responsiveness, but predictive biomarkers of ICI therapy efficacy in PM are still lacking. Methods: NIBIT-EPI-MESO is a retrospective, multicenter study, sponsored by the NIBIT Foundation, evaluating biological correlates of clinical outcomes in PM patients (pts) treated with ICI (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=82 pts), 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 tumor biology with clinical outcome of PM pts. Results. Unsupervised methylation profiling identified four PM subsets with increasingly global DNA methylation levels: demethylated (DEM), LOW, intermediate (INT), and CpG island methylator phenotype (CIMP). Methylation subtypes were significantly associated with response 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 subset exhibited the longest median overall survival (mOS) and the highest 3-year OS rate, expressed genes involved 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 OS rate, was characterized by genes enriched in developmental, morphogenetic and cell cycle-related processes, along with a “desert” TME. Functional characterization of the identified methylation classes of PM was validated in the MESOMICS dataset. Accordingly, a PM methylation subtype classifier was developed to predict response to ICI therapy. Conclusions: Tumor DNA methylation defines biologically and clinically distinct immune phenotypes in PM and robustly predicts clinical response and long-term survival in ICI-treated PM patients, regardless of tumor histology. Citation Format: Luana Calabrò, Francesca Pia Caruso, Alessia Covre, Teresa Maria Rosaria Noviello, Maria Fortunata Lofiego, Rossella Tufano, Luigi Ferraro, Piera Grisolia, Antonio De Falco, Vincenzo Lagano, Francesco Sgambelluri, Giovanna Sabella, Giulia Rossi, Giulia Gibilisco, Francesco Marzani, Emma Bello, Elena Simonetti, Vincenzo D'Alonzo, Michele Caraglia, Sandra Coral, Antonina De Angelis, Luigi Cerbone, Sara Delfanti, Federica Grosso, Anna Maria Di Giacomo, Massimo Milione, Roberta Mortarini, Andrea Anichini, Michele Ceccarelli, Michele Maio. Tumor methylation subtypes predict the clinical outcome to immunotherapy in pleural mesothelioma patients from the NIBIT-EPI-MESO 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-C009.

Patient-reported outcome measures (PROMs) have become essential in contemporary internal medicine, where chronic, multisystem diseases and comorbidities make traditional biomedical endpoints insufficient as sole indicators of therapeutic benefit. PROMs capture symptom burden, functional capacity, emotional well-being, social participation and overall quality of life, thereby complementing laboratory indices, imaging findings and clinician-rated scales. Their growing use in regulatory evaluation, health technology assessment and value-based care underscores the need for rigorous methodology in their development, validation and interpretation. This review outlines key conceptual and practical issues that must be addressed for PROMs to provide valid, interpretable and clinically meaningful information. It emphasizes the central importance of a clearly articulated conceptual framework grounded in literature, clinical expertise and qualitative research with patients. It summarizes best practices in item generation and refinement, scaling and response options and the assessment of psychometric properties, including reliability, validity, responsiveness and interpretability. Particular attention is given to cross-cultural adaptation, differential item functioning and longitudinal measurement, which are crucial in heterogeneous internal medicine populations. The review also addresses the integration of PROMs into clinical trials and routine care, focusing on issues of feasibility, respondent burden, missing data and the translation of score changes into clinically actionable decisions. By clarifying these methodological foundations, it aims to support clinicians, investigators and policymakers in choosing, implementing and interpreting PROMs so that they genuinely reflect patients' experiences and priorities and thereby advance the practice of patient-centered internal medicine.

ABSTRACTAging is a key driver of cardiac dysfunction, promoting structural remodeling, metabolic alterations, and loss of cellular resilience. In aged hearts, extracellular matrix remodeling and collagen accumulation reduce ventricular compliance, impairing both diastolic function and stress adaptability. Cardiomyocytes exhibit diminished regenerative capacity and dysregulated stress responses, with mitochondrial dysfunction emerging as a central contributor to energy imbalance, oxidative stress, and fibrosis. Traditional single‐omics approaches are insufficient to capture the complexity of these interconnected changes. To address this, we employed an integrative multi‐omics strategy—combining spatial transcriptomics, proteomics, and metabo‐lipidomics with electron microscopy—to investigate cardiac aging in mice at three life stages: adult (12 months), middle‐aged (24 months), and elderly (30 months). Electron microscopy revealed enlarged, structurally compromised mitochondria. Spatial transcriptomics showed reduced expression of cardioprotective genes (MANF, CISH, and BNP) and increased expression of profibrotic markers like CTGF. Proteomics revealed widespread mitochondrial dysregulation and impaired ATP production. Metabolic and lipidomic profiling identified reduced antioxidant metabolites and accumulation of lipotoxic species, such as ceramides and diacylglycerols. This multiscale analysis highlights key molecular and metabolic alterations driving cardiac aging, identifying potential therapeutic targets to mitigate age‐related functional decline. Overall, our findings highlight the value of integrated, system‐level approaches for uncovering the complex mechanisms that drive organ aging. Although our study was conducted in mice, validation in human models will be crucial to establish the translational relevance of these results and to guide future research with potential impact across diverse biomedical fields.

BackgroundGlioblastoma (GBM), the most common primary malignant brain tumor in adults, remains uniformly fatal due to the lack of effective targeted therapies. The epidermal growth factor receptor (EGFR) is the most frequently altered receptor tyrosine kinase oncogene in GBM with most alterations impacting the receptor ectodomain function, including gene amplification, mutation, rearrangement, and splicing site changes, which occur in approximately 50% of GBM tumors. Depatuxizumab mafodotin (Depatux-M; ABT-414), an antibody-drug conjugate composed of an EGFR-specific antibody (ABT-806) that recognizes the EGFR ectodomain linked to the cytotoxic agent monomethyl auristatin F, initially showed clinical promise. However, it failed to improve survival in phase III trials, highlighting an urgent need to understand mechanisms of resistance.MethodsWe generated in vivo ABT-414 resistant GBM models using patient-derived xenografts (PDXs) and performed genomics and transcriptomic profiling, including whole exome sequencing, bulk RNA sequencing, and single-cell RNA sequencing.ResultsABT-414-resistant tumors exhibited transcriptional reprogramming characterized by upregulation of synaptic and developmental gene networks and downregulation of biosynthetic processes, indicative of a plastic, therapy-adaptive state. Whole-exome sequencing revealed novel mutations exclusive to resistant tumors, including a recurrent TEK (TIE2) S466I point mutation present in all ABT-414 resistant GBM12 PDX tumors. Functional validation demonstrated that ectopic expression of TEK S466I and TEK WT in PDX models reduced EGFR levels, suggesting a novel feedback mechanism linking TEK signaling to EGFR downregulation and contributes to resistance.ConclusionOur findings demonstrate that resistance to ABT-414 arises through both adaptive transcriptional remodeling and newly acquired genetic alterations. TEK-mediated suppression of EGFR represents a previously unrecognized mechanism of resistance, with potential implications for overcoming antibody-drug conjugate failure in GBM.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12967-025-07216-5.

BackgroundMicroRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are key regulators of gene expression and play a crucial role in cancer progression. Recent studies have highlighted miR-423-5p as a potential modulator in hepatocellular carcinoma (HCC), especially in patients responding to sorafenib treatment. A functional interaction with the oncogenic lncRNA MALAT-1 has been hypothesized, suggesting a regulatory mechanism that may influence tumor aggressiveness.MethodsTo investigate this interaction, we analyzed in silico patient datasets to correlate miR-423-5p and MALAT-1 expression with overall survival (OS) and disease free survival (DFS). Stable overexpression of miR-423-5p and MALAT-1 was achieved in HCC cell lines (HepG2, Hep3B, and SNU387) using a lentiviral transduction system. Functional assays were performed to assess proliferation, migration, invasion, and clonogenic potential. The interaction between miR-423-5p and MALAT-1 was confirmed by RNA immunoprecipitation (RIP), followed by transcriptomic analysis using next-generation sequencing (NGS). Mitochondrial activity was evaluated using the Seahorse Mito Stress Test to measure oxygen consumption rate (OCR) and ATP production. In vivo experiments in orthotopic mouse models were performed to assess tumor growth.ResultsPatient data analysis revealed that high miR-423-5p expression correlated with a less aggressive tumor phenotype and improved survival, while MALAT-1 was associated with poorer prognosis. In vitro, miR-423-5p overexpression reduced MALAT-1 levels and significantly impaired proliferation, migration, and invasion. NGS analysis identified transcriptomic changes linked to tumor progression and metabolic shift. The Seahorse Mito Stress Test demonstrated decreased cellular respiration and ATP production upon miR-423-5p overexpression. In vivo, both tumors derived from miR-423-5p-overexpressing cells and MALAT-1 downregulation by ASO GapmeR evidenced a significantly reduced growth compared to controls.ConclusionThese findings suggest, for the first time, that miR-423-5p acts as a tumor suppressor affecting mitochondrial metabolism through MALAT-1 downregulation in HCC. This regulatory axis represents a potential therapeutic target for precision medicine approaches in liver cancer.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13046-025-03524-2.

1067 Background: ER expression is one of the main determinants of prognosis in patients (pts) with BC. Phenotypic conversion from ER+ (ER>=10%)/HER2- primary BC towards ER<10%/HER2- advanced BC has a well-known negative impact on outcome. However, in the specific context of phenotypically stable ER+/HER2- BC, the prognostic impact of ER expression in metastases or ER dynamics during disease evolution, remains largely understudied. Methods: We enrolled pts with advanced BC undergoing biopsy of a metastatic site. ER+ was defined as ER>=10%. ER expression was evaluated both as continuous and categorical variable (categories: 10-30%, 30-50%, 50-100%). Overall survival (OS) was the primary study endpoint. Cox multivariable models included covariates associated with OS in univariate analysis. Results: Among 1114 pts, 410 had ER+/HER2- phenotype in both primary and metastatic tumor specimens. In this subgroup, ER expression (both continuous and categoric) in metastases had significant prognostic value: for each 10% lower ER expression, the risk of death increased by 6.7% (p=0.011). Pts with ER 10-30% had significantly worse OS than those with ER 50-100% (HR 0.62, p=0.023), and numerically shorter than ER 30-50% (HR 0.51, p=0.063). The table shows the evolution of ER categories from primary BC to metastases. ER expression dynamics also had prognostic impact. Regarding continuous ER expression changes in paired primary vs. metastatic BC, each 10% ER increase corresponded to 5.9% decrease in the risk of death (p=0.008). Pts whose tumors showed an increased ER expression from 10-30% to 50-100% had the most favorable outcome overall, with better OS compared to pts with persistently low ER levels (HR 6.73, p=0.002), or to pts with decreased ER expression in metastases - particularly pts whose ER levels dropped from 50-100% to 10-30% (HR 2.89, p=014). These pts also had superior OS when compared to pts with persistently high ER levels (HR 2.08, p=0.043). The prognostic impact was preserved at the multivariate analysis (including age, grade, visceral/non-visceral disease, biopsy site). Conclusions: Intratumor ER expression and dynamics may in part explain the prognostic heterogeneity of pts with ER+/HER2- stable phenotype from primary to advanced BC. Pts with lower ER levels in metastases are prognostically disadvantaged. Dynamic changes in ER expression provide additional insights beyond those captured by single-point assessment. Interestingly, pts whose tumors shifted from ER 10–30% to ER 50–100% showed the most favorable prognosis, even outperforming those with consistently high ER levels. Metastases, ER 10-30% 30-50% 50-100% Total Primary BC, ER n % n % n % n % 10-30% 5 1.2 0 0 18 4.4 23 5.6 30-50% 7 1.7 4 1.0 11 2.7 22 5.4 50-100% 24 5.9 21 5.1 320 78.0 364 89 Total 36 8.8 25 6.1 349 85.1 410 100

Background: co-targeting of immune checkpoint inhibitors (ICI) CTLA-4 and PD-1 has recently become the new first-line standard of care therapy of pleural mesothelioma (PM) patients, with a significant improvement of overall survival (OS) over conventional chemotherapy. The analysis by tumor histotype demonstrated a greater efficacy of ICI therapy, compared to standard chemotherapy, in non-epithelioid (non-E) vs. epithelioid (E) PM, although some E PM patients also benefit from treatment. This evidence suggests that molecular tumor features, beyond histotype, could be relevant to improve the efficacy of ICI therapy in PM. Among these, tumor DNA methylation emerges as a promising factor to explore, due to its potential role in driving the immune phenotype of cancer cells. Therefore, we utilized a panel of cultured PM cells of different histotype to provide preclinical evidence supporting the role of the tumor methylation landscape and of its pharmacologic modulation, to prospectively improve the efficacy of ICI therapy of PM patients. Methods: the methylome profile (EPIC array) of distinct E (n=5) and non-E (n=9) PM cell lines was analyzed, followed by integrated analysis with their associated transcriptomic profile (Clariom S array), before and after in vitro treatment with the DNA hypomethylating agent (DHA) guadecitabine. The most variable methylated probes were selected to calculate the methylation score (CIMP index) for each cell line at baseline. Genes that were differentially expressed and methylated were then selected for gene ontology analysis. Results: the CIMP index stratified PM cell lines into two distinct classes, CIMP (hypermethylated; n=7) and LOW (hypomethylated; n=7), regardless of their E or non-E histotype. Integrated methylome and transcriptome analyses revealed that CIMP PM cells exhibited a substantial number of hypermethylated, silenced genes, which negatively impacted their immune phenotype compared to LOW PM cells. Treatment with DHA reverted the methylation-driven immune-compromised profile of CIMP PM cells and enhanced the constitutive immune-favorable profile of LOW PM cells. Conclusion: the study highlighted the relevance of DNA methylation in shaping the constitutive immune classification of PM cells, independent of their histological subtypes. The identified role of DHA in shifting the phenotype of PM cells towards an immune-favorable state supports its potential role in clinical trials of precision epigenetic therapy combined with ICI. Citation Format: Maria Fortunata Lofiego, Rossella Tufano, Emma Bello, Laura Solmonese, Francesco Marzani, Francesca Piazzini, Fabrizio Celesti, Francesca Pia Caruso, Teresa Maria Rosaria Noviello, Roberta Mortarini, Andrea Anichini, Michele Ceccarelli, Luana Calabrò, Michele Maio, Sandra Coral, Anna Maria Di Giacomo, Alessia Covre. DNA methylation status classifies pleural mesothelioma cells according to their immune profile: implication for precision epigenetic therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4841.

Cognitive impairment is a prevalent complication in chronic kidney disease (CKD), ranging from mild deficits in early stages to more severe conditions, such as mild cognitive impairment and dementia in advanced stages. CKD patients exhibit reduced performance in memory, attention, language, visuospatial abilities and executive functions. Contributing factors include uraemic toxins, structural brain changes, blood-brain barrier dysfunction, anaemia and comorbidities like diabetes mellitus. Malnutrition, affecting nearly half of CKD patients, exacerbates cognitive decline through inflammation, oxidative stress and protein-energy wasting. Nutritional deficiencies, particularly in protein, vitamin D, B vitamins, omega-3 fatty acids and antioxidants, are linked to impaired cognition. Emerging evidence highlights the role of the gut-brain axis, with gut-derived uraemic toxins and microbiome alterations contributing to cognitive dysfunction. Processed foods and microplastics further compound risks by promoting inflammation and neurotoxicity. Dialysis and kidney transplantation offer opportunities for cognitive recovery, though challenges remain, particularly in haemodialysis patients. Nutritional interventions, including tailored protein intake, micronutrient supplementation and dietary counselling, are critical for mitigating cognitive decline. Addressing CKD comorbidities, such as anaemia and diabetes through targeted nutritional and pharmacological strategies, improves outcomes. Integrating psychological and social support enhances quality of life, given the high prevalence of anxiety and depression in CKD patients. Future research should focus on personalized nutrition, gut microbiota modulation and routine cognitive assessments to optimise care. A holistic approach combining medical, nutritional and psychosocial strategies is essential for improving cognitive and overall health in CKD patients.