DHEA-S, Androstenedione, 17-ß-estradiol signature as novel biomarkers for early prediction of risk of malignant pleural mesothelioma linked to asbestos-exposure: A preliminary investigation.

17-ß-estradiol, involved in mesothelioma pathogenesis, and its precursors were explored as potential biomarkers for the early diagnosis of mesothelioma. Using enzyme-linked immunosorbent assay(ELISA) for 17-ß-estradiol and ultra-high performance liquid chromatography/tandem mass spectrometry(UHPLC-MS/MS) for 19 17-ß-estradiol precursors, a comprehensive analysis of 20steroid hormones was conducted in the serum of mesothelioma patients(n=67), asbestos-exposed healthy subjects(n=39), and non-asbestos-exposed healthy subjects(n=35). Bioinformatics analysis explored three potential serum biomarkers: 17-ß-estradiol, DHEA-S, and androstenedione. The results revealed significant differences in 17-ß-estradiol levels between mesothelioma patients and both non-asbestos-exposed and asbestos-exposed healthy subjects. No significant variations in serum 17-ß-estradiol levels were observed among mesothelioma patients at different stages, suggesting its potential as an early diagnostic marker. 17-ß-estradiol levels were similar in mesothelioma patients with environmental and occupational asbestos exposure, while males with occupational asbestos exposure exhibited significantly higher levels of 17-ß-estradiol compared to females. Significant reduction in androstenedione and an increase in DHEA-S were observed in asbestos-exposed individuals compared to non-asbestos-exposed individuals. The analysis of DHEA-S-androstenedione-17-ß-estradiol signature score showed an increase in asbestos-exposed individuals and mesothelioma patients compared to non-asbestos-exposed individuals, and this score effectively distinguished between the groups. The Cancer Genome Atlas data was utilized to analyze the expression of 5-α-reductase1 and hydroxysteroid-17ß-dehydrogenase2 genes. The findings indicated that mesothelioma patients with elevated gene values for 5-α-reductase1 and hydroxysteroid-17ß-dehydrogenase2 have a worse or better prognosis on overall survival, respectively. In conclusion, this study suggests 17-ß-estradiol, DHEA-S, and androstenedione as biomarkers for mesothelioma risk and early diagnosis of mesothelioma in asbestos-exposed individuals, aiding timely intervention and improved care.

Fetal chronic hypoxia does not affect urinary presepsin levels in newborns at birth.

OBJECTIVES: Early sepsis detection and diagnosis still constitutes an open issue since the accuracy of standard-of care parameters is biased by a series of perinatal factors including hypoxia. Therefore, we aimed at investigating the effect of fetal chronic hypoxia insult on urine levels of a promising new marker of sepsis, namely presepsin (P-SEP). METHODS: We conducted a prospective case-control study in 22 cases of early-intrauterine growth restriction (E-IUGR) compared with 22 small-for-gestational-age (SGA) newborns and 66 healthy controls. P-SEP urine samples were collected over the first 72 h from birth. Blood culture and C-reactive protein (CRP) blood levels were measured in E-IUGR and SGA infants. Perinatal standard monitoring parameters and main outcomes were also recorded. RESULTS: No significant urinary P-SEP differences (p>0.05, for all) were observed among studied groups. Moreover, no significant correlations (p>0.05, for both) between urinary P-SEP and blood CRP levels in both E-IUGR and SGA groups (R=0.08; R=0.07, respectively) were observed. CONCLUSIONS: The present results showing the lack of influence of fetal chronic hypoxia on urinary P-SEP levels offer additional data to hypothesize the possible use of urinary P-SEP measurement in neonates in daily clinical practice. Further multicenter prospective data are needed, including infants with early-onset sepsis.

Elevated S100B urine levels predict seizures in infants complicated by perinatal asphyxia and undergoing therapeutic hypothermia.

OBJECTIVES: Seizures (SZ) are one of the main complications occurring in infants undergoing therapeutic hypothermia (TH) due to perinatal asphyxia (PA) and hypoxic ischemic encephalopathy (HIE). Phenobarbital (PB) is the first-line therapeutic strategy, although data on its potential side-effects need elucidation. We investigated whether: i) PB administration in PA-HIE TH-treated infants affects S100B urine levels, and ii) S100B could be a reliable early predictor of SZ. METHODS: We performed a prospective case-control study in 88 PA-HIE TH infants, complicated (n=44) or not (n=44) by SZ requiring PB treatment. S100B urine levels were measured at 11 predetermined monitoring time-points from first void up to 96-h from birth. Standard-of-care monitoring parameters were also recorded. RESULTS: S100B significantly increased in the first 24-h independently from HIE severity in the cases who later developed SZ and requested PB treatment. ROC curve analysis showed that S100B, as SZ predictor, at a cut-off of 2.78 µg/L achieved a sensitivity/specificity of 63 and 84 %, positive/negative predictive values of 83 and 64 %. CONCLUSIONS: The present results offer additional support to the usefulness of S100B as a trustable diagnostic tool in the clinical daily monitoring of therapeutic and pharmacological procedures in infants complicated by PA-HIE.

Epigenetic investigation into circulating microRNA 197-3p in sera from patients affected by malignant pleural mesothelioma and workers ex-exposed to asbestos.

The epigenetic role of microRNAs is established at both physiological and pathological levels. Dysregulated miRNAs and their targets appear to be a promising approach for innovative anticancer therapies. In our previous study, circulating miR-197-3p tested dysregulated in workers ex-exposed to asbestos (WEA). Herein, an epigenetic investigation on this circulating miRNA was carried out in sera from malignant pleural mesothelioma (MPM) patients. MiR-197-3p was quantified in MPM (n = 75) sera and comparatively analyzed to WEA (n = 75) and healthy subject (n = 75) sera, using ddPCR and RT-qPCR techniques. Clinicopathological characteristics, occupational, non-occupational information and overall survival (OS) were evaluated in correlation studies. MiR-197-3p levels, analyzed by ddPCR, were significantly higher in MPM than in WEA cohort, with a mean copies/µl of 981.7 and 525.01, respectively. Consistently, RT-qPCR showed higher miR-197-3p levels in sera from MPM with a mean copies/µl of 603.7, compared to WEA with 336.1 copies/µl. OS data were significantly associated with histologic subtype and pleurectomy. Circulating miR-197-3p is proposed as a new potential biomarker for an early diagnosis of the MPM onset. Indeed, miR-197-3p epigenetic investigations along with chest X-ray, computed tomography scan and spirometry could provide relevant information useful to reach an early and effective diagnosis for MPM.

Diagnostics of BAP1-Tumor Predisposition Syndrome by a Multitesting Approach: A Ten-Year-Long Experience.

Germline mutations in the tumor suppressor gene BRCA1-associated protein-1 (BAP1) lead to BAP1 tumor predisposition syndrome (BAP1-TPDS), characterized by high susceptibility to several tumor types, chiefly melanoma, mesothelioma, renal cell carcinoma, and basal cell carcinoma. Here, we present the results of our ten-year experience in the molecular diagnosis of BAP1-TPDS, along with a clinical update and cascade genetic testing of previously reported BAP1-TPDS patients and their relatives. Specifically, we sequenced germline DNA samples from 101 individuals with suspected BAP1-TPDS and validated pathogenic variants (PVs) by assessing BAP1 somatic loss in matching tumor specimens. Overall, we identified seven patients (7/101, 6.9%) carrying six different germline BAP1 PVs, including one novel variant. Consistently, cascade testing revealed a total of seven BAP1 PV carriers. In addition, we explored the mutational burden of BAP1-TPDS tumors by targeted next-generation sequencing. Lastly, we found that certain tumors present in PV carriers retain a wild-type BAP1 allele, suggesting a sporadic origin of these tumors or a functional role of heterozygous BAP1 in neoplastic development. Altogether, our findings have important clinical implications for therapeutic response of BAP1-TPDS patients.

Sera from Patients with Malignant Pleural Mesothelioma Tested Positive for IgG Antibodies against SV40 Large T Antigen: The Viral Oncoprotein.

Malignant pleural mesothelioma (MPM), a fatal tumor, is mainly linked to the asbestos exposure. It has been reported that together with the inhalation of asbestos fibers, other factors are involved in the MPM onset, including simian virus 40 (SV40). SV40, a polyomavirus with oncogenic potential, induces (i) in vitro the mesenchymal cell transformation, whereas (ii) in vivo the MPM onset in experimental animals. The association between MPM and SV40 in humans remains to be elucidated. Sera (n = 415) from MPM-affected patients (MPM cohort 1; n = 152) and healthy subjects (HSs, n = 263) were investigated for their immunoglobulin G (IgG) against simian virus 40 large tumor antigen (Tag), which is the transforming protein. Sera were investigated with an indirect enzyme-linked immunosorbent assay (ELISA) using two synthetic peptides from SV40 Tag protein. SV40 Tag protein was evaluated by immunohistochemical (IHC) staining on MPM samples (MPM cohort 2; n = 20). Formalin-fixed and paraffin-embedded (FFPE) samples were obtained from MPM patients unrelated to MPM serum donors. The proportion of sera, from MPM patients, showing antibodies against SV40 Tag (34%) was significantly higher compared to HSs (20%) (odds ratio 2.049, CI 95% 1.32-3.224; p=0.0026). Immunohistochemical staining (IHS) assays showed SV40 Tag expression in 8/20, 40% of MPM specimens. These results indicate that SV40 is linked to a large fraction of MPM. It is worth noting that the prevalence of SV40 Tag antibodies detected in sera from cohort 1 of MPM patients is similar to the prevalence of SV40 Tag found to be expressed in FFPE tissues from MPM cohort 2.

Tumor treating fields affect mesothelioma cell proliferation by exerting histotype-dependent cell cycle checkpoint activations and transcriptional modulations.

Although clinical antitumor activity of Tumor Treating Fields (TTFields) has been reported in malignant pleural mesothelioma (MPM) patients, the mechanisms behind the different selectivity displayed by the various MPM histotypes to this physical therapy has not been elucidated yet. Taking advantage of the development of well characterized human MPM cell lines derived from pleural effusion and/or lavages of patients' thoracic cavity, we investigated the biological effects of TTFields against these cells, representative of epithelioid, biphasic, and sarcomatoid histotypes. Growth inhibition and cell cycle perturbations caused by TTFields were investigated side by side with RNA-Seq analyses at different exposure times to identify pathways involved in cell response to treatment. We observed significant differences of response to TTFields among the cell lines. Cell cycle analysis revealed that the most sensitive cells (epithelioid CD473) were blocked in G2M phase followed by formation of polyploid cells. The least sensitive cells (sarcomatoid CD60) were only slightly affected by TTFields with a general delay in all cell cycle phases. Apoptosis was present in all samples, but while epithelioid cell death was already observed during the first 24 h of treatment, sarcomatoid cells needed longer times before they engaged apoptotic pathways. RNA-Seq experiments demonstrated that TTFields induced a transcriptional response already detectable at early time points (8 h). The number of differentially expressed genes was higher in CD473 than in CD60 cells, involving several pathways, such as those pertinent to cell cycle checkpoints, DNA repair, and histone modifications. Our data provide further support to the notion that the antitumor effects of TTFields are not simply related to a non-specific reaction to a physical stimulus, but are dependent on the biological background of the cells and the particular sensitivity to TTFields observed in epithelioid MPM cells is associated with a higher transcriptional activity than that observed in sarcomatoid models.

Epithelioid Pleural Mesothelioma Is Characterized by Tertiary Lymphoid Structures in Long Survivors: Results from the MATCH Study.

Pleural mesothelioma (PM) is an aggressive tumor with few therapeutic options. Although patients with epithelioid PM (ePM) survive longer than non-epithelioid PM (non-ePM), heterogeneity of tumor response in ePM is observed. The role of the tumor immune microenvironment (TIME) in the development and progression of PM is currently considered a promising biomarker. A few studies have used high-throughput technologies correlated with TIME evaluation and morphologic and clinical data. This study aimed to identify different morphological, immunohistochemical, and transcriptional profiles that could potentially predict the outcome. A retrospective multicenter cohort of 129 chemonaive PM patients was recruited. Tissue slides were reviewed by dedicated pathologists for histotype classification and immunophenotype of tumor-infiltrating lymphocytes (TILs) and lymphoid aggregates or tertiary lymphoid structures (TLS). ePM (n = 99) survivors were further classified into long (>36 months) or short (<12 months) survivors. RNAseq was performed on a subset of 69 samples. Distinct transcriptional profiling in long and short ePM survivors was found. An inflammatory background with a higher number of B lymphocytes and a prevalence of TLS formations were detected in long compared to short ePM survivors. These results suggest that B cell infiltration could be important in modulating disease aggressiveness, opening a pathway for novel immunotherapeutic approaches.

Micro-RNA-215 and -375 regulate thymidylate synthase protein expression in pleural mesothelioma and mediate epithelial to mesenchymal transition.

The standard front-line treatment for pleural mesothelioma (PM) is pemetrexed-based chemotherapy, whose major target is thymidylate synthase (TS). In several cancer models, miR-215 and miR-375 have been shown to target TS, while information on these miRNAs in PM are still limited although suggest their role in epithelial to mesenchymal transition. Seventy-one consecutive PM tissues (4 biphasic, 7 sarcomatoid, and 60 epithelioid types) and 16 commercial and patient-derived PM cell lines were screened for TS, miR-215, and miR-375 expression. REN and 570B cells were selected for miR-215 and miR-375 transient transfections to test TS modulation. ZEB1 protein expression in tumor samples was also tested. Moreover, genetic profile was investigated by means of BAP1 and p53 immunohistochemistry. Expression of both miR-215 and miR-375 was significantly higher in epithelioid histotype. Furthermore, inverse correlation between TS protein and both miR-215 and miR-375 expression was found. Efficiently transfected REN and 570B cell lines overexpressing miR-215 and miR-375 showed decreased TS protein levels. Epithelioid PM with a mesenchymal component highlighted by reticulin stain showed significantly higher TS and ZEB1 protein and lower miRNA expression. A better survival was recorded for BAP1 lost/TS low cases. Our data indicate that miR-215 and miR-375 are involved in TS regulation as well as in epithelial-to-mesenchymal transition in PM.

SKP2 drives the sensitivity to neddylation inhibitors and cisplatin in malignant pleural mesothelioma.

BACKGROUND: The combination of pemetrexed and cisplatin remains the reference first-line systemic therapy for malignant pleural mesothelioma (MPM). Its activity is moderate because of tumor aggressiveness, immune-suppressive environment and resistance to chemotherapy-induced immunogenic cell death (ICD). Preliminary and limited findings suggest that MPM cells have deregulated ubiquitination and proteasome activities, although proteasome inhibitors achieved disappointing clinical results. METHODS: Here, we investigated the role of the E3-ubiquitin ligase SKP/Cullin/F-box (SCF) complex in cell cycle progression, endoplasmic reticulum (ER)/proteostatic stress and ICD in MPM, and the therapeutic potential of the neddylation/SCF complex inhibitor MLN4924/Pevonedistat. RESULTS: In patient-derived MPM cultures and syngenic murine models, MLN4924 and cisplatin showed anti-tumor effects, regardless of MPM histotype and BAP1 mutational status, increasing DNA damage, inducing S- and G2/M-cell cycle arrest, and apoptosis. Mechanistically, by interfering with the neddylation of cullin-1 and ubiquitin-conjugating enzyme UBE2M, MLN4924 blocks the SCF complex activity and triggers an ER stress-dependent ICD, which activated anti-MPM CD8+T-lymphocytes. The SKP2 component of SCF complex was identified as the main driver of sensitivity to MLN4924 and resistance to cisplatin. These findings were confirmed in a retrospective MPM patient series, where SKP2 high levels were associated with a worse response to platinum-based therapy and inferior survival. CONCLUSIONS: We suggest that the combination of neddylation inhibitors and cisplatin could be worth of further investigation in the clinical setting for MPM unresponsive to cisplatin. We also propose SKP2 as a new stratification marker to determine the sensitivity to cisplatin and drugs interfering with ubiquitination/proteasome systems in MPM.

Malignant pleural mesothelioma: Germline variants in DNA repair genes may steer tailored treatment.

INTRODUCTION: Malignant pleural mesothelioma (MPM) is a tumour associated with asbestos exposure. Approximately, 10% of patients with MPM carry a germline pathogenic variant (PV), mostly in DNA repair genes, suggesting the occurrence of inherited predispositions. AIM: This article aimed to 1) search for new predisposing genes and assess the prevalence of PVs in DNA repair genes, by next-generation sequencing (NGS) analysis of germline DNA from 113 unselected patients with MPM and 2) evaluate whether these patients could be sensitive to tailored treatments. METHODS: NGS was performed using a custom panel of 107 cancer-predisposing genes. To investigate the response to selected drugs in conditions of DNA repair insufficiency, we created a three-dimensional-MPM cell model that had a defect in ataxia telangiectasia mutated (ATM), the master regulator of DNA repair. RESULTS: We identified PVs in approximately 7% of patients with MPM (8/113) and a new PV in BAP1 in a further patient with familial MPM. Most of these PVs were in genes involved or supposedly involved in DNA repair (BRCA1, BRIP1, CHEK2, SLX4, FLCN and BAP1). In vitro studies showed apoptosis induction in ATM-silenced/inhibited MPM spheroids treated with an enhancer of zeste homologue 2 inhibitor (tazemetostat). CONCLUSIONS: Overall these data suggest that patients with MPM and DNA repair insufficiency may benefit from this treatment, which induces synthetic lethality.

S100B in cardiac surgery brain monitoring: friend or foe?

Recent advances in perioperative management of adult and pediatric patients requiring open heart surgery (OHS) and cardiopulmonary bypass (CPB) for cardiac and/or congenital heart diseases repair allowed a significant reduction in the mortality rate. Conversely morbidity rate pattern has a flat trend. Perioperative period is crucial since OHS and CPB are widely accepted as a deliberate hypoxic-ischemic reperfusion damage representing the cost to pay at a time when standard of care monitoring procedures can be silent or unavailable. In this respect, the measurement of neuro-biomarkers (NB), able to detect at early stage perioperative brain damage could be especially useful. In the last decade, among a series of NB, S100B protein has been investigated. After the first promising results, supporting the usefulness of the protein as predictor of short/long term adverse neurological outcome, the protein has been progressively abandoned due to a series of limitations. In the present review we offer an up-dated overview of the main S100B pros and cons in the peri-operative monitoring of adult and pediatric patients.

Circulating microRNA-197-3p as a potential biomarker for asbestos exposure.

Asbestos is considered the main cause of diseases in workers exposed to this mineral in the workplace as well as an environmental pollutant. The association between asbestos and the onset of different diseases has been reported, but asbestos exposure specific biomarkers are not known. MicroRNAs (miRNAs) are small, single-strand, non-coding RNAs, with potential value as diagnostic, prognostic, and predictive markers in liquid biopsies. Sera collected from workers ex-exposed to asbestos (WEA) fibers were compared with sera from healthy subjects (HS) of similar age, as liquid biopsies. The expression of the circulating miRNA 197-3p was investigated employing two different highly analytical PCR methods, i.e. RT-qPCR and ddPCR. MiR-197-3p levels were tested in sera from WEA compared to HS. MiR-197-3p tested dysregulated in sera from WEA (n = 75) compared to HS (n = 62). Indeed, miR-197-3p was found to be 2.6 times down-regulated in WEA vs. HS (p = 0.0001***). In addition, an inverse correlation was detected between miR-197-3p expression level and cumulative asbestos exposure, being this miRNA down-regulated 2.1 times in WEA, with high cumulative asbestos exposure, compared to WEA with low exposure (p = 0.0303*). Circulating miR-197-3p, found to be down regulated in sera from WEA, is proposed as a new potential biomarker of asbestos exposure.

Identification of novel COX-2 / CYP19A1 axis involved in the mesothelioma pathogenesis opens new therapeutic opportunities.

BACKGROUND: Based on previous studies highlighting that the induction of cyclooxygenase-2 (COX-2) and high prostaglandin E2 (PGE2) levels contribute to the pathogenesis of malignant pleural mesothelioma (MPM), and that aromatase (CYP19A1), an enzyme that plays a key role in estrogen biosynthesis, along with estradiol (E2) were expressed in MPM, this study aimed to investigate the possible interplay between COX-2 and CYP19A1 in the pathogenesis of mesothelioma, as well as the underlying mechanism. METHODS: The interaction between COX-2 and CYP19A1 was first investigated on different MPM lines upon PGE2, and COX-2 inhibitor (rofecoxib) treatment by western blot, RT-PCR. The key regulatory pathways involved in the COX-2 and CYP19A1 axis were further studied in MPM cells, after rofecoxib and exemestane (CYP19A1 inhibitor) treatment in monotherapy and in combination, by cell cycle distribution, western blot, and combination index analysis. To explore the role of COX-2/CYP19A1 axis in 3D preclinical models of MPM cells, we analyzed the effect of combination of COX-2 and CYP19A1 inhibitors in mesosphere formation. Immunohistochemical analysis of MPM mesosphere and specimens was utilized to evaluate the involvement of COX-2 on the CYP19A1 activity and the relationship between E2 and COX-2. RESULTS: PGE2 or rofecoxib treatment caused in MPM cells an increased or decreased, respectively, CYP19A1 expression at mRNA and protein levels. The effect of rofecoxib and exemestane combination in MPM cell proliferation was synergistic. Activation of caspase-3 and cleavage of PARP confirmed an apoptotic death for MPM cell lines. Increased expression levels of p53, p21, and p27, downregulation of cyclin D1 and inhibition of Akt activation (pAKT) were also found. The antagonistic effect of rofecoxib and exemestane combination found only in one cell line, was reverted by pretreatment with MK2206, a pAKT inhibitor, indicating pAKT as an actionable mediator in the COX-2-CYP19A1 axis. Reduction of size and sphere-forming efficiency in MPM spheres after treatment with both inhibitor and a decrease in COX-2 and E2 staining was found. Moreover, immunohistochemical analysis of 46 MPM samples showed a significant positive correlation between COX-2 and E2. CONCLUSIONS: Collectively, the results highlighted a novel COX-2/CYP19A1 axis in the pathogenesis of MPM that can be pharmacologically targeted, consequently opening up new therapeutic options.

New DNA Methylation Signals for Malignant Pleural Mesothelioma Risk Assessment.

Malignant pleural mesothelioma (MPM) is a rare and aggressive neoplasm. Patients are usually diagnosed when current treatments have limited benefits, highlighting the need for noninvasive tests aimed at an MPM risk assessment tool that might improve life expectancy. Three hundred asbestos-exposed subjects (163 MPM cases and 137 cancer-free controls), from the same geographical region in Italy, were recruited. The evaluation of asbestos exposure was conducted considering the frequency, the duration and the intensity of occupational, environmental and domestic exposure. A genome-wide methylation array was performed to identify novel blood DNA methylation (DNAm) markers of MPM. Multiple regression analyses adjusting for potential confounding factors and interaction between asbestos exposure and DNAm on the MPM odds ratio were applied. Epigenome-wide analysis (EWAS) revealed 12 single-CpGs associated with the disease. Two of these showed high statistical power (99%) and effect size (>0.05) after false discovery rate (FDR) multiple comparison corrections: (i) cg03546163 in FKBP5, significantly hypomethylated in cases (Mean Difference in beta values (MD) = -0.09, 95% CI = -0.12|-0.06, p = 1.2 × 10-7), and (ii) cg06633438 in MLLT1, statistically hypermethylated in cases (MD = 0.07, 95% CI = 0.04|0.10, p = 1.0 × 10-6). Based on the interaction analysis, asbestos exposure and epigenetic profile together may improve MPM risk assessment. Above-median asbestos exposure and hypomethylation of cg03546163 in FKBP5 (OR = 20.84, 95% CI = 8.71|53.96, p = 5.5 × 10-11) and hypermethylation of cg06633438 in MLLT1 (OR = 11.71, 95% CI = 4.97|29.64, p = 5.9 × 10-8) genes compared to below-median asbestos exposure and hyper/hypomethylation of single-CpG DNAm, respectively. Receiver Operation Characteristics (ROC) for Case-Control Discrimination showed a significant increase in MPM discrimination when DNAm information was added in the model (baseline model, BM: asbestos exposure, age, gender and white blood cells); area under the curve, AUC = 0.75; BM + cg03546163 at FKBP5. AUC = 0.89, 2.1 × 10-7; BM + cg06633438 at MLLT1. AUC = 0.89, 6.3 × 10-8. Validation and replication procedures, considering independent sample size and a different DNAm analysis technique, confirmed the observed associations. Our results suggest the potential application of DNAm profiles in blood to develop noninvasive tests for MPM risk assessment in asbestos-exposed subjects.

Evaluation of the Preclinical Efficacy of Lurbinectedin in Malignant Pleural Mesothelioma.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a highly aggressive cancer generally diagnosed at an advanced stage and characterized by a poor prognosis. The absence of alterations in druggable kinases, together with an immune-suppressive tumor microenvironment, limits the use of molecular targeted therapies, making the treatment of MPM particularly challenging. Here we investigated the in vitro susceptibility of MPM to lurbinectedin (PM01183), a marine-derived drug that recently received accelerated approval by the FDA for the treatment of patients with metastatic small cell lung cancer with disease progression on or after platinum-based chemotherapy. METHODS: A panel of primary MPM cultures, resembling the three major MPM histological subtypes (epithelioid, sarcomatoid, and biphasic), was characterized in terms of BAP1 status and histological markers. Subsequently, we explored the effects of lurbinectedin at nanomolar concentration on cell cycle, cell viability, DNA damage, genotoxic stress response, and proliferation. RESULTS: Stabilized MPM cultures exhibited high sensitivity to lurbinectedin independently from the BAP1 mutational status and histological classification. Specifically, we observed that lurbinectedin rapidly promoted a cell cycle arrest in the S-phase and the activation of the DNA damage response, two conditions that invariably resulted in an irreversible DNA fragmentation, together with strong apoptotic cell death. Moreover, the analysis of long-term treatment indicated that lurbinectedin severely impacts MPM transforming abilities in vitro. CONCLUSION: Overall, our data provide evidence that lurbinectedin exerts a potent antitumoral activity on primary MPM cells, independently from both the histological subtype and BAP1 alteration, suggesting its potential activity in the treatment of MPM patients.

Identification of Redox-Sensitive Transcription Factors as Markers of Malignant Pleural Mesothelioma.

Although asbestos has been banned in most countries around the world, malignant pleural mesothelioma (MPM) is a current problem. MPM is an aggressive tumor with a poor prognosis, so it is crucial to identify new markers in the preventive field. Asbestos exposure induces oxidative stress and its carcinogenesis has been linked to a strong oxidative damage, event counteracted by antioxidant systems at the pulmonary level. The present study has been focused on some redox-sensitive transcription factors that regulate cellular antioxidant defense and are overexpressed in many tumors, such as Nrf2 (Nuclear factor erythroid 2-related factor 2), Ref-1 (Redox effector factor 1), and FOXM1 (Forkhead box protein M1). The research was performed in human mesothelial and MPM cells. Our results have clearly demonstrated an overexpression of Nrf2, Ref-1, and FOXM1 in mesothelioma towards mesothelium, and a consequent activation of downstream genes controlled by these factors, which in turn regulates antioxidant defense. This event is mediated by oxidative free radicals produced when mesothelial cells are exposed to asbestos fibers. We observed an increased expression of Nrf2, Ref-1, and FOXM1 towards untreated cells, confirming asbestos as the mediator of oxidative stress evoked at the mesothelium level. These factors can therefore be considered predictive biomarkers of MPM and potential pharmacological targets in the treatment of this aggressive cancer.