BACKGROUND:Characterizing the immune landscape of thymic epithelial tumors (TETs) is essential for patient stratification for emerging immunotherapeutic approaches and for optimizing follow-up strategies. In this study, we examined the expression patterns and clinical significance of five immune-related markers in a large cohort of TETs. MATERIAL AND METHODS:Expression of TIM3, OX40L, GTF2I, XPO1, and GITR was assessed by immunohistochemistry (IHC) in the epithelial and lymphocytic compartments of 137 surgically resected TETs and correlated with clinicopathological parameters and patient outcomes. Tumors were grouped according to their immune profile using cluster analysis. RESULTS:TIM3, GTF2I, and XPO1 showed high and consistent expression in the epithelial compartment of TETs across histological subgroups, whereas GITR expression was mostly absent. In the lymphocytic compartment, only XPO1 was highly expressed, with no significant differences between TET subtypes. Epithelial and lymphocytic OX40L expressions were significantly higher in patients with myasthenia gravis (vs. those without this autoimmune disorder; p = 0.004 and p = 0.045, respectively). Importantly, hierarchical clustering identified four distinct TET subgroups based on their combined immune marker expression profiles, which displayed widely divergent survival outcomes (p < 0.0001). Among these, the subgroup characterized by elevated epithelial expression of GTF2I, XPO1, and TIM3 showed worse survival in our multivariate model (HR 6.37; p = 0.025). CONCLUSIONS:TIM3, GTF2I, and XPO1 are highly expressed in the epithelial compartment of TETs, providing a rationale for therapeutic targeting in future immunotherapy trials. Differential IHC expression of TIM3, OX40L, GTF2I, XPO1, and GITR defines distinct TET subtypes with independent prognostic relevance, enabling more accurate risk assessment in these highly heterogeneous thoracic malignancies.

Background/Objective: The identification of novel non-invasive diagnostic and prognostic biomarkers is urgently needed in pleural mesothelioma (PM). While soluble mesothelin-related peptides (SMRP) are the most established circulating biomarker, their prognostic value is limited. A wide range of microRNAs (miRs) play diverse roles in regulating gene expression in PM. MiR-21 has been shown to be upregulated in mesothelioma tissue; nevertheless, the diagnostic and prognostic utility of miR-21 in the circulation and its association with survival in PM have not been extensively investigated to date. The objective of the current study was to evaluate miR-21 as a potential blood-based diagnostic and prognostic biomarker in PM. Methods: Plasma samples from PM patients (n = 94) were collected at the time of diagnosis, prior to treatment. Sex- and age-matched healthy individuals (n = 30) served as controls. MiR-21 levels were measured using quantitative RT-PCR and normalized to miR-16, and potential correlations with clinicopathological data were analyzed. Serum SMRP levels were measured in matched patients (n = 84), and a direct comparative analysis of miR-21 and SMRP was conducted. In situ hybridization (ISH) was used to confirm the presence of miR-21 in tumor cells. Results: Plasma miR-21 levels were significantly elevated in PM patients compared to healthy controls (p < 0.001), demonstrating good diagnostic performance (AUC 0.81). The localization of miR-21 in PM cells was confirmed by ISH. High miR-21 levels were associated with significantly shorter median overall survival (12.4 vs. 24.3 months, p < 0.001). Elevated SMRP levels were also associated with reduced survival (12.4 vs. 19.5 months, p = 0.032); however, SMRP did not retain independent prognostic significance in multivariable analysis. In contrast, high-circulating miR-21 was confirmed as an independent predictor for poor survival (HR 3.12, p < 0.001). Conclusions: Our findings highlight that circulating miR-21 is a potential non-invasive biomarker with both diagnostic and independent prognostic value in pleural mesothelioma and outperforms SMRP in multivariable survival analysis. Further research is warranted to validate its role in the biology of this disease and to assess its correlation with outcome and treatment responses.

Patients with lung metastases frequently receive anti-angiogenic (AA) therapies based on the histological characteristics of the primary tumor. However, clinical benefit remains limited, suggesting the presence of therapeutic resistance. The contribution of intratumoral drug distribution to this limited therapeutic benefit remains insufficiently understood.We aimed to determine whether differences in therapeutic response are associated with differences in intratumoral drug distribution in matched primary and lung metastasis models of breast and renal cell carcinoma.Matched primary and lung metastasis models were established using 4T1, MDA-MB-231, and RENCA cells. Mice received sunitinib or vehicle, and therapeutic response was evaluated by complementary histopathological analyses of tumor growth, microvessel area (MVA), hypoxia, and necrosis, together with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) assessment of intratumoral sunitinib distribution.Sunitinib significantly inhibited tumor growth and reduced MVA in all primary tumor models. In metastasis models, MVA was also significantly reduced, whereas therapeutic response varied between tumor types. MALDI-MSI demonstrated higher normalized intratumoral sunitinib signal in metastases than in corresponding primary tumors, particularly in the breast cancer models. Drug signal also differed between viable, necrotic, and hypoxic tumor regions, demonstrating substantial intratumoral heterogeneity.These findings demonstrate that integrating MALDI-MSI with histopathological analyses enables comparative assessment of intratumoral drug distribution in matched primary and metastatic tumor models. Despite consistent anti-vascular effects and detectable intratumoral drug signal, additional mechanisms beyond inadequate drug delivery are likely to contribute to the limited response observed in breast cancer lung metastases.

Objectives: Lung squamous cell carcinoma (LUSC) is associated with a poor prognosis and a lack of specific treatment options. The dysregulation of activin A (ActA) has been reported in various malignancies. Herein, we investigated the diagnostic and prognostic significance of ActA in LUSC. Materials and methods: ActA concentrations were measured using ELISA in plasma samples of 128 LUSC patients (stage I-IV) and 73 controls, and correlated those values with clinicopathological parameters and survival. Results: ActA plasma levels were significantly higher in therapy-naive LUSC patients compared to controls (444.1 ± 310.9 pg/mL vs 338.9 ± 145.5 pg/mL, p = 0.010). ActA levels significantly correlated with advanced stage as well as with T and N factors. High circulating ActA levels were significantly increased in metastatic disease patients compared to M0 disease. Further, patients with ActA levels above a computationally established optimal cut-off value of 443.0 pg/mL had a significantly worse median overall (OS, 17.63 vs 64.77 months, HR 0.391, 95 % CI 0.200–0.762, p < 0.001) and median disease-/progression-free survival (DFS/PFS; 11.57 vs 30.20 months, HR 0.502, 95 % CI 0.248–1.019, p = 0.020). Multivariate analysis revealed that high ActA levels were an independent prognostic factor for shorter OS (p = 0.001) and DFS/PFS (p = 0.018). A newly developed score combining CRP and ActA levels was also an independent prognostic factor for OS and DFS/PFS. Conclusion: Measurement of circulating ActA levels may help identify advanced-stage LUSC patients, and this value could serve as a prognostic parameter in LUSC. Thus, ActA may be a novel blood-based biomarker for identifying LUSC patients with distant metastasis.

Background By being highly involved in the tumor evolution and disease progression of small cell lung cancer (SCLC), Myc family members (C-Myc, L-Myc, and N-Myc) might represent promising targetable molecules. Our aim was to investigate the expression pattern and prognostic relevance of these oncogenic proteins in an international cohort of surgically resected SCLC tumors. Methods Clinicopathological data and surgically resected tissue specimens from 104 SCLC patients were collected from two collaborating European institutes. Tissue sections were stained by immunohistochemistry (IHC) for all three Myc family members and the recently introduced SCLC molecular subtype-markers (ASCL1, NEUROD1, POU2F3, and YAP1). Results IHC analysis showed C-Myc, L-Myc, and N-Myc positivity in 48%, 63%, and 9% of the specimens, respectively. N-Myc positivity significantly correlated with the POU2F3-defined molecular subtype ( r = 0.6913, p = 0.0056). SCLC patients with C-Myc positive tumors exhibited significantly worse overall survival (OS) (20 vs. 44 months compared to those with C-Myc negative tumors, p = 0.0176). Ultimately, in a multivariate risk model adjusted for clinicopathological and treatment confounders, positive C-Myc expression was confirmed as an independent prognosticator of impaired OS (HR 1.811, CI 95% 1.054–3.113, p = 0.032). Conclusions Our study provides insights into the clinical aspects of Myc family members in surgically resected SCLC tumors. Notably, besides showing that positivity of Myc family members varies across the patients, we also reveal that C-Myc protein expression independently correlates with worse survival outcomes. Further studies are warranted to investigate the role of Myc family members as potential prognostic and predictive markers in this hard-to-treat disease.

BACKGROUND:Pleural mesothelioma (PM) is a relatively rare malignancy with limited treatment options and dismal prognosis. We have previously found elevated FGF18 expression in PM tissue specimens compared with normal mesothelium. The objective of the current study was to further explore the role of FGF18 in PM and evaluate its suitability as a circulating biomarker. METHODS:FGF18 mRNA expression was analyzed by real-time PCR in cell lines and in silico in datasets from the Cancer Genome Atlas (TCGA). Cell lines overexpressing FGF18 were generated by retroviral transduction and cell behavior was investigated by clonogenic growth and transwell assays. Plasma was collected from 40 PM patients, six patients with pleural fibrosis, and 40 healthy controls. Circulating FGF18 was measured by ELISA and correlated to clinicopathological parameters. RESULTS:FGF18 showed high mRNA expression in PM and PM-derived cell lines. PM patients with high FGF18 mRNA expression showed a trend toward longer overall survival (OS) in the TCGA dataset. In PM cells with low endogenous FGF18 expression, forced overexpression of FGF18 resulted in reduced growth but increased migration. Surprisingly, despite the high FGF18 mRNA levels observed in PM, circulating FGF18 protein was significantly lower in PM patients and patients with pleural fibrosis than in healthy controls. No significant association of circulating FGF18 with OS or other disease parameters of PM patients was observed. CONCLUSIONS:FGF18 is not a prognostic biomarker in PM. Its role in PM tumor biology and the clinical significance of decreased plasma FGF18 in PM patients warrant further investigation.

BackgroundNo targeted drugs are currently available against small cell lung cancer (SCLC). BCL-2 family members are involved in apoptosis regulation and represent therapeutic targets in many malignancies.MethodsExpression of BCL-2 family members in 27 SCLC cell lines representing all known four SCLC molecular subtypes was assessed by qPCR, Western blot and mass spectrometry-based proteomics. BCL-2 and MCL-1 inhibition (venetoclax and S63845, respectively) was assessed by MTT assay and flow cytometry and in mice bearing human SCLC tumours. Drug interactions were calculated using the Combenefit software. Ectopic BAX overexpression was achieved by expression plasmids.ResultsThe highest BCL-2 expression levels were detected in ASCL1- and POU2F3-driven SCLC cells. Although sensitivity to venetoclax was reflected by BCL-2 levels, not all cell lines responded consistently despite their high BCL-2 expression. MCL-1 overexpression and low BAX levels were both characteristic for venetoclax resistance in SCLC, whereas the expression of other BCL-2 family members did not affect therapeutic efficacy. Combination of venetoclax and S63845 resulted in significant, synergistic in vitro and in vivo anti-tumour activity and apoptosis induction in double-resistant cells; however, this was seen only in a subset with detectable BAX. In non-responding cells, ectopic BAX overexpression sensitised to venetoclax and S63845 and, furthermore, induced synergistic drug interaction.ConclusionsThe current study reveals the subtype specificity of BCL-2 expression and sheds light on the mechanism of venetoclax resistance in SCLC. Additionally, we provide preclinical evidence that combined BCL-2 and MCL-1 targeting is an effective approach to overcome venetoclax resistance in high BCL-2-expressing SCLCs with intact BAX.

Supplementary Figure S7. Coding variants detected in MPM cell cultures and their relation to TERT promoter status.

Background and Purpose: Pleural mesothelioma (PM) is a highly aggressive thoracic tumor with poor prognosis. Although reduced tissue drug accumulation is one of the key features of platinum (Pt) resistance, little is known about Pt distribution in human PM. Experimental Approach: We assessed Pt levels of blood samples and surgically resected specimens from 25 PM patients who had received neoadjuvant Pt-based chemotherapy (CHT). Pt levels and tissue distributions were measured by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) and correlated with clinicopathological features. Key Results: In surgically resected PM specimens, mean Pt levels were significantly higher in the non-tumorous (fibrotic) areas (vs. tumorous regions, p<0.001). No major heterogeneity of Pt distribution was seen within the tumorous areas. Pt levels correlated neither with microvessel area (MVA) nor with apoptosis rate in the tumorous or in the non-tumorous regions. A significant positive correlation was found between serum and both full tissue section and tumorous area average Pt levels (r=0.415, p=0.039 and r=0.532, p=0.006, respectively). Furthermore, a significant negative correlation was detected between serum Pt concentrations and elapsed time from the last cycle of CHT (r=-0.474, p=0.017). Serum Pt levels correlated negatively with overall survival (OS) (p=0.029). Conclusion and Implications: There are major differences in the drug distribution between tumorous and non-tumorous areas of PM specimens. Serum Pt levels significantly correlate with full section- and tumorous areas average Pt levels, elapsed time from the last CHT cycle, and OS. Further studies investigating clinicopathological factors that modulate tissue Pt concentration and distribution are warranted.

Supplementary Tables S1 to S9 with the exception of Supplementary Table S6

Supplementary Figure S4: TERT promoter activity and impact of ETS factors in human MPM.

BACKGROUND:Although immunotherapy has led to a paradigm shift in the treatment of lung cancer, the therapeutic approaches for lung neuroendocrine neoplasms (LNENs) are still limited. Our aim was to explore the immunological landscape and the expression of immune checkpoint markers in LNENs.METHODS:Surgically removed tumor samples of 26 atypical carcinoid (AC), 30 large cell neuroendocrine carcinoma (LCNEC) and 29 small cell lung cancer (SCLC) patients were included. The immune phenotype of each tumor type was assessed by using a panel of 15 immune-related markers. As these markers are potentially expressed by immune cells and/or tumor cells, they might serve as putative targets for immunotherapy. Expression patterns were measured by immunohistochemistry and correlated with clinicopathological parameters and prognosis.RESULTS:Unsupervised hierarchical clustering revealed distinct immunologic profiles across tumor types. Specifically, AC tumors were characterized by high tumor cell CD40 expression and low levels of immune infiltrates whereas SCLC samples had a high CD47 and Inducible T Cell Costimulator (ICOS) expression in tumor cells and immune cells, respectively. High CD70 and CD137 expression by tumor cells as well as elevated expression of CD27, Lymphocyte Activation Gene 3 (LAG3), and CD40 by immune cells were characteristic for LCNEC samples. Overall, SCLC and LCNEC tumors had a more immunogenic phenotype than AC samples. High tumor cell CD47 and CD40 expressions were associated with impaired and improved survival outcomes, respectively.CONCLUSIONS:By providing insights into the widely divergent immunologic profiles of LNENs, our results might serve as a basis for the development of novel immunotherapy-related approaches in these devastating malignancies.

Trabectedin synergizes with the bcl-2 inhibitor obatoclax in a pleural mesothelioma cell model

Supplementary Figure S5: Impact of TERT promoter status on sensitivity of MPM cell models against pharmacological telomerase and ETS factor inhibition.

The effect of nintedanib treatment on the migratory activity of the SPC212 cells

AimsPleural mesothelioma (PM) is a highly aggressive thoracic tumour with poor prognosis. Although reduced tissue drug accumulation is one of the key features of platinum (Pt) resistance, little is known about Pt distribution in human PM. MethodsWe assessed Pt levels of blood samples and surgically resected specimens from 25 PM patients who had received neoadjuvant Pt-based chemotherapy (CHT). Pt levels and tissue distributions were measured by laser ablation-inductively coupled plasma-mass spectrometry and correlated with clinicopathological features. ResultsIn surgically resected PM specimens, mean Pt levels of nontumourous (fibrotic) areas were significantly higher (vs tumourous regions, P = 0.0031). No major heterogeneity of Pt distribution was seen within the tumourous areas. Pt levels correlated neither with the microvessel area nor with apoptosis rate in the tumourous or nontumourous regions. A significant positive correlation was found between serum and both full tissue section and tumourous area mean Pt levels (r = 0.532, P = 0.006, 95% confidence interval [95% CI] 0.161-0.771 and r = 0.415, P = 0.039, 95% CI 0.011-0.702, respectively). Furthermore, a significant negative correlation was detected between serum Pt concentrations and elapsed time from the last cycle of CHT (r = -0.474, P = 0.017, 95% CI -0.738--0.084). Serum Pt levels correlated negatively with overall survival (OS) (P = 0.029). ConclusionsThere are major differences in drug distribution between tumourous and nontumourous areas of PM specimens. Serum Pt levels significantly correlate with full section and tumourous area average Pt levels, elapsed time from the last CHT cycle, and OS. Further studies investigating clinicopathological factors that modulate tissue Pt concentration and distribution are warranted.

Supplementary Figure S1. Representative sequencing charts of the TERT promoter region harboring activating mutations.

Supplementary Figure S8. BAP1 expression in MPM samples and its relation to TERT promoter status and patient overall survival.

Supplementary Material and Methods

Supplementary Figure S3: The impact of TERT promoter mutation status on telomere parameters.