C-Myc protein expression indicates unfavorable clinical outcome in surgically resected small cell lung cancer.

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.

Laser ablation-inductively coupled plasma-mass spectrometry analysis reveals differences in chemotherapeutic drug distribution in surgically resected pleural mesothelioma.

AIMS: Pleural 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. METHODS: 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 and correlated with clinicopathological features. RESULTS: In 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). CONCLUSIONS: There 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.

Comparative expression analysis of immune-related markers in surgically resected lung neuroendocrine neoplasms.

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.

Circulating FGF18 is decreased in pleural mesothelioma but not correlated with disease prognosis.

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.

Dual targeting of BCL-2 and MCL-1 in the presence of BAX breaks venetoclax resistance in human small cell lung cancer.

BACKGROUND: No 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. METHODS: Expression 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. RESULTS: The 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. CONCLUSIONS: The 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.

Protein Expression of immune checkpoints STING and MHCII in small cell lung cancer.

BACKGROUND: SCLC is an aggressive malignancy where immunotherapies show limited efficacy. We aimed to characterize the SCLC microenvironment according to the expression patterns of SCLC subtype markers and novel immune checkpoints to identify therapeutic vulnerabilities. METHODS: We included SCLC tissue samples from 219 surgically resected, limited-stage patients in this cross-sectional study. We performed immunohistochemistry for STING and MHCII, as well as for the novel subtype markers (ASCL1, NEUROD1, POU2F3, YAP1). Moreover, we assessed CD45 + , CD8 + and CD68 + immune cell infiltration. RESULTS: 36% of SCLC tumors showed significant stromal or intraepithelial CD45 + immune cell infiltration. These patients exhibited significantly increased overall survival (OS) (vs. patients with immune-deserted tumors). High CD8 expression was associated with increased median OS. We found STING expression on cancer-associated fibroblasts in the stroma and on T-cells and macrophages in both tumorous and stromal compartments. STING expression positively correlated with immune cell infiltration. Increased STING-positivity in tumor nests was an independent favorable prognosticator for OS. ASCL1 was the most frequently expressed subtype-specific protein. Concomitant expression of three or four subtype-defining markers was seen in 13.8% of the included samples, whereas 24.1% of the cases were classified as quadruple negative tumors. YAP1 expression was associated with increased immune infiltrates. Tumor cell MHCII expression positively correlated with immune cell infiltration and with STING- and YAP1 expressions. CONCLUSIONS: STING and MHCII are expressed in SCLC. The majority of immune-infiltrated SCLCs exhibit increased STING expression. Immune infiltration and STING expression are prognostic in limited-stage SCLC, making STING a potential therapeutic target.

In-depth proteomic analysis reveals unique subtype-specific signatures in human small-cell lung cancer.

BACKGROUND: Small-cell lung cancer (SCLC) molecular subtypes have been primarily characterized based on the expression pattern of the following key transcription regulators: ASCL1 (SCLC-A), NEUROD1 (SCLC-N), POU2F3 (SCLC-P) and YAP1 (SCLC-Y). Here, we investigated the proteomic landscape of these molecular subsets with the aim to identify novel subtype-specific proteins of diagnostic and therapeutic relevance. METHODS: Pellets and cell media of 26 human SCLC cell lines were subjected to label-free shotgun proteomics for large-scale protein identification and quantitation, followed by in-depth bioinformatic analyses. Proteomic data were correlated with the cell lines' phenotypic characteristics and with public transcriptomic data of SCLC cell lines and tissues. RESULTS: Our quantitative proteomic data highlighted that four molecular subtypes are clearly distinguishable at the protein level. The cell lines exhibited diverse neuroendocrine and epithelial-mesenchymal characteristics that varied by subtype. A total of 367 proteins were identified in the cell pellet and 34 in the culture media that showed significant up- or downregulation in one subtype, including known druggable proteins and potential blood-based markers. Pathway enrichment analysis and parallel investigation of transcriptomics from SCLC cell lines outlined unique signatures for each subtype, such as upregulated oxidative phosphorylation in SCLC-A, DNA replication in SCLC-N, neurotrophin signalling in SCLC-P and epithelial-mesenchymal transition in SCLC-Y. Importantly, we identified the YAP1-driven subtype as the most distinct SCLC subgroup. Using sparse partial least squares discriminant analysis, we identified proteins that clearly distinguish four SCLC subtypes based on their expression pattern, including potential diagnostic markers for SCLC-Y (e.g. GPX8, PKD2 and UFO). CONCLUSIONS: We report for the first time, the protein expression differences among SCLC subtypes. By shedding light on potential subtype-specific therapeutic vulnerabilities and diagnostic biomarkers, our results may contribute to a better understanding of SCLC biology and the development of novel therapies.

Malignant pleural mesothelioma nodules remodel their surroundings to vascularize and grow.

Background: The microanatomical steps of malignant pleural mesothelioma (MPM) vascularization and the resistance mechanisms to anti-angiogenic drugs in MPM are unclear. Methods: We investigated the vascularization of intrapleurally implanted human P31 and SPC111 MPM cells. We also assessed MPM cell's motility, invasion and interaction with endothelial cells in vitro. Results: P31 cells exhibited significantly higher two-dimensional (2D) motility and three-dimensional (3D) invasion than SPC111 cells in vitro. In co-cultures of MPM and endothelial cells, P31 spheroids permitted endothelial sprouting (ES) with minimal spatial distortion, whereas SPC111 spheroids repealed endothelial sprouts. Both MPM lines induced the early onset of submesothelial microvascular plexuses covering large pleural areas including regions distant from tumor colonies. The development of these microvascular networks occurred due to both intussusceptive angiogenesis (IA) and ES and was accelerated by vascular endothelial growth factor A (VEGF-A)-overexpression. Notably, SPC111 colonies showed different behavior to P31 cells. P31 nodules incorporated tumor-induced capillary plexuses from the earliest stages of tumor formation. P31 cells deposited a collagenous matrix of human origin which provided "space" for further intratumoral angiogenesis. In contrast, SPC111 colonies pushed the capillary plexuses away and thus remained avascular for weeks. The key event in SPC111 vascularization was the development of a desmoplastic matrix of mouse origin. Continuously invaded by SPC111 cells, this matrix transformed into intratumoral connective tissue trunks, providing a route for ES from the diaphragm. Conclusions: Here, we report two distinct growth patterns of orthotopically implanted human MPM xenografts. In the invasive pattern, MPM cells invade and thus co-opt peritumoral capillary plexuses. In the pushing/desmoplastic pattern, MPM cells induce a desmoplastic response within the underlying tissue which allows the ingrowth of a nutritive vasculature from the pleura.

Expression patterns and prognostic relevance of subtype-specific transcription factors in surgically resected small-cell lung cancer: an international multicenter study.

The tissue distribution and prognostic relevance of subtype-specific proteins (ASCL1, NEUROD1, POU2F3, YAP1) present an evolving area of research in small-cell lung cancer (SCLC). The expression of subtype-specific transcription factors and P53 and RB1 proteins were measured by immunohistochemistry (IHC) in 386 surgically resected SCLC samples. Correlations between subtype-specific proteins and in vitro efficacy of various therapeutic agents were investigated by proteomics and cell viability assays in 26 human SCLC cell lines. Besides SCLC-A (ASCL1-dominant), SCLC-AN (combined ASCL1/NEUROD1), SCLC-N (NEUROD1-dominant), and SCLC-P (POU2F3-dominant), IHC and cluster analyses identified a quadruple-negative SCLC subtype (SCLC-QN). No unique YAP1-subtype was found. The highest overall survival rates were associated with non-neuroendocrine subtypes (SCLC-P and SCLC-QN) and the lowest with neuroendocrine subtypes (SCLC-A, SCLC-N, SCLC-AN). In univariate analyses, high ASCL1 expression was associated with poor prognosis and high POU2F3 expression with good prognosis. Notably, high ASCL1 expression influenced survival outcomes independently of other variables in a multivariate model. High POU2F3 and YAP1 protein abundances correlated with sensitivity and resistance to standard-of-care chemotherapeutics, respectively. Specific correlation patterns were also found between the efficacy of targeted agents and subtype-specific protein abundances. In conclusion, we investigated the clinicopathological relevance of SCLC molecular subtypes in a large cohort of surgically resected specimens. Differential IHC expression of ASCL1, NEUROD1, and POU2F3 defines SCLC subtypes. No YAP1-subtype can be distinguished by IHC. High POU2F3 expression is associated with improved survival in a univariate analysis, whereas elevated ASCL1 expression is an independent negative prognosticator. Proteomic and cell viability assays of human SCLC cell lines revealed distinct vulnerability profiles defined by transcription regulators. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Clinical relevance of circulating activin A and follistatin in small cell lung cancer.

OBJECTIVES: Circulating levels of activin A (ActA) and follistatin (FST) have been investigated in various disorders including malignancies. However, to date, their diagnostic and prognostic relevance is largely unknown in small cell lung cancer (SCLC). Our aim was to evaluate circulating ActA and FST levels as potential biomarkers in this devastating disease. METHODS: Seventy-nine Caucasian SCLC patients and 67 age- and sex-matched healthy volunteers were included in this study. Circulating ActA and FST concentrations were measured by ELISA and correlated with clinicopathological parameters and long-term outcomes. RESULTS: Plasma ActA and FST concentrations were significantly elevated in SCLC patients when compared to healthy volunteers (p < 0.0001). Furthermore, extensive-stage SCLC patients had significantly higher circulating ActA levels than those with limited-stage disease (p = 0.0179). Circulating FST concentration was not associated with disease stage (p = 0.6859). Notably, patients with high (≥548.8 pg/ml) plasma ActA concentration exhibited significantly worse median overall survival (OS) compared to those with low (<548.8 pg/ml) ActA levels (p = 0.0009). Moreover, Cox regression analysis adjusted for clinicopathological parameters revealed that high ActA concentration is an independent predictor of shorter OS (HR: 1.932; p = 0.023). No significant differences in OS have been observed with regards to plasma FST levels (p = 0.1218). CONCLUSION: Blood ActA levels are elevated and correlate with disease stage in SCLC patients. Measurement of circulating ActA levels might help in the estimation of prognosis in patients with SCLC.

Characterization of Tumor-Associated Macrophages and the Immune Microenvironment in Limited-Stage Neuroendocrine-High and -Low Small Cell Lung Cancer.

This study aims to characterize tumor-infiltrating macrophages (TAMs), myeloid-derived suppressor cells (MDSC), and the related molecular milieu regulating anti-tumor immunity in limited-stage neuroendocrine (NE)-high and NE-low small cell lung cancer. Primary tumors and matched lymph node (LN) metastases of 32 resected, early-stage SCLC patients were analyzed by immunohistochemistry (IHC) with antibodies against pan-macrophage marker CD68, M2-macrophage marker CD163, and MDSC marker CD33. Area-adjusted cell counting on TMAs showed that TAMs are the most abundant cell type in the TME, and their number in tumor nests exceeds the number of CD3 + T-cells (64% vs. 38% in NE-low and 71% vs. 18% in NE-high). Furthermore, the ratio of CD163-expressing M2-polarized TAMs in tumor nests was significantly higher in NE-low vs. NE-high tumors (70% vs. 31%). TAM density shows a strong positive correlation with CD45 and CD3 in tumor nests, but not in the stroma. fGSEA analysis on a targeted RNAseq oncological panel of 2560 genes showed that NE-high tumors exhibited increased enrichment in pathways related to cell proliferation, whereas in NE-low tumors, immune response pathways were significantly upregulated. Interestingly, we identified a subset of NE-high tumors representing an immune-oasis phenotype, but with a different gene expression profile compared to NE-low tumors. In contrast, we found that a limited subgroup of NE-low tumors is immune-deserted and express distinct cellular pathways from NE-high tumors. Furthermore, we identified potential molecular targets based on our expression data in NE-low and immune-oasis tumor subsets, including CD70, ANXA1, ITGB6, TP63, IFI27, YBX3 and CXCR2.

Down-regulation of A20 promotes immune escape of lung adenocarcinomas.

Inflammation is a well-known driver of lung tumorigenesis. One strategy by which tumor cells escape tight homeostatic control is by decreasing the expression of the potent anti-inflammatory protein tumor necrosis factor alpha-induced protein 3 (TNFAIP3), also known as A20. We observed that tumor cell intrinsic loss of A20 markedly enhanced lung tumorigenesis and was associated with reduced CD8+ T cell-mediated immune surveillance in patients with lung cancer and in mouse models. In mice, we observed that this effect was completely dependent on increased cellular sensitivity to interferon-γ (IFN-γ) signaling by aberrant activation of TANK-binding kinase 1 (TBK1) and increased downstream expression and activation of signal transducer and activator of transcription 1 (STAT1). Interrupting this autocrine feed forward loop by knocking out IFN-α/ß receptor completely restored infiltration of cytotoxic T cells and rescued loss of A20 depending tumorigenesis. Downstream of STAT1, programmed death ligand 1 (PD-L1) was highly expressed in A20 knockout lung tumors. Accordingly, immune checkpoint blockade (ICB) treatment was highly efficient in mice harboring A20-deficient lung tumors. Furthermore, an A20 loss-of-function gene expression signature positively correlated with survival of melanoma patients treated with anti-programmed cell death protein 1. Together, we have identified A20 as a master immune checkpoint regulating the TBK1-STAT1-PD-L1 axis that may be exploited to improve ICB therapy in patients with lung adenocarcinoma.

EGFR Alterations Influence the Cetuximab Treatment Response and c-MET Tyrosine-Kinase Inhibitor Sensitivity in Experimental Head and Neck Squamous Cell Carcinomas.

Background: Anti-EGFR antibody therapy is still one of the clinical choices in head and neck squamous cell carcinoma (HNSCC) patients, but the emergence of cetuximab resistance questioned its effectiveness and reduced its applicability. Although several possible reasons of resistance against the antibody treatment and alternative therapeutic proposals have been described (EGFR alterations, activation of other signaling pathways), there is no method to predict the effectiveness of anti-EGFR antibody treatments and to suggest novel therapeutics. Our study investigated the effect of EGFR R521K alteration on efficiency of cetuximab therapy of HNSCC cell lines and tried to find alternative therapeutic approaches against the resistant cells. Methods: After genetic characterization of HNSCC cells, we chose one wild type and one R521K+ cell line for in vitro proliferation and apoptosis tests, and in vivo animal models using different therapeutic agents. Results: Although the cetuximab treatment affected EGFR signalization in both cells, it did not alter in vitro cell proliferation or apoptosis. In vivo cetuximab therapy was also ineffective on R521K harboring tumor xenografts, while blocked the tumor growth of EGFR-wild type xenografts. Interestingly, the cetuximab-resistant R521K tumors were successfully treated with c-MET tyrosine kinase inhibitor SU11274. Conclusion: Our results suggest that HNSCC cell line expressing the R521K mutant form of EGFR does not respond well to cetuximab treatment in vitro or in vivo, but hopefully might be targeted by c-MET tyrosine kinase inhibitor treatment.

[Predictive relevance of KRAS mutational status in bone metastatic lung adenocarcinoma treated with bisphosphonate therapy]. / A KRAS-mutációs státusz prediktív szerepe biszfoszfonáttal kezelt, csontáttétet képzo tüdo-adenokarcinómában.

The therapeutic impact of KRAS mutations remains controversial in bone metastatic lung adenocarcinoma (LADC). Therefore, our aim was to investigate the effects of KRAS mutational status on overall survival (OS) in these patients according to bisphosphonate therapy (BTx) and radiation therapy (RTx). In total, 134 LADC patients diagnosed with simultaneous bone metastasis were included in this study. The results of the univariate (p=0.008) and multivariate (p=0.004) survival analyses indicated that KRAS mutation is a negative prognostic factor. Both BTx and RTx can increase the OS with a pronounced benefit for patients with KRAS wild-type tumors. Importantly, the concomitant use of BTx and RTx might increase the OS irrespective of KRAS status compared to BTx or RTx alone. In summary, our results might contribute to the development of new therapeutic approaches with regards to KRAS mutational status in bone metastatic LADC.

Prognostic impact of PD-1 and PD-L1 expression in malignant pleural mesothelioma: an international multicenter study.

BACKGROUND: Programmed cell death 1/programmed death ligand 1 (PD-1/PD-L1) immune-checkpoint blockade is a promising new therapeutic strategy in cancer. However, expression patterns and prognostic significance of PD-L1 and PD-1 are still controversial in human malignant pleural mesothelioma (MPM). METHODS: Formalin-fixed paraffin-embedded (FFPE) tumor samples from 203 MPM patients receiving standard treatment without immunotherapy were collected from 5 European centers. PD-L1 and PD-1 expression of tumor cells (TCs) and tumor-infiltrating lymphocytes (TILs) were measured by immunohistochemistry and correlated with clinical parameters and long-term outcome. RESULTS: High (>10%) PD-L1 TC and PD-1 TILs expressions were found in 18 (8%) and 39 (24%) patients, respectively. PD-L1 was rarely expressed by TILs [≥1%, n=13 (8%); >10%, n=1]. No significant associations were found between the PD-L1 or PD-1 expression of TCs or TILs and clinicopathological parameters such as stage or histological subtype. Notably, patients with high (>10%) TC-specific PD-L1 expression exhibited significantly worse median overall survival (OS) (6.3 vs. 15.1 months of those with low TC PD-L1 expression; HR: 2.51, P<0.001). In multivariate cox regression analysis adjusted for clinical parameters, high TC PD-L1 expression (>10%) proved to be an independent negative prognostic factor for OS (HR: 2.486, P=0.005). There was no significant correlation between PD-L1 or PD-1 expression of TILs and OS. CONCLUSIONS: In this multicenter cohort study, we demonstrate that high (>10%) PD-L1 expression of TCs independently predicts worse OS in MPM. Further studies are warranted to investigate the value of PD-L1/PD-1 expression as a marker for treatment response in MPM patients receiving immunotherapy.

The effects of bisphosphonate and radiation therapy in bone-metastatic lung adenocarcinoma: the impact of KRAS mutation.

BACKGROUND: KRAS mutation is the most common genetic alteration in lung adenocarcinoma (LADC) in Western countries and is associated with worse outcome in bone-metastatic cases. Yet, to date, no effective treatment guidelines were developed for these patients. Accordingly, our aim was to investigate the impact of KRAS mutation on bisphosphonate (BTx) and radiation therapy (RTx) in bone-metastatic LADC patients. METHODS: Clinicopathological variables of 134 consecutive LADC patients with bone metastases at diagnosis and known KRAS status were retrospectively analyzed. The effects of BTx, RTx and KRAS mutation on overall survival (OS) were investigated. RESULTS: Of the total cohort, 93 patients were identified as KRAS wild-type (WT) (69.4%) and 41 (30.6%) as KRAS mutant patients. The presence of KRAS mutation was associated with significantly reduced median OS (5.1 vs. 10.2 months in KRAS WT patients; P=0.008). Irrespective of KRAS mutational status both BTx (P=0.007) and RTx (P=0.021) conferred a significant benefit for OS. Notably, however, when analyzing the patients with KRAS-mutant and KRAS WT tumors separately, the benefit from BTx and RTx on OS remained statistically significant only in KRAS WT patients (P=0.032 and P=0.031, respectively). CONCLUSIONS: KRAS mutation is a strong negative prognostic factor in bone-metastatic LADC patients. Both BTx and RTx can increase the OS with a pronounced benefit for patients with KRAS WT tumors. Altogether, KRAS mutational status should be considered during therapeutic decision making in bone-metastatic LADC patients.

EGFR variant allele frequency predicts EGFR-TKI efficacy in lung adenocarcinoma: a multicenter study.

BACKGROUND: Although lung adenocarcinoma (LADC) with sensitizing mutations of the epidermal growth factor receptor (EGFR) is highly sensitive to EGFR tyrosine kinase inhibitors (EGFR-TKIs), in most cases disease progression inevitably occurs. Our aim was to investigate the predictive and prognostic significance of adjusted tumoral EGFR variant allele frequency (EGFR-aVAF) in the above setting. METHODS: Eighty-nine Caucasian advanced-stage LADC patients with known exon-specific EGFR mutations undergoing EGFR-TKI treatment were included. The correlations of EGFR-aVAF with clinicopathological variables including progression-free and overall survival (PFS and OS, respectively) were retrospectively analyzed. RESULTS: Of 89 EGFR-mutant LADC patients, 46 (51.7%) had exon 19 deletion, while 41 (46.1%) and 2 (2.2%) patients had exon 21- and exon 18-point mutations, respectively. Tumoral EGFR-aVAF was significantly higher in patients harboring EGFR exon 19 mutations than in those with exon 21-mutant tumors (P<0.001). Notably, patients with EGFR exon 19 mutant tumors demonstrated significantly improved PFS (P=0.003) and OS (P=0.02) compared to patients with exon 21 mutations. Irrespective of specific exon mutations, a statistically significant positive linear correlation was found between EGFR-aVAF of tumoral tissue and PFS (r=0.319; P=0.002). High (≥70%) EGFR-aVAF was an independent predictor of longer PFS [vs. low (<70%) EGFR-aVAF; median PFSs were 52 vs. 26 weeks, respectively; P<0.001]. Additionally, patients with high EGFR-aVAF also had significantly improved OS than those with low EGFR-aVAF (P=0.011). CONCLUSIONS: Our study suggests that high (≥70%) EGFR-aVAF of tumoral tissue predicts benefit from EGFR-TKI treatment in advanced LADC and, moreover, that exon 19 EGFR mutation is associated with high EGFR-aVAF and improved survival outcomes.

Molecular profiles of small cell lung cancer subtypes: therapeutic implications.

Small cell lung cancer (SCLC; accounting for approximately 13%-15% of all lung cancers) is an exceptionally lethal malignancy characterized by rapid doubling time and high propensity to metastasize. In contrast to the increasingly personalized therapies in other types of lung cancer, SCLC is still regarded as a homogeneous disease and the prognosis of SCLC patients remains poor. Recently, however, substantial progress has been made in our understanding of SCLC biology. Advances in genomics and development of new preclinical models have facilitated insights into the intratumoral heterogeneity and specific genetic alterations of this disease. This worldwide resurgence of studies on SCLC has ultimately led to the development of novel subtype-specific classifications primarily based on the neuroendocrine features and distinct molecular profiles of SCLC. Importantly, these biologically distinct subtypes might define unique therapeutic vulnerabilities. Herein, we summarize the current knowledge on the molecular profiles of SCLC subtypes with a focus on their potential clinical implications.

Apelin promotes blood and lymph vessel formation and the growth of melanoma lung metastasis.

Apelin, a ligand of the APJ receptor, is overexpressed in several human cancers and plays an important role in tumor angiogenesis and growth in various experimental systems. We investigated the role of apelin signaling in the malignant behavior of cutaneous melanoma. Murine B16 and human A375 melanoma cell lines were stably transfected with apelin encoding or control vectors. Apelin overexpression significantly increased melanoma cell migration and invasion in vitro, but it had no impact on its proliferation. In our in vivo experiments, apelin significantly increased the number and size of lung metastases of murine melanoma cells. Melanoma cell proliferation rates and lymph and blood microvessel densities were significantly higher in the apelin-overexpressing pulmonary metastases. APJ inhibition by the competitive APJ antagonist MM54 significantly attenuated the in vivo pro-tumorigenic effects of apelin. Additionally, we detected significantly elevated circulating apelin and VEGF levels in patients with melanoma compared to healthy controls. Our results show that apelin promotes blood and lymphatic vascularization and the growth of pulmonary metastases of skin melanoma. Further studies are warranted to validate apelin signaling as a new potential therapeutic target in this malignancy.

The landscape of small cell lung cancer metastases: Organ specificity and timing.

BACKGROUND: Early metastasis is a hallmark of small cell lung cancer (SCLC). However, the mechanisms and resulting patterns of SCLC dissemination are unclear. Our aim was thus to investigate the organ specificity and timing of blood-borne metastases in a comprehensive large cohort of SCLC patients. METHODS: In this retrospective non-interventional cross-sectional study of 1009 Caucasian SCLC patients, we investigated the correlation between the distinct locations of the primary tumor and metastatic sites. RESULTS: The onset of bone (p < 0.001), brain (p < 0.001), and pericardial (p = 0.02) metastases were late events, whereas adrenal gland (p = 0.005) and liver (p < 0.001) metastases occurred earlier. No significant difference was found in the distribution of early versus late metastases when comparing central and peripheral primary tumors. Patients with bone metastases had a higher than expected likelihood of having liver metastases, while brain metastases tended to appear together with adrenal gland metastases. Pleural and both lung and pericardial metastases also tended to co-metastasize together more frequently than expected if metastatic events occurred independently. Notably, patients with central primary tumors had decreased median overall survival (OS) compared to those with peripheral tumors, although this tendency does not appear to be significant (p = 0.072). CONCLUSION: Our results are suggestive for particular site- and sequence-specific metastasis patterns in human SCLC. SCLC bone metastases tend to appear together with liver metastases, while brain metastases occur together with adrenal gland metastases. Better understanding of metastasis distribution patterns might help to improve the diagnosis and therapeutic decision-making in SCLC patients.