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Supplementary Figure S5 shows role of MFGE8 in MPM.

Supplementary Figure Legends

(A) WB analysis of HER2 expression after exposure of the indicated SCLC cells to CDDP at IC10 and IC50 concentrations for the indicated times. (B) WB analysis of HER2 expression after exposure of SBC-3 and SBC-5 cells to ETP and CPT-11 at their IC50 concentrations. All experiments were repeated at least three times with similar results. ETP: etoposide, CPT-11: irinotecan.

Supplementary Figure S4 shows that MFGE8 regulates the FAK pathway.

WB analysis for downstream intracellular signaling of HER2 after exposure to CDDP (IC50 concentration), trastuzumab (1 μg/ml), and CDDP plus trastuzumab.

Supplementary Table S1 shows the selected genes with statistically significant changes in transcriptome analysis using two different shRNA constructs.

The primer sequences for qRT-PCR analysis

Supplementary Figure S6 shows that restoring MFGE8 abolishes apoptosis by re-activating the FAK-AKT pathway.

Supplementary Figure S1 shows that TGFβ1 treatment in MPM cells.

Small cell lung cancer (SCLC) accounts for 15% of all lung cancer cases and is a highly lethal disease. For the last several decades, the standard treatment for SCLC has been deadlocked, and new therapeutic strategies are urgently needed. HER2 is a member of the HER family and has been reported to be overexpressed in 30% of SCLC cases with poor prognosis. However, the clinical relevance of HER2-targeted therapy for SCLC remains unclear. Here, we first identify that cytotoxic drugs induce significant HER2 overexpression through microRNA-125a (miR-125a) and miR-125b downregulation, which in turn act as a novel therapeutic target for trastuzumab-mediated cellular cytotoxicity in SCLC. In this study, we showed that treatment of the HER2-positive SCLC cells, SBC-3 and SBC-5, with cytotoxic drugs induced a significant upregulation of HER2. Cisplatin (CDDP) treatment of SCLC cells resulted in a significant downregulation of miR-125a and miR-125b. We confirmed that miR-125a and miR-125b bound to the 3′-untranslated regions of HER2 mRNA, and that downregulation of miR-125a and miR-125b resulted in upregulation of HER2 in SCLC cells, suggesting a relationship between cytotoxic drug exposure and miR-125/HER2 dysregulation. Furthermore, using a calcein assay, we demonstrated a significantly enhanced cytotoxic effect of CDDP and trastuzumab that was mediated via antibody-dependent cellular cytotoxicity. Finally, we clearly demonstrated the enhanced antitumor effect of these agents in an orthotopic lung cancer model in vivo. Our results offer a novel therapeutic strategy for HER2-positive SCLCs by using trastuzumab combined with cytotoxic drugs. Mol Cancer Ther; 14(6); 1414–23. ©2015 AACR.

Supplementary Table S2 shows the chromatin accessibility with statistically significant changes in genome-wide ATAC-seq.

Supplementary Figure S3 shows that LSD1 suppression attenuated the mesenchymal phenotype.

Malignant pleural mesothelioma (MPM) is a highly aggressive tumor that has a low overall survival; however, no significant treatment advances have been made in the past 15 years. Large-scale molecular studies have identified a poor prognostic subset of MPM linked to the epithelial–mesenchymal transition (EMT) that may contribute toward resistance to chemotherapy, suggesting that EMT could be targeted to treat patients with MPM. Previously, we reported that histone modifiers regulating EMT could be therapeutic targets; therefore, in this study, we investigated whether targeting lysine-specific demethylase 1 (LSD1/KDM1), a histone-modifying enzyme responsible for demethylating histone H3 lysine 4 and lysine 9, could represent a novel therapeutic strategy for MPM. We suppressed LSD1 and investigated the EMT phenotype using EMT marker expression and wound-healing assay; and chemosensitivity using apoptosis assay. We found that suppressing LSD1 induces an epithelial phenotype in sarcomatoid MPM cells, while attenuating the mesenchymal phenotype sensitized MPM cells to cisplatin-induced apoptosis. Subsequent genome-wide identification, comprehensive microarray analysis, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) to assess genome-wide changes in chromatin accessibility suggested that LSD1 directly regulates milk fat globulin protein E8 (MFGE8), an integrin ligand that is involved in the FAK pathway. Furthermore, we found that LSD1 regulates the mesenchymal phenotype and apoptosis by activating the FAK–AKT–GSK3β pathway via a positive feedback loop involving MFGE8 and Snail expression, thereby leading to cisplatin resistance.Implications:This study suggests that LSD1 regulates the mesenchymal phenotype and apoptosis, and that LSD1 inhibitors could be combined with the cisplatin as a novel therapy for patients with MPM.

Supplementary Figure S2 shows that Snail-1 induces a mesenchymal phenotype in MPM cells.

The clinicopathological differences among high-grade fetal lung adenocarcinomas completely comprising tumor cells that resemble fetal lung epithelium (pure type) and those with fetal lung-like components admixed with conventional adenocarcinoma cells (mixed type) remain undetermined. Here, we examined the clinicopathological, immunohistochemical, and molecular features of 11 lung adenocarcinomas with fetal lung-like morphology among 3895 consecutive cases of primary lung cancer based on the expression pattern of transcription factors. According to the current WHO classification, two cases (0.05%) were categorized as low-grade fetal adenocarcinoma, two cases (0.05%) were pure-type high-grade fetal adenocarcinoma, five cases (0.1%) were mixed-type high-grade fetal adenocarcinoma, and the remaining two cases (0.05%) were lung adenocarcinoma with high-grade fetal features (fetal lung-like morphology occupied less than 50%). CTNNB1 mutations were exclusively identified in low-grade fetal adenocarcinomas. In contrast, mixed-type high-grade fetal adenocarcinoma or lung adenocarcinoma with high-grade fetal features frequently harbored mitogenic drivers including EGFR mutations. Furthermore, almost all tumor cells expressed CDX2 and HNF4α in both cases of pure-type high-grade fetal lung adenocarcinoma, but lacked TTF-1 positivity. In contrast, TTF-1 was frequently expressed in mixed-type high-grade fetal lung adenocarcinoma and in lung adenocarcinoma with high-grade fetal features. Our data suggest similar prevalence of low-grade fetal lung adenocarcinoma and pure-type high-grade fetal lung adenocarcinoma, and indicate that pure- and mixed-type high-grade fetal lung adenocarcinomas are distinct, with the former akin to low-grade fetal adenocarcinoma with respect to purely embryonic morphology and absence of common lung adenocarcinoma mitogenic drivers, and the latter being genetically and transcriptionally related to conventional lung adenocarcinoma.

A distinct subset of lung adenocarcinomas (LADs), arising from a series of peripheral lung cells defined as the terminal respiratory unit (TRU), is characterised by thyroid transcription factor 1 (TTF-1) expression. The clinical relevance of transcription factors (TFs) other than TTF-1 remains unknown in LAD and was explored in the present study.Seventy-one LAD samples were subjected to high-throughput transcriptome screening of LAD using cap analysis gene expression (CAGE) sequencing data; CAGE provides genome-wide expression levels of the transcription start sites (TSSs). In total, 1083 invasive LAD samples were subjected to immunohistochemical examination for paired box 9 (PAX9) and TTF-1 expression levels. PAX9 is an endoderm development-associated TF that most strongly and inversely correlates with the expression of TTF-1 TSS subsets. Immunohistochemically, PAX9 expression was restricted to the nuclei of ciliated epithelial and basal cells in the bronchi and bronchioles and the nuclei of epithelial cells of the bronchial glands; moreover, PAX9 expression was observed in 304 LADs (28%). PAX9-positive LADs were significantly associated with heavy smoking, non-lepidic subtype, EGFR wild-type tumours and PD-L1 expression (all P 0.0001). All these characteristics were opposite to those of TRU-type LADs with TTF-1 expression. PAX9 expression was an independent prognostic factor for decreased overall survival (P = 0.022).Our results revealed that PAX9 expression defines an aggressive subset of LADs preferentially occurring in smokers that may arise from bronchial or bronchiolar cells.

ALK, ROS1, and RET kinase fusions are important predictive biomarkers of tyrosine kinase inhibitors (TKIs) in non‐small‐cell lung cancer (NSCLC). Analysis of cell‐free DNA (cfDNA) provides a noninvasive method to identify gene changes in tumor cells. The present study sought to use cfRNA and cfDNA for identifying fusion genes. A reliable protocol was established to detect fusion genes using cfRNA and assessed the analytical validity and clinical usefulness in 30 samples from 20 cases of fusion‐positive NSCLC. The results of cfRNA‐based assays were compared with tissue biopsy and cfDNA‐based liquid biopsy (Guardant360 plasma next‐generation sequencing [NGS] assay). The overall sensitivity of the cfRNA‐based assay was 26.7% (8/30) and that of cfDNA‐based assay was 16.7% (3/18). When analysis was limited to the samples collected at chemo‐naïve or progressive disease status and available for both assays, the sensitivity of the cfRNA‐based assay was 77.8% (7/9) and that of cfDNA‐based assay was 33.3% (3/9). Fusion gene identification in cfRNA was correlated with treatment response. These results suggest that the proposed cfRNA assay is a useful diagnostic test for patients with insufficient tissues to facilitate effective administration of first‐line treatment and is a useful tool to monitor the progression of NSCLC for consideration of second‐line treatments., cfRNA‐ and cfDNA‐based assays are evaluated in 20 cases of fusion‐positive NSCLC. cfRNA assay was superior to cfDNA assay for the detection of gene fusions. The results of the cfRNA assay were consistent with the therapeutic effect.

Various genetic alterations of the fibroblast growth factor receptor (FGFR) family have been detected across a wide range of cancers. However, inhibition of FGFR signaling by kinase inhibitors demonstrated limited clinical effectiveness. Herein, we evaluated the transforming activity and sensitivity of 160 nonsynonymous FGFR mutations and ten fusion genes to seven FGFR tyrosine kinase inhibitors (TKI) using the mixed-all-nominated-in-one (MANO) method, a high-throughput functional assay. The oncogenicity of 71 mutants was newly discovered in this study. The FGFR TKIs showed anti-proliferative activities against the wild-type FGFRs and their fusions, while several hotspot mutants were relatively resistant to those TKIs. The drug sensitivities assessed with the MANO method were well concordant with those evaluated using in vitro and in vivo assays. Comprehensive analysis of published FGFR structures revealed a possible mechanism through which oncogenic FGFR mutations reduce sensitivity to TKIs. It was further revealed that recurrent compound mutations within FGFRs affect the transforming potential and TKI-sensitivity of corresponding kinases. In conclusion, our study suggests the importance of selecting suitable inhibitors against individual FGFR variants. Moreover, it reveals the necessity to develop next-generation FGFR inhibitors, which are effective against all oncogenic FGFR variants.

Extremely low alanine aminotransferase (ALT) may reflect aging, frailty, sarcopenia, and malnutrition in several cardiovascular diseases, but the association between low ALT and patient characteristics, cardiovascular and all-cause mortality is not well investigated in the population with atrial fibrillation. We conducted a post hoc analysis of a prospective, observational multicenter study. Patients with nonvalvular AF in the SAKURA AF Registry (n = 3156) were classified into 3 tertiles according to baseline ALT: first (ALT ≤ 15 U/L, n = 1098), second (15 P P P

Background Zinc‐finger E‐box‐binding homeobox 1 (ZEB1) is an important regulator of epithelial‐mesenchymal transition (EMT) and is involved in the maintenance of cancer stem cells (CSCs) via miR‐200c and BMI1 pathway. Recent studies revealed that ZEB1 contributes to the EMT‐mediated acquired resistance to gefitinib in EGFR‐mutant non‐small cell lung cancer (NSCLC). However, the precise role of ZEB1 in the maintenance of lung CSCs that lead to acquired resistance to gefitinib remains unclear. Methods PC9 and HCC827 NSCLC cell lines were treated with high concentrations of gefitinib, and surviving cells were referred to as “gefitinib‐resistant persisters” (GRPs). ZEB1 knockdown or overexpression was performed to determine the biological significance of ZEB1 in the CSC features of GRPs, and animal models were studied for in vivo validation. Expression of ZEB1, BMI1, and ALDH1A1 was analyzed by immunohistochemistry in tumor specimens from NSCLC patients with acquired resistance to gefitinib. Results GRPs had characteristic features of mesenchymal and CSC phenotypes with high expression of ZEB1 and BMI1, and decreased miR‐200c, in vitro and in vivo. ZEB1 silencing attenuated the suppression of miR‐200c, resulting in the reduction in BMI1 and reversed the mesenchymal and CSC features of GRPs. Furthermore, ZEB1 overexpression induced EMT and increased the levels of CD133‐ and BMI1‐positive GRPs in vitro and gefitinib resistance in vivo. Finally, ZEB1, BMI1, and ALDH1A1 were highly expressed in tumor specimens from EGFR‐mutant NSCLC patients with gefitinib resistance. Conclusions ZEB1 plays an important role in gefitinib‐resistant lung CSCs with EMT features via regulation of miR‐200c and BMI1., Gefitinib‐resistant persisters (GRPs) of EGFR‐mutant NSCLC cells have characteristic features of mesenchymal and CSC phenotypes. ZEB1 is highly expressed in GRPs and involved in the maintenance of lung CSCs resistant to gefitinib via miR‐200c‐BMI1 pathway.

Liquid biopsy analyzes the current status of primary tumors and their metastatic regions. We aimed to develop an optimized protocol for single‐cell sequencing of floating tumor cells (FTCs) in pleural effusion as a laboratory test. FTCs were enriched using a negative selection of white blood cells by a magnetic‐activated cell sorting system, and CD45‐negative and cytokeratin‐positive selection using a microfluidic cell separation system with a dielectrophoretic array. The enriched tumor cells were subjected to whole‐genome amplification (WGA) followed by genome sequencing. The FTC analysis detected an EGFR exon 19 deletion in Case 1 (12/19 cells, 63.2%), and EML4‐ALK fusion (17/20 cells, 85%) with an alectinib‐resistant mutation of ALK (p.G1202R) in Case 2. To eliminate WGA‐associated errors and increase the uniformity of the WGA product, the protocol was revised to sequence multiple single FTCs individually. An analytical pipeline, accurate single‐cell mutation detector (ASMD), was developed to identify somatic mutations of FTCs. The large numbers of WGA‐associated errors were cleaned up, and the somatic mutations detected in FTCs by ASMD were concordant with those found in tissue specimens. This protocol is applicable to circulating tumor cells analysis of peripheral blood and expands the possibility of utilizing molecular profiling of cancers., An optimized protocol is established for single‐cell sequencing of floating tumor cells (FTCs) in pleural effusion as a laboratory test that could be utilized for patient care. The FTC analysis detected an EGFR exon 19 deletion in Case 1 (12/19 cells, 63.2%) and EML4‐ALK fusion in Case 2 (17/20 cells, 85%). A novel analytical pipeline, accurate single‐cell mutation detector (ASMD), was developed to identify somatic mutations in single cells accurately.

Pulmonary emphysema is predominantly caused by chronic exposure to cigarette smoke (CS). Novel tobacco substitutes, such as heated tobacco products (HTPs), have emerged as healthier alternatives to cigarettes. IQOS, the most popular HTP in Japan, is advertised as harmless compared with conventional cigarettes. Although some studies have reported its toxicity, few in vivo studies have been conducted. Here, 12-wk-old C57BL6/J male mice were divided into three groups and exposed to air (as control), IQOS aerosol, or CS for 6 mo. After exposure, the weight gain was significantly suppressed in the IQOS and CS groups compared with the control (−4.93 g; IQOS vs. air and −5.504 g; CS vs. air). The serum cotinine level was significantly higher in the IQOS group than in the control group. The neutrophils and lymphocyte count increased in the bronchoalveolar lavage fluid of the IQOS and CS groups compared with those in the control group. Chronic IQOS exposure induced pulmonary emphysema similar to that observed in the CS group. Furthermore, expression levels of the genes involved in the apoptosis-related pathways were significantly upregulated in the lungs of the IQOS-exposed mice. Cytochrome c, cleaved caspase-3, and cleaved poly (ADP-ribose) polymerase-1 were overexpressed in the IQOS group compared with the control. Single-stranded DNA and TdT-mediated dUTP nick-end labeling-positive alveolar septal cell count significantly increased in the IQOS group compared with the control. In conclusion, chronic exposure to IQOS aerosol induces pulmonary emphysema predominantly via apoptosis-related pathways. This suggests that HTPs are not completely safe tobacco products.

Cancer cachexia is associated with poor immunotherapeutic outcomes. This prospective observational study longitudinally evaluated the role of cachexia-related circulating cytokines in predicting the risk and benefit of PD-1/PD-L1 blockade in advanced lung cancer. Forty-one circulating cytokines at baseline and after one cycle of PD-1/PD-L1 blockade treatment were measured in patients with advanced lung cancer between 2019 and 2020. The cachexia-related cytokines were identified by comparing the levels of circulating cytokines between cachectic and non-cachectic patients. Among 55 patients, 49.1% were diagnosed with cachexia at the beginning of PD-1/PD-L1 blockade therapy. Baseline levels of the circulating cytokines IL-6, IL-8, IL-10, IL-15, and IP-10 were significantly higher in cachectic patients. In contrast, the level of eotaxin-1 was lower in cachectic patients than in those without cachexia. Higher IL-6 at baseline and during treatment was associated with a greater risk of immune-related adverse events, while higher IL-10 at baseline was linked to worse overall survival. More importantly, increased eotaxin-1 after one cycle of PD-1/PD-L1 blockade treatment was associated with higher objective response and better overall survival. A blood-based, cachexia-related cytokine assay may yield potential biomarkers for the early prediction of clinical response to PD-1/PD-L1 blockade and provide clues for improving the outcomes of cachectic patients.

Purpose Idiopathic pulmonary fibrosis (IPF) is characterized by the accumulation of extracellular matrix (ECM) protein in the lungs. Transforming growth factor (TGF) β-induced ECM protein synthesis contributes to the development of IPF. Tranilast, an anti-allergy drug, suppresses TGFβ expression and inhibits interstitial renal fibrosis in animal models. However, the beneficial effects of tranilast or its mechanism as a therapy for pulmonary fibrosis have not been clarified. Methods We investigated the in vitro effect of tranilast on ECM production and TGFβ/SMAD2 pathway in TGFβ2-stimulated A549 human alveolar epithelial cells, using quantitative polymerase chain reaction, Western blotting, and immunofluorescence. In vitro observations were validated in the lungs of a murine pulmonary fibrosis model, which we developed by intravenous injection of bleomycin. Results Treatment with tranilast suppressed the expression of ECM proteins, such as fibronectin and type IV collagen, and attenuated SMAD2 phosphorylation in TGFβ2-stimulated A549 cells. In addition, based on a wound healing assay in these cells, tranilast significantly inhibited cell motility, with foci formation that comprised of ECM proteins. Histological analyses revealed that the administration of tranilast significantly attenuated lung fibrosis in mice. Furthermore, tranilast treatment significantly reduced levels of TGFβ, collagen, fibronectin, and phosphorylated SMAD2 in pulmonary fibrotic tissues in mice. Conclusion These findings suggest that tranilast inhibits pulmonary fibrosis by suppressing TGFβ/SMAD2-mediated ECM protein production, presenting tranilast as a promising and novel anti-fibrotic agent for the treatment of IPF.

Programmed death ligand 1 and 2 (PD-L1 and PD-L2) bind programmed death 1 (PD-1). PD-L1 is an established predictive biomarker of response to immunotherapies targeting PD-1 and PD-L1 in lung adenocarcinoma (LUAD). However, the clinical relevance of PD-L2 expression in patients with LUAD remains unclear; we aimed to examine this aspect using LUAD specimens.PD-L2 expression status was immunohistochemically evaluated in 980 surgically resected LUAD specimens. PD-L2 expression status was classified based on the tumor proportion score (TPS) as negative (1%), weakly positive (1-49%), or strongly positive (≥50%). Correlations between PD-L2 and PD-L1 expression status, clinicopathological features, driver oncogene alterations (EGFR, KRAS, ALK, ROS1, and RET), and prognosis were also analyzed.PD-L2 expression was negative in 720 (73%) of 980 LUADs, weakly positive in 190 (19%), and strongly positive in 70 (7%). The concordance rate between PD-L1 and PD-L2 expression was 60%. Male sex, smokers, tumors 3 cm in size, high-grade tumors, tumors without EGFR mutation or ALK fusion, and tumors with KRAS mutation were more common in patients with PD-L2-positive tumors (TPS ≥ 1%) than in patients with PD-L2-negative tumors (TPS 1%). PD-L2 expression was not associated with overall survival (OS) or relapse-free survival (RFS). However, positive PD-L2 expression tended to be associated with better OS/RFS in PD-L1-positive patients and worse OS/RFS in PD-L1-negative patients.PD-L2-positive LUADs showed biologically aggressive characteristics. PD-L2 expression status was not associated with survival outcomes, but tended to show contrasting prognostic impacts based on PD-L1 expression status.

Summary The extensive clinical applications of next-generation sequence analyzers have made uncommon and compound EGFR mutations more prevalent than previously described. However, clinicopathological impacts of compound EGFR mutations in lung adenocarcinoma remain unclear. We earlier examined the presence of compound EGFR mutations primarily in the cis allele by EGFR exon sequencing and droplet digital polymerase chain reaction in 462 completely resected EGFR-mutated adenocarcinomas of the lung and identified 64 tumors with compound mutations. We evaluated clinicopathological characteristics of lung adenocarcinomas with compound EGFR mutations in comparison with cases with common or uncommon single mutations. Among 64 compound EGFR mutations, L858R/E709G (9%) was the most frequent mutation type, followed by L858R/S768I (8%), L858R/T790M (8%), and L858R/L833V (6%). We observed both single and compound mutations frequently in women, never or light smokers; their adenocarcinomas showed thyroid transcription factor-1 immunoreactivity. In contrast, compound mutations were significantly associated with lymph node metastases (p = 0.0242; single vs. compound cases) and the presence of tumor cells with clear cytoplasm (p = 0.0014; single vs. compound cases). Furthermore, patients with compound mutations had significantly poorer prognoses than cases with single EGFR mutations (p = 0.043). Overall, clinicopathological features of common, uncommon, and compound EGFR mutations are similar; however, tumors with compound mutations may be characterized by aggressive behavior and histological findings of clear cell features.

Background In the Japanese clinical setting, the prevalence, potential cofounders of gastrointestinal (GI) bleeding from anticoagulant therapy, including direct oral anticoagulants (DOACs) and warfarin, and prognosis after GI bleeding are unclear.Methods and Results:We examined about GI bleeding from anticoagulant therapy using data from the SAKURA AF Registry, a prospective multicenter registry in Japan. Among 3,237 enrollees, 48.8% (n=1,561) were warfarin users and 51.2% (n=1,676) DOAC users. GI bleeding was identified in 68 patients (2.1%). No incidental differences in GI bleeding were observed between the DOAC and warfarin users (32 [1.9%] patients [0.67 events per 100 person-years] vs. 36 [2.3%] patients [0.75 events per 100 person-years], respectively; P=0.43). Multivariate Cox proportional hazard model analysis revealed that creatinine (hazard ratio [HR] 1.379, 95% confidence interval [CI] 1.091-1.743 P=0.007) and hemoglobin (HR 0.814, 95% CI 0.705-0.941, P=0.005) remained independent determinants of GI bleeding. Patients experiencing GI bleeding events had a higher risk of all-cause death (18%) than those without GI bleeding (6%) (P=0.045). Conclusions GI bleeding was strongly associated with anemia and renal impairment. Patients experiencing GI bleeding had higher risk for all-cause death than those without GI bleeding.

Atrial fibrillation (AF) and heart failure (HF) often coexist. The aims of this study were to explore the factors associated with the serum levels of N-terminal pro-brain natriuretic peptide (NT-proBNP), and the association between prognosis and a history of HF or the serum NT-proBNP level in Japanese patients with AF.The present sub-study was based on the SAKURA AF Registry, a Japanese multicenter observational registry that included 3267 AF patients (median follow-up period: 39 months). All the patients were receiving warfarin or any of four direct oral anticoagulants. Serum NT-proBNP levels were available for 2417 patients, and the median value was 508 (interquartile range 202-1095) pg/mL at the time of enrollment. Log NT-proBNP was associated with non-paroxysmal AF, creatinine clearance > 60 mL/minute, history of HF and ischemic heart disease, antiarrhythmic drug use, anemia, being elderly female, and history of AF ablation. The relative risk of adverse clinical events, except major bleeding, was significantly higher in the highest NT-proBNP quartile as compared to the lowest quartile (adjusted hazard ratios: 2.87 for death, 2.39 for stroke), but a history of HF was associated only with a higher incidence of all-cause death.Concomitant HF was associated with a higher mortality, but the high NT-proBNP was associated with higher mortality and stroke events. In Japanese AF patients receiving anticoagulant treatment, high serum NT-proBNP levels predict the risk for both stroke events and deaths, and intensive follow-up is needed in such patients.

There is minimal evidence of EGFR-mutated lung adenocarcinoma transforming to small cell lung carcinoma (SCLC) without the administration of EGFR-tyrosine kinase inhibitor (TKI). Here, we present a case of EGFR/PTEN co-mutated lung adenocarcinoma with lymph node metastases, which comprised adenocarcinoma admixed with SCLC. EGFR L858R and PTEN R130Q mutations were shared between the primary tumor and lymph node metastasis. Additionally, EGFR I744M mutation was shared between the adenocarcinoma and SCLC components in the lymph node metastasis, confirming spontaneous transformation from adenocarcinoma to SCLC. Furthermore, TP53 and RB1 mutations were detected only in the SCLC components of the lymph node metastasis. Immunohistochemically, complete absence of Rb expression in SCLC was observed, suggesting the loss of function of RB1. Our case clearly shows that EGFR/PTEN co-mutated lung adenocarcinoma transformed to SCLC in the lymph node without TKI-mediated evolutionary selection pressures.

Background Osimertinib (AZD9291) is a third-generation EGFR-tyrosine kinase inhibitor (TKI) that selectively inhibits the activating EGFR mutation and T790M mutation, and is currently used globally to treat EGFR-mutant non-small cell lung cancer (NSCLC). However, acquired resistance to osimertinib is inevitable. Methods We established osimertinib-resistant cells (PC9/T790M/AZDR and H1975/AZDR) derived from EGFR-mutant NSCLC cells harboring T790M mutation, and investigated the mechanism of acquired resistance to osimertinib by whole-exome sequencing and multiple phospho-receptor tyrosine kinase (RTK) array. A tumor specimen from an EGFR-mutant NSCLC patient with acquired resistance to osimertinib was also subjected to immunohistochemical analysis. Results Whole-exome sequencing analysis demonstrated that genetic alterations, such as acquisition of EGFR C797S, loss of T790M mutation, MET amplification, or mutated KRAS, MEK, BRAF, PIK3CA, were not detected. Analysis of phospho-RTK array revealed that insulin-like growth factor-1 receptor (IGF1R) was activated in PC9/T790M/AZDR and H1975/AZDR cells. Knockdown of IGF1R by siRNA as well as inhibition of IGF1R activation by linstinib (IGF1R inhibitor) significantly restored the sensitivity to osimertinib. Immunohistochemical analysis revealed that the expression level of phosphorylated IGF1R was higher in the tumor specimen from the EGFR-mutant NSCLC patient with acquired resistance to osimertinib than in the specimen collected prior to the treatment. Conclusions IGF1R activation could occur following treatment with osimertinib in EGFR-mutant NSCLC with T790M mutation, and might be one of the mechanisms underlying osimertinib resistance. Combined treatment of osimertinib and IGF1R inhibitor might be effective in overcoming the acquired resistance to osimertinib induced by IGF1R activation. Key points Significant findings of the study: Using osimertinib-resistant cells, we found that IGF1R activation induced by osimertinib treatment in EGFR-mutant NSCLC with T790M mutation is involved in resistance. Increased phosphorylation of IGF1R was observed in the tumor specimen from an EGFR-mutant NSCLC patient with acquired osimertinib resistance. What this study adds IGF1R activation might be one of the mechanisms of osimertinib resistance. A combination therapy with osimertinib and an IGF1R inhibitor might be an optimal approach for overcoming the acquired resistance to osimertinib induced by IGF1R activation.

Background Relations between characteristics and outcomes of patients in Japan with atrial fibrillation (AF) and the type of medical facility providing their outpatient care are unclear. Methods and results We compared patient characteristics and outcomes between 2 university hospitals (n = 1178), 20 general hospitals (n = 1308), and 41 private clinics (n = 751) (follow-up: 39.3 months) in the prospective SAKURA AF Registry. Private clinic patients were significantly older than university hospital and general hospital patients (73.4 ± 9.2 vs. 70.3 ± 9.8 and 72.6 ± 8.9 years; p Conclusions Adverse clinical events at small to large hospitals appear to be higher than those at private clinics, suggesting that careful attention for preventing stroke/SE and cardiovascular events should be paid to patients at a university or general hospital.

Background Immunodeficient patients are recommended to receive pneumococcal vaccination. However, there is limited evidence showing effectiveness of the polysaccharide vaccine. Polysaccharide vaccination has shown an association with cardiovascular event risk reduction. We assessed the efficacy of the 23-valent pneumococcal polysaccharide vaccine (PPSV23) in relation to the risk of hospitalization and death due to pneumonia and acute cardiac events. Methods The medical records of all dialysis patients attending our 8 study centers in 2010 were studied, and we selected 1038 consecutive patients. One-to-one propensity score matching was used to correct for potential selection bias in a PPSV23-vaccinated group versus a non-vaccinated group, and a total of 510 patients were identified for outcome analysis. Time to first admission, or deaths due to all-cause pneumonia or cardiac events until 2015 were compared between both groups. Results The all-cause death rate was significantly decreased in the PPSV23-vaccinated group, (hazard ratio [HR] 0.62, 95% confidence interval [CI]; 0.46–0.83, P = 0.002). All-cause death was considered to be a competing risk for the other outcomes. Further outcomes were evaluated by competing risk analysis adjusting for mortality. There was no statistically significant difference in the hospitalization rate for pneumonia; however, the hospitalization rate due to cardiac events was significantly lower in the PPSV23-vaccinated group than in the non-vaccinated group (HR 0.44, 95% CI; 0.20–0.96, P = 0.040). There was no statistically significant difference in the death rate due to pneumonia; however, the rate of cardiac death was significantly lower in the PPSV23-vaccinated group than in the non-vaccinated group (HR 0.36, 95% CI; 0.18–0.71, P = 0.003). Conclusions The PPSV23 vaccination is associated with a good prognosis and a low-risk of cardiac events in dialysis patients; however, there was no evidence indicating enhanced protective efficacy against pneumonia, suggesting the PPSV23 vaccination might improve the prognosis by directly preventing cardiovascular events.

Background The prognosis of patients with NSCLC harboring oncogenic driver gene alterations, such as EGFR gene mutations or ALK fusion, has improved dramatically with the advent of corresponding molecularly targeted drugs. As patients were followed up for about five years in most clinical trials, the long-term outcomes beyond 5 years are unclear. The objectives of this study are to explore the clinical course beyond five years of chemotherapy initiation and to investigate factors that lead to long-term survival. Methods One hundred and seventy-seven patients with advanced, EGFR-mutated or ALK-rearranged NSCLC who received their first chemotherapy between December 2008 and September 2015 were included. Kaplan Meier curves were drawn for the total cohort and according to subgroups of patients’ characteristics. Results Median OS in the total cohort was 40.6 months, the one-year survival rate was 89%, the three-year survival rate was 54%, and the five-year survival rate was 28%. Median OS was 36.9 months in EGFR-mutated patients and 55.4 months in ALK-rearranged patients. The OS curve seemed to plateau after 72 months, and most of the patients who were still alive after more than five years are on treatment. Female sex, age under 75 years, an ECOG PS of 0 to 1, ALK rearrangement, postoperative recurrence, and presence of brain metastasis were significantly associated with longer OS. Conclusions A tail plateau was found in the survival curves of patients with advanced, EGFR-mutated and ALK-rearranged NSCLC, but most were on treatment, especially with EGFR-mutated NSCLC.

Small cell lung cancer (SCLC) is a type of high-grade neuroendocrine carcinoma. It initially responds to chemotherapy but rapidly becomes chemoresistant and it is highly proliferative. The prognosis in SCLC is poor. We have established a novel SCLC cell line, SCLC-J1, from a malignant pleural effusion in a patient with advanced SCLC. SCLC-J1 cells express ganglioside GD2, CD276, and Delta-like protein 3. RB1 is lost. These features of the new SCLC cell line may be useful in understanding the cellular and molecular biology of SCLC and in designing better treatment., Highlight • A novel small lung cancer cell line, SCLC-J1, was successfully established. • SCLC-J1 cells express the tumor-specific antigens ganglioside GD2, CD276, and Delta-like protein 3. RB1 is lost. • SCLC-J1 will provide insights into SCLC biology that may permit better therapeutic targeting.

Programmed death-ligand 1 (PD-L1) expression is a predictive biomarker of response to immunotherapies targeting programmed death-1/PD-L1 in advanced-stage lung adenocarcinoma. The aim of this study was to investigate the associations between PD-L1 expression and clinicopathological features, prognosis, and driver oncogene alterations in patients with lung adenocarcinoma.We evaluated PD-L1 expression in 1,005 surgically resected lung adenocarcinoma specimens, by immunohistochemistry using the 22C3 antibody. PD-L1 positivity was defined based on the proportion of stained tumor cells (TPS) on tissue microarrays:1% (negative), 1-49% (weakly positive), and ≥ 50% (strongly positive). Correlations between PD-L1 expression and clinicopathological features, prognosis, and driver oncogene (EGFR, KRAS, ALK, ROS1, and RET) alterations in lung adenocarcinoma were analyzed.PD-L1 expression was negative in 726 (72%) of 1,005 tumors, weakly positive in 161 (16%), and strongly positive in 118 (12%). Male sex, smoking, elevated serum carcinoembryonic antigen levels, advanced pathological stages, high-grade tumors, predominantly solid tumors, tumors with lymphatic permeation or vascular or pleural invasion, tumors without EGFR mutations, and tumors with KRAS mutations were more common in patients with PD-L1-positive tumors (TPS ≥ 1%) than in those with PD-L1-negative tumors (TPS 1%). PD-L1 positivity was not associated with ALK, ROS1, or RET fusion status. Although PD-L1 positivity was associated with poor overall survival and poor relapse-free survival in all patients, this was not statistically significant after adjusting for prognostic factors in the multivariate analysis. In the subgroup analysis according to driver oncogene alterations, PD-L1 positivity was associated with poor relapse-free survival only in patients with EGFR-mutated tumors.Surgically resected lung adenocarcinomas with increased PD-L1 expression were biologically aggressive tumors that frequently occurred in male smokers. PD-L1 expression and its prognostic significance differed according to driver oncogene alterations.

Nausea and diarrhea are the most common adverse effects of nintedanib in patients with idiopathic pulmonary fibrosis (IPF). However, the clinical risk factors for these side effects remain unknown. In the present study, we investigated the characteristics of patients who developed gastrointestinal side effects during nintedanib treatment for IPF and determined the risk factors for these side effects. We enrolled 77 patients with IPF who received nintedanib between October 2015 and March 2018. Performance status (PS) as a patient’s general condition, body mass index (BMI), modified Medical Research Council Dyspnea Scale score, severity of IPF at nintedanib initiation, and gastrointestinal toxicity of nintedanib were evaluated. In total, 25 and 27 patients exhibited nausea and diarrhea, respectively, during the follow-up period. A poor PS, low BMI, and full dosage of nintedanib at treatment initiation were risk factors for nausea. A low BMI was a significant risk factor for diarrhea, which could be prevented by combination treatment with nintedanib and prednisolone. In addition, the mean annual rate of decline in forced vital capacity was significantly greater in patients with nausea than in patients without nausea. In conclusion, our findings suggest that patients with a low BMI and/or poor PS and those who receive the full nintedanib dosage at treatment initiation are more susceptible to gastrointestinal adverse effects during nintedanib treatment. Addition of prednisolone to the treatment regimen may prevent the development of diarrhea during treatment.

Tumor molecular profiling is becoming a standard of care for patients with cancer, but the optimal platform for cancer sequencing remains undetermined. We established a comprehensive assay, the Todai OncoPanel (TOP), which consists of DNA and RNA hybridization capture-based next-generation sequencing panels. A novel method for target enrichment, named the junction capture method, was developed for the RNA panel to accurately and cost-effectively detect 365 fusion genes as well as aberrantly spliced transcripts. The TOP RNA panel can also measure the expression profiles of an additional 109 genes. The TOP DNA panel was developed to detect single nucleotide variants and insertions/deletions for 464 genes, to calculate tumor mutation burden and microsatellite instability status, and to infer chromosomal copy number. Clinically relevant somatic mutations were identified in 32.2% (59/183) of patients by prospective TOP testing, signifying the clinical utility of TOP for providing personalized medicine to cancer patients.

The discovery of oncogenic driver gene mutations, including epidermal growth factor receptor (EGFR) mutation, anaplastic lymphoma kinase (ALK) fusion, ROS proto-oncogene 1 (ROS1) fusion, and ret proto-oncogene (RET) fusion, has led to the development of molecularly targeted therapy for non-small-cell lung cancer (NSCLC). This therapy has changed the standard of care for NSCLC. Despite the dramatic response to molecularly targeted therapy, almost all patients ultimately develop resistance to the drugs. To understand the mechanisms of resistance to molecularly targeted agents, it is essential to understand the molecular pathways of NSCLC. Here, we review the mechanisms of resistance to molecularly targeted therapy and discuss strategies to overcome drug resistance.

Synovial sarcoma (SS) is a malignant soft tissue lesion and most commonly arises in young adults. Chromosomal translocation t(X;18)(p11;q11) results in the formation of SS18/SSX by gene fusion of the SS18 gene on chromosome 18 to either SSX1, SSX2, or SSX4 gene located on chromosome X, which is detected in more than 95% of SSs. Although multiple lines of evidence suggest that the SS18/SSX fusion is the oncogene in this tumor, the protein expression profiles associated with SS18/SSX have yet to be elucidated. In this study, we conducted proteomic studies using SS18/SSX knockdown in three SS cell lines to identify the regulated proteins associated with SS18/SSX in SS. Isobaric tags for relative and absolute quantitation (i-TRAQ) analyses identified approximate 1700–2,000 proteins regulated by the SS18/SSX fusion in each SS cell line. We also analyzed the three profiles to identify proteins that were similarly altered in all 3 cell lines and found 17 consistently upregulated and 18 consistently downregulated proteins, including TAGLN and ACTN4. In addition, network analyses identified several critical pathways including RUNX2 and SMARCA4. RUNX2 and SMARCA4 had the highest ranking in these identified pathways. In addition, we found that expression of TAGLN inhibited cell viability in SS cell lines. Our data suggest that the differentiation and cell growth of SS may be enhanced by the identified proteins induced by SS18/SSX. We believe that the findings obtained in the present functional analyses will help to improve our understanding of the relationship between SS18/SSX and malignant behavior in SS.

Autocrine and paracrine factors, including glutamate and epidermal growth factor (EGF), are potent inducers of brain tumor cell invasion, a pathological hallmark of malignant gliomas. System xc(-) consists of xCT and CD98hc subunits and functions as a plasma membrane antiporter for the uptake of extracellular cystine in exchange for intracellular glutamate. We previously showed that the EGF receptor (EGFR) interacts with xCT and thereby promotes the activity of system xc(-) in a kinase-independent manner, resulting in enhanced glutamate release in glioma cells. However, the molecular mechanism underlying EGFR-mediated glioma progression in a glutamate-rich microenvironment has remained unclear. Here we show that the GluN2B subunit of the N-methyl-d-aspartate-sensitive glutamate receptor (NMDAR) is a substrate of EGFR in glioma cells. In response to EGF stimulation, EGFR phosphorylated the COOH-terminal domain of GluN2B and thereby enhanced glutamate-NMDAR signaling and consequent cell migration in EGFR-overexpressing glioma cells. Treatment with the NMDAR inhibitor MK-801 or the system xc(-) inhibitor sulfasalazine suppressed EGF-elicited glioma cell migration. The administration of sulfasalazine and MK-801 also synergistically suppressed the growth of subcutaneous tumors formed by EGFR-overexpressing glioma cells. Furthermore, shRNA-mediated knockdown of xCT and GluN2B cooperatively prolonged the survival of mice injected intracerebrally with such glioma cells. Our findings thus establish a central role for EGFR in the signaling crosstalk between xCT and GluN2B-containing NMDAR in glioma cells.

Malignant pleural mesothelioma (MPM) is a highly aggressive tumor that has a low overall survival; however, no significant treatment advances have been made in the past 15 years. Large-scale molecular studies have identified a poor prognostic subset of MPM linked to the epithelial–mesenchymal transition (EMT) that may contribute toward resistance to chemotherapy, suggesting that EMT could be targeted to treat patients with MPM. Previously, we reported that histone modifiers regulating EMT could be therapeutic targets; therefore, in this study, we investigated whether targeting lysine-specific demethylase 1 (LSD1/KDM1), a histone-modifying enzyme responsible for demethylating histone H3 lysine 4 and lysine 9, could represent a novel therapeutic strategy for MPM. We suppressed LSD1 and investigated the EMT phenotype using EMT marker expression and wound-healing assay; and chemosensitivity using apoptosis assay. We found that suppressing LSD1 induces an epithelial phenotype in sarcomatoid MPM cells, while attenuating the mesenchymal phenotype sensitized MPM cells to cisplatin-induced apoptosis. Subsequent genome-wide identification, comprehensive microarray analysis, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) to assess genome-wide changes in chromatin accessibility suggested that LSD1 directly regulates milk fat globulin protein E8 (MFGE8), an integrin ligand that is involved in the FAK pathway. Furthermore, we found that LSD1 regulates the mesenchymal phenotype and apoptosis by activating the FAK–AKT–GSK3β pathway via a positive feedback loop involving MFGE8 and Snail expression, thereby leading to cisplatin resistance. Implications: This study suggests that LSD1 regulates the mesenchymal phenotype and apoptosis, and that LSD1 inhibitors could be combined with the cisplatin as a novel therapy for patients with MPM.

Aims In the evolving era of precision medicine, increasing emphasis is placed on detecting molecular alterations driving the development of specific cancers and targeting them with matched therapies that can yield the best outcomes for patients. Lung adenocarcinomas with uncommon actionable alterations, including MET exon 14 skipping (METex14), ERBB2 and BRAF mutations, are rare and poorly characterised cancers. Methods and results To more clearly understand the histopathological features of lung adenocarcinoma with uncommon actionable alterations, we compared the histological features of 678 cases with mitogenic driver alterations from 996 surgically resected lung adenocarcinomas. Genomic data from our cohort revealed METex14, ERBB2 and BRAF mutations in 13, 16 and 15 cases, respectively. Patients who had lung adenocarcinoma with METex14 were often elderly females. Histological features such as clear cell features (23%), hyaline globules (31%) and nuclear pleomorphism (39%) were the most frequently identified in METex14-positive cases; among those, three cases (23%) had tumour cells with bizarre giant or multilobulated nuclei. Additionally, the micropapillary pattern was the most frequently identified in ERBB2-mutated lung adenocarcinoma (31%). Lung adenocarcinoma with BRAF mutations tended to be less invasive, and the BRAF V600E mutation was identified in only one case with lepidic adenocarcinoma. Immunohistochemically, all METex14, ERBB2 and BRAF-positive tumours, except for invasive mucinous adenocarcinoma, were positive for thyroid transcription factor 1 (TTF-1). Conclusions Our data from Japanese patients showed that lung adenocarcinoma with METex14 had unique clinicopathological characteristics: tumour cells with marked nuclear pleomorphism, hyaline globules and expression of TTF-1 in elderly women who never or lightly smoked.

There are multiple transcription start sites (TSSs) in agreement with multiple transcript variants encoding different isoforms of NKX2‐1/TTF‐1 (thyroid transcription factor 1); however, the clinicopathological significance of each transcript isoform of NKX2‐1/TTF‐1 in lung adenocarcinoma (LAD) is unknown. Herein, TSS‐level expression of NKX2‐1/TTF‐1 isoforms was evaluated in 71 LADs using bioinformatic analysis of cap analysis of gene expression (CAGE)‐sequencing data, which provides genome‐wide expression levels of the 5′‐untranslated regions and the TSSs of different isoforms. Results of CAGE were further validated in 664 LADs using in situ hybridisation. Fourteen of 17 TSSs in NKX2‐1/TTF‐1 (80% of known TSSs in FANTOM5, an atlas of mammalian promoters) were identified in LADs, including TSSs 1–13 and 15; four isoforms of NKX2‐1/TTF‐1 transcripts (NKX2‐1_001, NKX2‐1_002, NKX2‐1_004, and NKX2‐1_005) were expressed in LADs, although NKX2‐1_005 did not contain a homeodomain. Among those, six TSSs regulated NKX2‐1_004 and NKX2‐1_005, both of which contain exon 1. LADs with low expression of isoforms from TSS region 11 regulating exon 1 were significantly associated with poor prognosis in the CAGE data set. In the validation set, 62 tumours (9.3%) showed no expression of NKX2‐1/TTF‐1 exon 1; such tumours were significantly associated with older age, EGFR wild‐type tumours, and poor prognosis. In contrast, 94 tumours, including 22 of 30 pulmonary invasive mucinous adenocarcinomas (IMAs) exhibited exon 1 expression without immunohistochemical TTF‐1 protein expression. Furthermore, IMAs commonly exhibited higher exon 1 expression relative to that of exon 4/5, which contained a homeodomain in comparison with EGFR‐mutated LADs. These transcriptome and clinicopathological results reveal that LAD use at least 80% of NKX2‐1 TSSs and expression of the NKX2‐1/TTF‐1 transcript isoform without exon 1 (NKX2‐1_004 and NKX2‐1_005) defines a distinct subset of LAD characterised by aggressive behaviour in elder patients. Moreover, usage of alternative TSSs regions regulating NKX2‐1_005 may occur in subsets of LADs.

Invasive mucinous adenocarcinoma (IMA) of the lung is a unique variant of lung adenocarcinoma. Aberrant mucin expression is associated with cancer development and metastasis. However, the clinicopathological significance of mucin expression in IMA is not fully understood. Herein, we evaluated the clinicopathological, immunohistochemical, and molecular characteristics of 70 IMA tumors. EGFR, KRAS, GNAS, and TP53 mutations were assessed by PCR-based sequencing. Next-generation sequencing was used to assess cases without EGFR/KRAS mutations. A NanoString-based screening for fusions was performed in all IMAs without mitogenic driver mutations. Expression of mucins (MUC1, MUC2, MUC4, MUC5AC, and MUC6) was evaluated by immunohistochemistry and categorized as follows: negative (

Purpose Idiopathic pulmonary fibrosis (IPF) is characterized by the accumulation of extracellular matrix (ECM) protein in the lungs. Transforming growth factor (TGF) β-induced ECM protein synthesis contributes to the development of IPF. Tranilast, an anti-allergy drug, suppresses TGFβ expression and inhibits interstitial renal fibrosis in animal models. However, the beneficial effects of tranilast or its mechanism as a therapy for pulmonary fibrosis have not been clarified. Methods We investigated the in vitro effect of tranilast on ECM production and TGFβ/SMAD2 pathway in TGFβ2-stimulated A549 human alveolar epithelial cells, using quantitative polymerase chain reaction, Western blotting, and immunofluorescence. In vitro observations were validated in the lungs of a murine pulmonary fibrosis model, which we developed by intravenous injection of bleomycin. Results Treatment with tranilast suppressed the expression of ECM proteins, such as fibronectin and type IV collagen, and attenuated SMAD2 phosphorylation in TGFβ2-stimulated A549 cells. In addition, based on a wound healing assay in these cells, tranilast significantly inhibited cell motility, with foci formation that comprised of ECM proteins. Histological analyses revealed that the administration of tranilast significantly attenuated lung fibrosis in mice. Furthermore, tranilast treatment significantly reduced levels of TGFβ, collagen, fibronectin, and phosphorylated SMAD2 in pulmonary fibrotic tissues in mice. Conclusion These findings suggest that tranilast inhibits pulmonary fibrosis by suppressing TGFβ/SMAD2-mediated ECM protein production, presenting tranilast as a promising and novel anti-fibrotic agent for the treatment of IPF.

Atrial fibrillation (AF) and heart failure (HF) often coexist. The aims of this study were to explore the factors associated with the serum levels of N-terminal pro-brain natriuretic peptide (NT-proBNP), and the association between prognosis and a history of HF or the serum NT-proBNP level in Japanese patients with AF.The present sub-study was based on the SAKURA AF Registry, a Japanese multicenter observational registry that included 3267 AF patients (median follow-up period: 39 months). All the patients were receiving warfarin or any of four direct oral anticoagulants. Serum NT-proBNP levels were available for 2417 patients, and the median value was 508 (interquartile range 202-1095) pg/mL at the time of enrollment. Log NT-proBNP was associated with non-paroxysmal AF, creatinine clearance60 mL/minute, history of HF and ischemic heart disease, antiarrhythmic drug use, anemia, being elderly female, and history of AF ablation. The relative risk of adverse clinical events, except major bleeding, was significantly higher in the highest NT-proBNP quartile as compared to the lowest quartile (adjusted hazard ratios: 2.87 for death, 2.39 for stroke), but a history of HF was associated only with a higher incidence of all-cause death.Concomitant HF was associated with a higher mortality, but the high NT-proBNP was associated with higher mortality and stroke events. In Japanese AF patients receiving anticoagulant treatment, high serum NT-proBNP levels predict the risk for both stroke events and deaths, and intensive follow-up is needed in such patients.

Background: Liver diseases drive the development and progression of atrial fibrillation (AF). The Fibrosis-4 (FIB4) index is a non-invasive scoring method for detecting liver fibrosis, but the prognostic impact of using it for AF patients is still unknown. Herein, we evaluated using the FIB4 index as a risk assessment tool for cardiovascular events and mortality in patients with AF. Methods: We performed a post-hoc analysis of a prospective, observational multicenter study. A total of 3067 patients enrolled in a multicenter Japanese registry were grouped as first tertile (FIB4 index &lt, 1.75, n = 1022), second tertile (1.75 &le, FIB4 index &lt, 2.51, n = 1022), and third tertile (FIB4 index &ge, 2.51, n = 1023). Results: The third tertile had statistically significant results: older age, lower body mass index, increased heart failure prevalence, and lower clearances of hemoglobin and creatinine (all p &lt, 0.05). During the follow-up period, incidences of major bleeding, cardiovascular events, and all-cause mortality were significantly higher for the third tertile (all p &lt, 0.05). After multivariate adjustment, the third tertile associated independently with cardiovascular events (HR 1.72, 95% CI 1.31&ndash, 2.25) and all-cause mortality (HR 1.43, 95% CI 1.06&ndash, 1.95). Adding the FIB4 index to a baseline model with CHA2DS2-VASc score improved the prediction of cardiovascular events and all-cause mortality, as shown by the significant increase in the C-statistic (all p &lt, 0.05), net reclassification improvement (all p &lt, 0.001), and integrated discrimination improvement (all p &lt, 0.001). A FIB4 index &ge, 2.51 most strongly associated with cardiovascular events and all-cause mortality in AF patients with high CHADS2 scores (all p &lt, 0.001). Conclusions: The FIB4 index is independently associated with risks of cardiovascular events and all-cause mortality in AF patients.

Histologically tubulopapillary structures with glycogen-rich clear cytoplasm in gastric adenocarcinoma with enteroblastic differentiation (GAED) are well known, but a solid growth pattern can also be observed as a minor component. In contrast, hepatoid adenocarcinoma (HAC) of the stomach shows many overlapping features, including solid pattern and α-fetoprotein expression. In this study, we employed next-generation sequencing (NGS) to establish a molecular/clinicopathological concept of GAED and clarify whether these two tumors should be grouped together in one category. Among 2273 primary gastric cancers treated in our hospital between 2008 and 2017, we defined 51 cases as GAEDs showing tubulopapillary or solid patterns that express at least one of the following markers: α-fetoprotein, glypican-3, or spalt-like transcription factor 4. All cases previously diagnosed as HAC in our hospital had clear cytoplasm and were included as GAEDs by histological re-evaluation and immunohistochemical findings. We performed NGS for 24 histologically typical GAEDs and Sanger sequencing for the remaining cases. The most frequently mutated gene was TP53, and almost all cases with missense mutation showed p53 overexpression. An analysis of copy number variation revealed that ERBB2 amplification was the most frequent in GAED. Additionally, HER2 immunohistochemistry and fluorescence in situ hybridization confirmed that 22% of informative cases were HER2 positive. There was no correlation between molecular/clinicopathological parameters and α-fetoprotein expression or growth patterns in GAED. Our analysis showed that GAED frequently harbors TP53 mutations and ERBB2 amplification. As with conventional gastric adenocarcinoma, trastuzumab may be effective for GAED. Furthermore, HAC may be subcategorized as a solid-type GAED.