Taishi Harada

Association of PD-L1 overexpression with activating EGFR mutations in surgically resected nonsmall-cell lung cancer

BACKGROUND Recent clinical trials have shown that immune-checkpoint blockade yields a clinical response in a subset of individuals with advanced nonsmall-cell lung cancer (NSCLC). We examined whether the expression of programmed death-ligand 1 (PD-L1) is related to clinicopathologic or prognostic factors in patients with surgically resected NSCLC. PATIENTS AND METHODS The expression of PD-L1 was evaluated by immunohistochemical analysis in 164 specimens of surgically resected NSCLC. Cell surface expression of PD-L1 in NSCLC cell lines was quantified by flow cytometry. RESULTS Expression of PD-L1 in tumor specimens was significantly higher for women than for men, for never smokers than for smokers, and for patients with adenocarcinoma than for those with squamous cell carcinoma. Multivariate analysis revealed that the presence of epidermal growth factor receptor gene (EGFR) mutations and adenocarcinoma histology were significantly associated with increased PD-L1 expression in a manner independent of other factors. Cell surface expression of PD-L1 was also significantly higher in NSCLC cell lines positive for activating EGFR mutations than in those with wild-type EGFR. The EGFR inhibitor erlotinib downregulated PD-L1 expression in the former cell lines but not in the latter, suggesting that PD-L1 expression is increased by EGFR signaling conferred by activating EGFR mutations. A high level of PD-L1 expression in resected tumor tissue was associated with a significantly shorter overall survival for NSCLC patients. CONCLUSIONS High expression of PD-L1 was associated with the presence of EGFR mutations in surgically resected NSCLC and was an independent negative prognostic factor for this disease.

Hypomethylation Status of CpG Sites at the Promoter Region and Overexpression of the Human MDR1 Gene in Acute Myeloid Leukemias

Selection of human cells for resistance to vincristine or doxorubicin often induces overexpression of the multidrug resistance 1 gene (MDR1), which encodes the cell surface P-glycoprotein, as a result of gene amplification or transcriptional activation. Moreover, overexpression of the MDR1 gene has been shown to be associated closely with clinical outcome in various hematological malignancies, including acute myeloid leukemia (AML). However, the precise mechanism underlying overexpression of the MDR1 gene during acquisition of drug resistance remains unclear. We recently described an inverse correlation between the methylation status of CpG sites at the promoter region and expression of the MDR1 gene in malignant cell lines. In this study, we expanded this analysis to 42 clinical AML samples. We adapted a quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay for gene expression and a quantitative PCR after digestion by Hpa II for methylation status of the MDR1 gene. We observed a statistically significant inverse correlation between methylation and MDR1 expression in clinical samples. The hypomethylation status of the MDR1 promoter region might be a necessary condition for MDR1 gene overexpression and establishment of P-glycoprotein-mediated multidrug resistance in AML patients.

Induction of PD-L1 Expression by the EML4–ALK Oncoprotein and Downstream Signaling Pathways in Non–Small Cell Lung Cancer

Purpose: Therapies targeted to the immune checkpoint mediated by PD-1 and PD-L1 show antitumor activity in a subset of patients with non–small cell lung cancer (NSCLC). We have now examined PD-L1 expression and its regulation in NSCLC positive for the EML4–ALK fusion gene. Experimental Design: The expression of PD-L1 at the protein and mRNA levels in NSCLC cell lines was examined by flow cytometry and by reverse transcription and real-time PCR analysis, respectively. The expression of PD-L1 in 134 surgically resected NSCLC specimens was evaluated by immunohistochemical analysis. Results: The PD-L1 expression level was higher in NSCLC cell lines positive for EML4–ALK than in those negative for the fusion gene. Forced expression of EML4–ALK in Ba/F3 cells markedly increased PD-L1 expression, whereas endogenous PD-L1 expression in EML4–ALK–positive NSCLC cells was attenuated by treatment with the specific ALK inhibitor alectinib or by RNAi with ALK siRNAs. Furthermore, expression of PD-L1 was downregulated by inhibitors of the MEK–ERK and PI3K–AKT signaling pathways in NSCLC cells positive for either EML4–ALK or activating mutations of the EGFR. Finally, the expression level of PD-L1 was positively associated with the presence of EML4–ALK in NSCLC specimens. Conclusions: Our findings that both EML4–ALK and mutant EGFR upregulate PD-L1 by activating PI3K–AKT and MEK–ERK signaling pathways in NSCLC reveal a direct link between oncogenic drivers and PD-L1 expression. Clin Cancer Res; 21(17); 4014–21. ©2015 AACR.

Expression of TrkB and BDNF is associated with poor prognosis in non-small cell lung cancer

High expression levels of TrkB and BDNF are associated with aggressive malignant behavior in tumor cells and a poor prognosis in patients with various types of cancer. In this study, we aimed to identify the relationship between TrkB and BDNF expression and clinicopathological variables and prognosis in non-small cell lung cancer (NSCLC). We evaluated TrkB and BDNF expression in the tumor cells of 102 NSCLC patients by immunohistochemistry. Out of all clinicopathological factors examined, only vascular invasion was significantly correlated with TrkB (P=0.010) and BDNF (P=0.015) expression. TrkB-positive tumors had significantly worse disease-free survival (P=0.0094) and overall survival (P=0.0019) than TrkB-negative tumors, and TrkB expression was an independent prognostic factor for disease-free survival (HR 3.735, 95% CI 1.560-11.068, P=0.002) and overall survival (HR 4.335, 95% CI 1.534-15.963, P=0.004) in multivariate analysis. Finally, our analysis revealed that co-expression of TrkB and BDNF conferred poorer prognosis compared with overexpression of either protein alone. Our results indicate that expression of TrkB and BDNF is associated with poor prognosis in NSCLC patients.

Rapid decrease in forced vital capacity in patients with idiopathic pulmonary upper lobe fibrosis

BACKGROUND We are occasionally presented with patients with unclassifiable interstitial pneumonia of unknown etiology. Idiopathic pulmonary upper lobe fibrosis (IPUF) does not fit any of the currently defined subsets of idiopathic interstitial pneumonias (IIPs). This study was performed to examine clinical, functional, and pathological characteristics of IPUF. METHODS We present 9 cases of histologically confirmed IPUF. The clinical and histological characteristics of the 9 patients were evaluated. The baseline respiratory function of all patients was measured. There were 7 patients whose forced vital capacity (FVC) had been monitored for at least a year who were selected to quantify the yearly decline in FVC. RESULTS All patients were slender, with a body mass index of 16.0-19.8 kg/m(2). Seven patients had a history of pneumothorax. Six patients died 1.8 to 5.7 years after the onset of the first symptoms. Fundamental histological features were intraalveolar collagen deposition and densely packed elastic fibers in the subpleural areas. These findings are the same as those seen in pleuroparenchymal fibroelastosis. However, the visceral pleura was thickened with dense collagen in only 2 patients, and pleural thickening was localized, if present, in the remaining 7 patients. Ventilatory impairment was also a characteristic. The time course decline of FVC was rapid and almost linear. The median yearly decline in FVC was -20.3% (range, -7.7% to -26.5%), which was more rapid than that reported for chronic fibrosing interstitial pneumonias such as idiopathic pulmonary fibrosis. CONCLUSIONS IPUF is a unique pulmonary fibrosis that results in rapid deterioration of ventilatory function and poor prognosis.

Role and Relevance of TrkB Mutations and Expression in Non―Small Cell Lung Cancer

Purpose: TrkB has been involved in poor cancer outcome. TrkB mutations have been reported in non–small cell lung cancer. In this study, we aimed at characterizing the role of three potentially sensitizing TrkB mutations previously reported in lung cancer. Experimental Design: We characterized three activation loop mutants of TrkB (M713I, R715G, and R734C) in terms of pathway activation/phosphorylation, migration, anchorage-independent growth, and sensitivity to a Trk inhibitor, using NIH3T3 cells and Baf3 cells. We also sequenced the tyrosine kinase domain of TrkB in a large number of lung cancer samples of East-Asian origin and cell lines. Results: None of the mutants were constitutively active in NIH3T3 transformation and migration assays. M713I and R734C mutants showed low levels of autophosphorylation in comparison with wild-type TrkB. Although R715G showed similar level of autophosphorylation to wild-type TrkB on brain-derived neurotrophic factor stimulation, the mutant was not as competent as wild-type TrkB in supporting interleukin-3–independent growth of Baf3 cells. In addition, the Trk inhibitor AZD6918 inhibited wild-type TrkB-induced cell migration and cell growth, whereas the mutants were relatively resistant to the Trk inhibitor compared with wild-type TrkB. We could not confirm the presence of nonsynonymous mutation in 78 lung cancer samples and 29 cell lines. Conclusions: Wild-type, but not mutant, TrkB enhances cell migration and transformation. Our study suggests that TrkB mutations should not be used for selection of patients with lung cancer treated with Trk inhibitors. High expression of wild-type TrkB might be beneficial for studies of Trk inhibitors. Clin Cancer Res; 17(9); 2638–45. ©2011 AACR.

The thoracic cage becomes flattened in the progression of pleuroparenchymal fibroelastosis

To the Editor: Pleuroparenchymal fibroelastosis (PPFE) was first reported by Frankel et al. [1]. PPFE can occur without any aetiology or underlying diseases (idiopathic PPFE), or with underlying diseases or conditions. Idiopathic PPFE has been listed as one of the rare idiopathic interstitial pneumonias (IIPs) in the revised international multidisciplinary consensus classification of IIPs [2]. The natural history of PPFE is variable, some are slowly progressive and others sometimes show rapid deterioration resulting in poor prognosis, like idiopathic pulmonary fibrosis (IPF). Idiopathic pulmonary upper lobe fibrosis (PULF), first proposed by Amitani et al. [3], is currently considered to be almost identical to idiopathic PPFE [1, 4, 5], which is now globally accepted as a representative nomenclature for this disorder. Therefore, we use the term PPFE to describe the same disease as PULF. Amitani et al. [3] recognised a characteristic constitution in patients with PPFE: they are slender and their thoracic cage is flattened, i.e. the ratio of the anteroposterior diameter of the thoracic cage (APDT) to the transverse diameter of the thoracic cage (TDT) is abnormally lower than in normal populations. Herein, we have provisionally named this deformity of the thoracic cage as “flat chest”. Other investigators have also noticed this deformity in idiopathic PPFE [6–8]. Flat chest may result from a congenital disposition or …

Addition of bevacizumab enhances antitumor activity of erlotinib against non-small cell lung cancer xenografts depending on VEGF expression

PurposeErlotinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), and bevacizumab, an anti-vascular endothelial growth factor (VEGF) agent, are promising therapies for advanced non-small cell lung cancer (NSCLC). Our study was aimed to determine whether there were conditions under which the addition of bevacizumab would enhance the antitumor activity of erlotinib against NSCLC tumors in vitro and in vivo.MethodsMTS was for NSCLC cell (PC9, 11–18, H1975, H157, H460 and A549) growth assay in vitro. ELISA was for VEGF protein assay in cells and tumor tissues. Mouse xenograft models were established with H157, H460 and A549 with primary resistance to erlotinib and treated with erlotinib plus bevacizumab or each agent alone. Erlotinib concentrations in tumors were determined by high-performance liquid chromatography.ResultsBevacizumab alone did not inhibit NSCLC cell growth in vitro. In primarily erlotinib-resistant NSCLC cells, the levels of VEGF protein were highest in H157 cell followed in order by H460 and A549 cells. In vivo, bevacizumab alone significantly inhibited tumor growth only in xenograft models with high (H157) and/or moderate (H460) levels of VEGF protein. A combination of erlotinib and bevacizumab partially reversed resistance to erlotinib in H157 xenografts (high VEGF level) with increasing intratumoral erlotinib concentrations, but not in H460 (moderate) or A549 (low) xenografts.ConclusionsThese results support that combined with anti-VEGF therapy could enhance antitumor activity of anti-EGFR therapy and/or partially reverse resistance to EGFR TKI, by increasing EGFR TKI concentration in specific tumors that express high levels of VEGF protein.

Epithelial–mesenchymal transition in human lungs with usual interstitial pneumonia: Quantitative immunohistochemistry

Fibroblastic foci, a major histological feature of usual interstitial pneumonia (UIP), play a critical role in the development of UIP. The mechanisms involved in the formation of these foci, however, including cellular origin, remain unclear. Recent in vitro and animal studies suggested epithelial–mesenchymal transition (EMT) of alveolar epithelial cells during pulmonary fibrogenesis. The aim of the present study was to investigate the presence of EMT in patients with UIP on quantitative immunohistochemistry using pathological tissue sections. The study subjects were 13 patients with UIP pattern among 52 patients with interstitial pneumonia who underwent lung biopsy. Alveolar epithelial cells overlying fibroblastic foci expressed epithelial markers less frequently and mesenchymal markers more frequently compared with those in non‐diseased control lung tissues (n= 10). Moreover, double immunostaining showed that some epithelial cells stained for both epithelial and mesenchymal markers. Furthermore, significantly higher numbers of epithelial marker‐positive fibroblastic cells were found in fibroblastic foci in UIP as well as in other non‐UIP fibrosing diseases than in control lung tissues. The results showed that some epithelial cells overlying fibroblastic foci lose the epithelial phenotype and gain the mesenchymal phenotype, and that some fibroblastic cells in fibroblastic foci originate from epithelial cells. But this EMT may not be specific for UIP.

Histological evolution of pleuroparenchymal fibroelastosis

To investigate the histological evolution in the development of pleuroparenchymal fibroelastosis (PPFE).