Koichi Takayama

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.

Randomized Phase III Trial of Platinum-Doublet Chemotherapy Followed by Gefitinib Compared With Continued Platinum-Doublet Chemotherapy in Japanese Patients With Advanced Non–Small-Cell Lung Cancer: Results of a West Japan Thoracic Oncology Group Trial (WJTOG0203)

PURPOSE Gefitinib is a small molecule inhibitor of the epidermal growth factor receptor tyrosine kinase. We conducted a phase III trial to evaluate whether gefitinib improves survival as sequential therapy after platinum-doublet chemotherapy in patients with advanced non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS Chemotherapy-naïve patients with advanced stage (IIIB/IV) NSCLC, Eastern Cooperative Oncology Group performance status of 0 to 1, and adequate organ function were randomly assigned to either platinum-doublet chemotherapy up to six cycles (arm A) or platinum-doublet chemotherapy for three cycles followed by gefitinib 250 mg orally once daily, until disease progression (arm B). Patients were stratified by disease stage, sex, histology, and chemotherapy regimens. The primary end point was overall survival; secondary end points included progression-free survival, tumor response, safety, and quality of life. Results Between March 2003 and May 2005, 604 patients were randomly assigned. There was a statistically significant improvement in progression-free survival in arm B (hazard ratio [HR], 0.68; 95% CI, 0.57 to 0.80; P < .001); however, overall survival results did not reach statistical significance (HR, 0.86; 95% CI, 0.72 to 1.03; P = .11). In an exploratory subset analysis of overall survival by histologic group, patients in arm B with adenocarcinoma did significantly better than patients in arm A with adenocarcinoma (n = 467; HR, 0.79; 95% CI, 0.65 to 0.98; P = .03). CONCLUSION This trial failed to meet the primary end point of OS in patients with NSCLC. The exploratory subset analyses demonstrate a possible survival prolongation for sequential therapy of gefitinib, especially for patients with adenocarcinoma.

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.

Anti-tumor angiogenesis therapy using soluble receptors: enhanced inhibition of tumor growth when soluble fibroblast growth factor receptor-1 is used with soluble vascular endothelial growth factor receptor

We have shown that a soluble receptor for vascular endothelial growth factor (sVEGFR), which adsorbs VEGF and may function as a dominant-negative receptor, suppresses tumor angiogenesis and enhances apoptosis of cancer cells, thereby inhibiting tumor growth [Cancer Res 60 (2000) 2169–2177]. In the present study, using as many as 11 cancer cell lines, we tested two hypotheses: (a) that a soluble fibroblast growth factor receptor-1 (sFGFR1) might inhibit tumor angiogenesis and growth in sVEGFR-resistant cancers, and (b) that combining sFGFR1 with sVEGFR might produce an enhanced inhibitory effect. In two cell lines derived from human lung cancer, H460 and A549, both of which produce a considerable amount of FGF-2, sVEGFR and a soluble receptor for angiopoietin-1 were both ineffective; however, sFGFR1 inhibited tumor angiogenesis and growth, demonstrating the critical role that FGFs play in some cancers. In three cell lines (QG56 from lung cancer, T3M4 and Panc1 from pancreatic cancer), which produced both VEGF and FGF-2 at detectable levels, combined sVEGFR and sFGFR1 produced an enhanced inhibitory effect compared to their individual effects. The combined usage of sVEGFR plus sFGFR1 suppressed tumor growth in all cancer cell lines tested, suggesting possible effectiveness of this strategy against a wide range of cancers.

Coxsackievirus B3 Is an Oncolytic Virus with Immunostimulatory Properties That Is Active against Lung Adenocarcinoma

Although oncolytic virotherapy is a promising anticancer therapy, antitumor efficacy is hampered by low tumor selectivity. To identify a potent and selective oncolytic virotherapy, we carried out large-scale two-step screening of 28 enteroviral strains and found that coxsackievirus B3 (CVB3) possessed specific oncolytic activity against nine human non-small cell lung cancer (NSCLC) cell lines. CVB3-mediated cytotoxicity was positively correlated with the expression of the viral receptors, coxsackievirus and adenovirus receptor, and decay-accelerating factor, on NSCLC cells. In vitro assays revealed that the CVB3 induced apoptosis and phosphoinositide 3-kinase/Akt and mitogen-activated protein (MAP)/extracellular signal-regulated (ERK) kinase (MEK) survival signaling pathways, leading to cytotoxicity and regulation of CVB3 replication. Intratumoral injections of CVB3 elicited remarkable regression of preestablished NSCLC tumors in vivo. Furthermore, administrations of CVB3 into xenografts on the right flank resulted in significantly durable regression of uninjected xenografts on the left flank, where replication-competent CVB3 was detected. All treatments with CVB3 were well tolerated without treatment-related deaths. In addition, after CVB3 infection, NSCLC cells expressed abundant cell surface calreticulin and secreted ATP as well as translocated extranuclear high-mobility group box 1, which are required for immunogenic cell death. Moreover, intratumoral CVB3 administration markedly recruited natural killer cells and granulocytes, both of which contributed to the antitumor effects as shown by depletion assays, macrophages, and mature dendritic cells into tumor tissues. Together, our findings suggest that CVB3 is a potent and well-tolerated oncolytic agent with immunostimulatory properties active against both localized and metastatic NSCLC.

Interleukin-8 participates in angiogenesis in non-small cell, but not small cell carcinoma of the lung

We examined interleukin-8 (IL-8) production in 17 lung cancer cell lines, IL-8 expression in tumor specimens and IL-8s contribution to tumor-induced angiogenesis in vivo. Eight of 13 non-small cell lung cancer cell lines constitutively produced high levels of IL-8. Four small cell lung cancer cell lines produced little or no IL-8. Immunohistochemical analysis of transbronchial biopsy specimens revealed IL-8 staining within adenocarcinomas (22/32), squamous cell carcinomas (12/21) and large cell carcinomas (2/3), but not within most small cell carcinomas (1/22). Anti-IL-8 antisera blocked tumor angiogenesis by two IL-8 producing cell lines in a mouse model.

Interleukin-6 induces both cell growth and VEGF production in malignant mesotheliomas.

Malignant mesothelioma (MM), an incurable tumor, is reportedly an interleukin‐6 (IL‐6) secreting tumor. The pathological significance of IL‐6 overexpression in this tumor, however, has remained unclear. We investigated the biological functions of IL‐6 in mesotheliomas. Five mesothelioma cell lines were analyzed for IL‐6 production and IL‐6 receptor (IL‐6R) expression. Of them, 2 produced high levels of IL‐6, 2 produced intermediate levels and 1 cell line showed no secretion. All mesothelioma cell lines used in this study expressed very small amounts of IL‐6R mRNA. We compensated for this low level of IL‐6R expression in mesotheliomas by adding recombinant soluble IL‐6R (sIL‐6R) to mediate the IL‐6 signal. IL‐6 together with sIL‐6R was found to promote cell growth of H2052 and H226 MMs classified as high‐level IL‐6 producers in a dose‐dependent manner. Moreover, a humanized anti‐IL‐6R antibody (MRA) capable of blocking IL‐6 signaling suppressed the cell growth of mesotheliomas induced by IL‐6/sIL‐6R. These findings demonstrate that IL‐6 serves as an autocrine growth factor in the development of mesothelioma. In addition, IL‐6/sIL‐6R stimulation increased the expression of vascular endothelial growth factor (VEGF) in 4 out of 5 cell lines, and this induction was inhibited by MRA treatment. The involvement of the signal transducer and activator of transcription 3 (STAT3) pathway in both cell growth and VEGF induction by IL‐6/sIL‐6R was verified by dominant negative STAT3 transduction combined with adenovirus gene‐delivery methods. Although IL‐6 induces VEGF through the JAK2/STAT3 pathway, anti‐VEGF antibody could not inhibit the IL‐6‐induced cell growth observed in H2052 and H226. We concluded that IL‐6‐dependent growth does not occur via VEGF induction. These results suggest that treatment with anti‐IL‐6R antibody may constitute a potential molecular targeting therapy for MMs.

NQO1, MPO, and the risk of lung cancer: a HuGE review.

The aim of this study is to summarize the available molecular epidemiologic studies of lung cancer and metabolic genes, such as NAD(P)H quinone reductase 1 (NQO1) and myeloperoxidase (MPO). NQO1 plays a dual role in the detoxification and activation of procarcinogens whereas MPO has Phase I activity by converting lipophilic carcinogens into hydrophilic forms. Variant genotypes of both NQO1 Pro187 Ser and MPO G-463A polymorphisms may be related to low enzyme activity. The Pro/Ser and Ser/Ser genotypes combined of NQO1 was significantly associated with decreased risk of lung cancer in Japanese [random effects odds ratio (OR) = 0.70, 95% confidence interval (CI) = 0.56—0.88] among whom the variant allele is common. The variant genotype of MPO was associated with decreased risk of lung cancer among Caucasians (random effects OR = 0.70, 95% CI = 0.47--1.04). Gene-environment interactions in both polymorphisms may be hampered by inaccurate categorization of tobacco exposure. Evidence on gene-gene interactions is extremely limited. As lung cancer is a multifactorial disease, an improved understanding of such interactions may help identify individuals at risk for developing lung cancer. Such a study should include larger sample size and other polymorphisms in the metabolism of tobacco-derived carcinogens and address interactions with smoking status. The effects of polymorphisms are best represented by their haplotypes. In future studies on lung cancer, the development of haplotype-based approaches will facilitate the evaluation of haplotypic effects, either for selected polymorphisms physically close to each other or for multiple genes within the same drug-metabolism pathway.

The relationship between CYP1A1 aryl hydrocarbon hydroxylase activity and lung cancer in a Japanese population.

Because aryl hydrocarbon hydroxylase (AHH) is considered to be responsible for the activation of benzo(a)pyrene and other polyaromatic hydrocarbons in cigarette smoke to carcinogens, it is important to examine CYP1A1 (AHH) activity in the determination of susceptibility to lung cancer. We investigated AHH activity in peripheral mitogen-treated lymphocytes in 108 lung cancer patients and 95 healthy control individuals. Non-induced AHH activity was detectable in all the samples. AHH inducibility (3-methylcholanthrene-induced/non-induced AHH activity) showed a very wide interindividual variation as well as non-induced AHH activity. No significant associations were found between adjusted AHH activity and histologic type of tumor among lung cancer patients. Adjusted AHH inducibility of genotype C [geometric mean and 95% confidence interval (CI); 15.56 and 11.69-20.71] in MspI polymorphism was significantly higher than those of the other two genotypes (P = 0.0001), while no significant difference was observed between genotypes A (4.76 and 3.82-5.93) and B (5.60 and 4.57-6.86). On the other hand, non-induced AHH activity of genotype Val/Val (0.121 and 0.082-0.178 pmol/min/10(6) cells) in isoleucine-valine (Ile-Val) polymorphism was significantly higher than those of genotypes Ile/Ile (0.042 and 0.034-0.052 pmol/min/10(6) cells) and Ile/Val (0.040 and 0.030-0.053 pmol/min/10(6) cells) (P < 0.0001). Even after controlling for age, cigarettes smoked per day and season of the year, high AHH inducibility (7.0 < versus 0 < < or = 3.0: OR and 95 %CI, 12.4 and 2.88-53.4) was an independent risk factor for lung cancer. The data indicate that high AHH inducibility may strongly associate with the susceptibility to lung carcinogenesis.

EPHX1 polymorphisms and the risk of lung cancer: a HuGE review.

Background: Microsomal epoxide hydrolase 1 (EPHX1) plays an important role in both the activation and detoxification of tobacco-derived carcinogens. Polymorphisms at exons 3 and 4 of the EPHX1 gene have been reported to be associated with variations in EPHX1 activity. The aim of this study is to review and summarize the available molecular epidemiologic studies of lung cancer and EPHX1. Methods: We searched MEDLINE, Current Contents, and Web of Science databases for studies published before August 2004. We conducted a systematic review and meta-analysis of 13 case–control studies. Summary odds ratios and summary prevalence of the variant allele (genotype) of both polymorphisms in the EPHX1 gene were calculated using the DerSimonian and Laird method. Results: The low-activity (variant) genotype of EPHX1 polymorphism at exon 3 was associated with decreased risk of lung cancer (odds ratio = 0.65; 95% confidence interval = 0.44–0.96) in lung cancer risk among whites. In white populations, the high-activity (variant) genotype of EPHX1 polymorphism at exon 4 was associated with a modest increase in risk of lung cancer (1.22; 0.79–1.90) and the predicted low activity was associated with a modest decrease in risk (0.72; 0.43–1.22). Conclusions: EPHX1 enzyme may act as a phase I enzyme in lung carcinogenesis. The low-activity genotype of EPHX1 gene is associated with decreased risk of lung cancer among whites.