Effective treatment for advanced lung cancer and idiopathic interstitial pneumonia (IIP) remains an unmet medical need. The relationship between chemotherapy’s effectiveness in advanced lung cancer and the risk of acute exacerbation of IIP is poorly investigated. There is limited evidence that patients who experience an acute exacerbation of IIPs during cytotoxic chemotherapy have poorer outcomes than those who do not. Among 1004 patients with advanced lung cancer and IIPs enrolled in our published multi-centre retrospective study from 110 Japanese institutions, 708 patients (male: female, 645:63; mean age, 70.4) received first-line chemotherapy. The occurrence of chemotherapy-triggered acute exacerbations of IIPs and overall survival (OS) were analysed. The OS between groups of patients with and without the occurrence of acute exacerbation was compared at four landmark time points (30, 60, 90, and 120 days), starting from the first-line chemotherapy, using the landmark method. The incidence of acute exacerbation in patients who received first-line chemotherapy with small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) was more frequent in NSCLC patients than in SCLC (4.2% vs 12.6%; odds ratio [OR]: 3.316; 95% confidence interval [CI] 1.25–8.8). Median survival time was 9.9 months (95% CI 9.2–10.7). Patients who experienced acute exacerbation had significant worse survival outcomes than those who did not at various time points (30 days, hazard ratio [HR]: 5.191, 95% CI 2.889–9.328; 60 days, HR: 2.351, 95% CI 1.104–5.009; 90 days, HR: 2.416, 95% CI 1.232–4.739; and 120 days, HR: 2.521, 95% CI 1.357–4.681). Acute exacerbation during first-line chemotherapy can predict poor survival. Trial Registration number: UMIN000018227.

Background Liquid biopsy-based next-generation sequencing (NGS) is clinically useful as a less invasive multi-gene analysis for precision cancer medicine. However, performance of NGS using plasma cell-free DNA (cfDNA) compared to tumor tissue has not been evaluated in a large-scale sample. Methods A liquid biopsy study is additionally conducted in the nation-wide lung cancer genome screening project (LC-SCRUM-Japan), targeting 2000 NSCLC patients. Tumor genetic alterations are evaluated by cfDNA- and tissue-based NGS tests. Plasma is collected within 4 weeks of the corresponding tissue biopsy. cfDNA is analyzed using Guardant360Ⓡ (investigational device exemption version, Guardant Health); tumor tissue is analyzed by Oncomine Comprehensive AssayⓇ version 3 (Thermo Fisher Scientific). We assessed the detectability of 8 oncogene drivers: mutations of EGFR, KRAS, BRAF, ERBB2 and MET, and fusions of ALK, RET and ROS1. Results As of March 2019, 270 advanced NSCLC patients were enrolled. Median age was 67 (range, 30-87) years, 169 patients (63%) were men, 86 (32%) were nonsmokers, 215 (80%) had adenocarcinoma, 206 (76%) were stage IV, and 224 (83%) received no prior chemotherapy. An oncogene driver was detected in 156 patients (58%) by at least one NGS method, including 138 (51%) in tissue and 107 (40%) in cfDNA. Using the tissue assay as a control, the positive percent agreement (PPA) of gene mutations in the cfDNA assay was 75% (91 of 121), including 74% (53 of 72) for EGFR, 78% (31 of 40) for KRAS, 100% (3 of 3) for BRAF, 75% (3 of 4) for ERBB2 and 50% (1 of 2) for MET. The PPA of gene fusions in the cfDNA assay was 24% (4 of 17), including 33% (2 of 6) for ALK, 33% (2 of 6) for RET and 0% (0 of 5) for ROS1. Of 132 patients in whom no oncogene driver was detected by the tissue assay, 17 had the drivers detected in cfDNA: 8 EGFR, 5 KRAS, 1 BRAF, 1 ERBB2, 1 MET and 1 ALK. Conclusions In the cfDNA-based NGS, the detectability of gene mutations was comparable with the previously reported single-gene tests using cfDNA, whereas that of gene fusions was low. The cfDNA-based assay may be useful for patients in whom oncogene driver detection was failed by the tissue-based assay. This study is now ongoing and we will present updated results. Clinical trial identification UMI30496. Legal entity responsible for the study National Cancer Center. Funding Guardant Health, Merck Serono. Disclosure S. Matsumoto: Honoraria (self), Research grant / Funding (institution): Novartis; Research grant / Funding (institution): Chugai Pharma; Honoraria (self): Pfizer; Honoraria (self): AstraZeneca; Research grant / Funding (institution): MSD; Research grant / Funding (institution): Merck Serono. H. Udagawa: Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): AbbVie; Honoraria (self), Research grant / Funding (self): Daiichisankyo; Honoraria (self), Research grant / Funding (self): MSD; Research grant / Funding (self): AMGEN; Advisory / Consultancy: Boehringer Ingelheim; Honoraria (self): AstraZeneca; Honoraria (self): Chugai Pharma; Honoraria (self): Taiho; Honoraria (self): Ono; Honoraria (self): Bristol-Myers Squibb; Honoraria (self): Amco. K. Nishino: Honoraria (self): Chugai Pharma. S. Kuyama: Advisory / Consultancy, Speaker Bureau / Expert testimony: AstraZeneca; Speaker Bureau / Expert testimony: Eli Lilly; Speaker Bureau / Expert testimony: Chugai pharma; Speaker Bureau / Expert testimony: Boehringer Ingelheim; Speaker Bureau / Expert testimony: Pfizer; Speaker Bureau / Expert testimony: MSD; Speaker Bureau / Expert testimony: Taiho; Speaker Bureau / Expert testimony: Meiji. Y. Tsubata: Speaker Bureau / Expert testimony, Research grant / Funding (institution): AstraZeneca; Speaker Bureau / Expert testimony: Chugai Pharma; Speaker Bureau / Expert testimony, Research grant / Funding (institution): Daiichi Sankyo; Research grant / Funding (institution): Ono Pharmaceutical . T. Kato: Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): AstraZeneca; Advisory / Consultancy: MSD; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Eli Lilly; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Chugai Pharma; Advisory / Consultancy: Nitto Denko; Advisory / Consultancy, Research grant / Funding (institution): AbbVie; Advisory / Consultancy, Research grant / Funding (institution): Merck Serono; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Pfizer; Honoraria (self): Roche; Honoraria (self): Boehringer Ingelheim; Honoraria (self), Research grant / Funding (institution): Ono Pharmaceutical; Honoraria (self), Research grant / Funding (institution): Taiho Pharmaceutical; Honoraria (self): Bristol-Myers Squibb Japan; Honoraria (self), Research grant / Funding (institution): Merck Sharp & Dohme; Honoraria (self): Novartis; Honoraria (self): Sumitomo Dainippon Pharma; Honoraria (self): Quintiles; Honoraria (self): Takeda; Research grant / Funding (institution): Kyowa Hakko Kirin; Research grant / Funding (institution): Astellas Pharma; Research grant / Funding (self): Kyorin; Research grant / Funding (self): Regeneron. Y. Ohe: Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): AstraZeneca; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Chugai Pharma; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Eli Lilly Japan; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Ono Pharmaceutical; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Novartis; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Kyorin; Honoraria (self), Advisory / Consultancy: Celltrion; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Takeda; Honoraria (self), Advisory / Consultancy: Amgen; Honoraria (self), Research grant / Funding (institution): Bristol-Myers Squibb Japan; Honoraria (self): Boehringer Ingelheim; Honoraria (self): Bayer; Honoraria (self), Research grant / Funding (institution): Pfizer; Honoraria (self): MSD; Honoraria (self), Research grant / Funding (institution): Taiho Pharmaceutical; Honoraria (self): Kyowa Hakko Kirin; Research grant / Funding (institution): Sumitomo Dainippon Pharma; Research grant / Funding (institution): Ignyta. K. Goto: Honoraria (self), Advisory / Consultancy: Otsuka; Honoraria (self), Research grant / Funding (self): Bristol-Myers Squibb; Honoraria (self), Research grant / Funding (self): AstraZeneca ; Honoraria (self), Research grant / Funding (self): Pfizer ; Honoraria (self), Research grant / Funding (self): Chugai Pharma; Honoraria (self), Research grant / Funding (self): Taiho Pharmaceutical; Honoraria (self), Research grant / Funding (self): Ono Pharmaceutical; Honoraria (self), Research grant / Funding (self): Novartis; Honoraria (self), Research grant / Funding (self): Eli Lilly; Honoraria (self), Research grant / Funding (self): Boehringer Ingelheim; Honoraria (self): Quintiles ; Honoraria (self), Research grant / Funding (self): Merck Serono; Honoraria (self), Research grant / Funding (self): Life Technologies; Honoraria (self), Research grant / Funding (self): MSD; Honoraria (self): AbbVie ; Honoraria (self), Research grant / Funding (self): Riken Genesis ; Honoraria (self): Nippon Kayaku; Honoraria (self), Research grant / Funding (self): Takeda; Research grant / Funding (self): Sumitomo Dainippon Pharma ; Honoraria (self): SRL Diagnostics; Research grant / Funding (self): Daiichi Sankyo; Research grant / Funding (self): Kyowa Hakko Kirin; Research grant / Funding (self): Astellas Pharma; Research grant / Funding (self): Eisai ; Research grant / Funding (self): Ignyta; Research grant / Funding (self): RTI Health Solutions; Research grant / Funding (self): Janssen; Research grant / Funding (self): Xcoo; Research grant / Funding (self): Loxo. All other authors have declared no conflicts of interest.

Background Immune checkpoint inhibitors (ICPi) have dramatically changed the treatment of non-small cell lung cancer (NSCLC), but the effect of these agents on brain metastases (BM) has not been clearly elucidated. We retrospectively evaluated the effect of ICPi on BM. Methods NSCLC patients who harbored BM treated with ICPi in out institution were enrolled. History of radiation therapy (RT) to the brain was not included in the selection criteria. The primary endpoint was overall survival after the initiation of ICPi (OS), and the secondary endpoints were duration of ICPi administration, causes of withdrawal, responses of BM, time to the first response of BM, and the incidence of radiation necrosis (RN). Results 35 NSLC with BM were treated with ICPi: nivolmab in 21, pembrolizumab in 13, and athezolizumab in 1 case. Among these cases, BM were progressive in 25 but stable in 10 cases at the initiation of ICPi. At the time of evaluation, ICPi were already withdrawn in 26 cases and the median administration time was 2.4 (0.5 to 25.9) months. The cause of withdrawal was progression of extracranial lesions in 8, progression of BM in 6, and adverse events or PS deterioration in the others. In competing risk analysis, the times to progression of extra- and intra-cranial lesions were not different. The responses of BM were evaluable in 28 cases: CR in 3, PR in 9, SD in 10 and PD in 6 cases. ICPi had been sequentially administrated after RT in one of the 3 CR and all of the 9 PR cases. The objective response rate was 76.9% after sequential RT/ICPi, but only 13.3% after ICPi alone (P = 0.002). The median time to the first response of BM was 1.0 (0.4 to 3.0) months after RT/ICPi and 1.3 (0.3 to 3.6) months after ICPi alone (n.s.). The median OS was 14.8 (95% CI: 8.8 to 22.7) months after the initiation of ICPi, and majority (90.5%) of the deaths were owing to the progression of extracranial disease. Among the 24 cases with history of RT to the brain, RN was observed in 7 (29.2%) and required necrotomy in 2 cases, even though ICPi could safely be continued after craniotomy in these 2 cases. Conclusions ICPi alone showed only limited efficacy on BM and were recommended to be administrated sequentially with RT. RN was the major neurological adverse event, and necrotomy was one of the choices to manage RN during ICPi. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

Background: Bevacizumab (Bev) combined with platinum-based chemotherapy is a standard treatment for advanced non-squamous non-small-cell lung cancer (non-Sq NSCLC). Cisplatin + pemetrexed (CisPem) is suggested as the most promising chemotherapy regimen combined with Bev. However, no study has been conducted to evaluate the efficacy and safety of CisPemBev compared with carboplatin + paclitaxel + bevacizumab (CarPacBev) for advanced non-Sq NSCLC. Methods: Treatment-naïve patients with advanced or recurrent EGFR/ALK-negative non-Sq NSCLC from 55 sites across Japan were randomly assigned in a 2:1 ratio to either CisPemBev (4 cycles of Cis [75 mg/m2] + Pem [500 mg/m2] + Bev [15 mg/kg] q3wk, followed by maintenance Pem + Bev q3wk until progression) or CarPacBev (4 cycles of Car [AUC 6] + Pac [200 mg/m2] + Bev q3wk, followed by maintenance Bev q3wk until progression). The primary endpoint was progression-free survival (PFS) by central review. The secondary endpoints were overall survival (OS), overall response rate (ORR) and safety profile. The target numbers of patients and events were determined to be 210 and 170, respectively, to observe a point estimate of HR for PFS (CisPemBev/CarPacBev) <0.83 with a high probability (80%) when the true HR was 0.72. The data were cutoff in July, 2017. OS data were updated for this presentation in April, 2018. Results: Between May 2014 and May 2016, 199 patients were randomly assigned to receive CisPemBev (N = 132) or CarPacBev (N = 67). In the primary analysis, PFS events occurred in 171 patients. The HR for PFS by central review (CisPemBev/CarPacBev) was 0.825 (95% CI 0.600-1.134, median PFS, 7.6 vs 7.0 months), and the ORR was 57% for CisPemBev and 55% for CarPacBev. OS events occurred in 119 patients. The median survival follow-up duration was 28.3 months. The median OS was 23.4 months for CisPemBev and 21.6 months for CarPacBev (HR 0.845, 95% CI 0.583-1.242). Conclusions: PFS was prolonged with CisPemBev compared with CarPacBev. However, there was no difference in OS between two arms. CisPem is the most effective chemotherapy regimen combined with Bev for advanced non-Sq NSCLC. Legal entity responsible for the study: National Cancer Center Hospital East. Funding: Chugai Pharmaceutical. Disclosure: H. Udagawa: Honoraria: Chugai Pharmaceutical. M. Mori: Honorarium for speech: AstraZeneca, Boehringer Ingelheim, Chugai, Eli Lilly, MSD, Ono Pharmaceutical, Shionogi; Research fund: Chugai. T. Yokoyama: Personal fees: Chugai Pharmaceutical, Eli Lilly Japan, outside the submitted work. T. Sasaki: Research grant: Boehringer Ingelheim and Pfizer. A. Sekine: Lecture fees: Boehringer Ingelheim, Chugai Pharmaceutical, AstraZeneca; Institutional financial support for contracted research: Boehringer Ingelheim. K. Goto: Honoraria: Chugai, Lilly Japan; Reserch grants: Chugai and Lilly Japan. All other authors have declared no conflicts of interest.

Background: Diagnosis of leptomeningeal carcinomatosis (LMC) is made based upon the findings on MRI (LMCi), and/or cytologically confirmed cancer cells in the cerebrospinal fluid (CSF) (LMCc). However, in EGFR-mutated (EGFRm) cases, brain metastasis (BM) has a tendency to arise close to CSF space, and discrepancies between imaging and cytological results makes it difficult to construct treatment strategy. Although the effect of tyrosine kinase inhibitors (TKIs) on LMC has been expected, a little is known regarding the effect and limitation of TKIs. Methods: Overall survival (OS) after diagnosis of BM were compared between patients with and without LMC at diagnosis, and prognostic value of LMCi and LMCc were retrospectively compared to clarify the reliable diagnostic method of LMC. Survival differences of patients with LMC after treatment with and without TKIs were also compared to evaluate the efficacy of TKIs on LMC. Results: From Aug. 2006 to now, 215 BMs from EGFRm non-small cell cancers were treated. At diagnosis of BMs, LMCi was diagnosed in 140 cases. Among these cases, CSF cytology was evaluated in 113 cases, but malignant cells were detected only in 47 cases (41.6%). TKIs were administrated in 159 patients. Without TKIs, OS after BM with LMCi (9.5m) was shorter than that without LMCi (26.9m) (HR: 1.75, P=0.096), but this difference was reduced in patients treated with TKIs (18.1m vs. 20.9m, P=0.123). On the other hand, without TKIs, OS after BM with LMCc (4.9m) was extremely worse than that without LMCc (16.6m) (HR:7.76, P=0.001), but this significant difference was lost by TKIs (HR:1.16, P=0.671). During the follow up, 152 deaths were observed, and 45 were owing to the progression of intracranial lesions (CNS death). Not LMCi but LMCc was a significant risk factor of CNS death (OR: 0.59 and 6.26, P=0.148 and 0.002, respectively). TKI had decreased the incidence of CNS death from 100% to 64% in LMCc cases, but LMCc was still a significant risk factor of CNS death even with TKIs (OR: 5.13, P=0.017), and disappearance of cancer cells in CSF by TKIs was observed only in 41.2% of patients. Conclusions: MRI was insufficient to diagnose LMC. TKIs were strongly recommended for patients with LMCc, but its’ effect was still inadequate in half of the patients. Legal entity responsible for the study: Chiba Cancer Center Funding: None Disclosure: All authors have declared no conflicts of interest.

Background: Several actionable gene alterations, such as EGFR and KRAS mutations, for lung cancer are different in frequency between Caucasian and East Asian. NTRK fusions have been recently identified as a therapeutic target in a rare fraction of Caucasian patients with lung adenocarcinoma (3.3%). However, their frequency in East Asian has not yet been examined. Methods: A nationwide lung cancer genomic screening project in Japan (LC-SCRUM-Japan) was initiated from February 2013, and a total of 4118 lung cancer patients have been enrolled as of April 2017. A total of 2668 patients with lung cancer (non-squamous/squamous/small=2088/275/305) were screened by using a next generation sequencing platform, Oncomine™ Focus Assay (OFA) for detecting NTRK1/2/3 fusions or Oncomine™ Comprehensive Assay (OCA) for detecting NTRK1/3 fusions. Results: Only one patient with ETV6-NTRK3 fusion (0.04%) was detected. The patient harboring ETV6-NTRK3 fusion was a 60-year-old man with a 35 pack-year smoking history, and pathologically diagnosed with adenocarcinoma. We also evaluated NTRK1 gene fusion–induced imbalances in the expression of the 5’ and 3’ regions of the transcripts using the OFA sequencing data, and there was no case with 5’/3’ expression imbalances of NTRK1 gene transcripts. Conclusions: The frequency of NTRK fusions in Japanese patients with lung cancer is extremely rare (0.04%) compared with Caucasian. Legal entity responsible for the study: National Cancer Center Funding: Taiho, Chugai, Ono, Astra Zeneka, Bristol-Myers Squibb, MSD, Astellas, Eisai, DAIICHI SANKYO, Pfizer, Kyowa Hakko Kirin, Novartis, Takeda, Eli Lilly Japan, Merck Serono, Boehringer Ingelheim, Amgen Disclosure: T. Hida: Ignyta, Y. Ohe: HONORARIA: AstraZeneca, Chugai, Lilly, ONO, BMS, Daiichi-Sankyo, Nipponkayaku, Boehringer Ingelheim, Bayer, Pfizer, MSD, Taiho RESEARCH FUNDING: AstraZeneca, Chugai, Lilly, ONO, BMS, Kyorin, Dainippon- Sumitomo, Pfizer, Taiho, Novartis, Merck Serono, CONSULTING OR ADVISORY ROLE: AstraZeneca, Chugai, Lilly, ONO, Novartis, S. Kuyama: HONORARIA: AstraZeneca, Meiji Seika, Boehringer Ingelheim, Lilly, Chugai, ONO, MSD, K. Yoh: Dr. Yoh has received personal fees as honoraria from Chugai, Eli Lilly, AstraZeneca, Taiho, Boehringer Ingelheim, Bristol-Myers Squibb, and Ono. His institution has received research funding from Pfizer, Taiho, Eli Lilly, AstraZeneca, Bayer and Novartis. K. Goto: Corporate-sponsored research: Taiho, Chugai, Ono, Astra Zeneka, Sumitomo Dainippon, Bristol-Myers Squibb, MSD, Astellas, Eisai, DAIICHI SANKYO, Pfizer, Kyowa Hakko Kirin, Novartis, Takeda, Eli Lilly Japan, Merck Serono, OxOnc, AbbVie Stemcentrx, Ignyta, Boehringer Ingelheim All other authors have declared no conflicts of interest.

Immune checkpoint inhibitors have shown clinical efficacy in non-small-cell lung cancer (NSCLC). However, data for efficacy in brain metastasis is limited because patients with untreated brain metastases were excluded in clinical trials.

Brain metastases (BMs) are a frequent complication of non-small-cell lung cancer (NSCLC). Whole brain radiation therapy (WBRT) is a standard treatment for BMs, but favorable response of BMs with mutant epidermal growth factor receptor (EGFR) against tyrosine kinase inhibitors (TKIs) have also been reported. The aim of this study is to clarify the effect of TKIs alone without radiation therapy for BMs from EGFR-mutant NSCLC. BMs from NSCLC with EGFR-mutation were enrolled. Gefitinib was administrated first, and erlotinib or afatinib was used at tumor progression. Erlotinib (or afatinib) was also selected for patients whose BMs had developed after gefitinib (or erlotinib). The primary endpoint was overall survival after BM (OS), and the secondary endpoints were maximum response to TKIs, time to progression of intracranial lesions, and time to salvage radiation therapy (RT). In this study, 108 patients with BMs were enrolled. The types of mutations were as follows: exon 19 deletion in 62, exon 20 L858R in 39, and other types of mutations in 7 cases. During the follow-up period, 70 deaths were observed but only 17 of these deaths were owing to the progression of intracranial lesions. The medium OS was 20.9 months. The response rates of first-line and second-line TKIs were 76.9% and 70.6%, respectively. The medium time to progression of intracranial lesions was 14.5 months, and medium time to salvage RT was 19.0 months. Among the patients, gefitinib was administrated in 83 (first-line) and erlotinib in 59 (fist-line:22, second-line, 37) cases. The response rates of gefitnib and erlotinib were 76.8% and 78.6%, and the time to progression of intracranial lesions after gefitinib and erlotinib were 13.9 and 20.3 months, respectively. We could not find any significantly different response of BMs to TKIs owing to the types of mutations. TKIs showed favorable control of BMs harboring EGFR-mutation without RT. At progression, other types of TKIs still showed excellent effect. TKIs first treatment could postpone RT, and feasible for BMs from EGFR-mutant NSCLC.

DNA ‐mediated immunization of a tumour antigen is a possible immunotherapy for cancer, and interleukin ( IL )‐27 has diverse functions in adaptive immunity. In this study, we examined whether IL ‐27 DNA administration enhanced antitumour effects in mice vaccinated with DNA encoding a putative tumour antigen, β ‐galactosidase ( β ‐gal). An intramuscular injection of cardiotoxin before DNA administration facilitated the exogenous gene expression. In mice received β ‐gal and IL ‐27 DNA , growth of β ‐gal‐positive P815 tumours was retarded and survival of the mice was prolonged. Development of β ‐gal‐positive Colon 26 tumours was suppressed by vaccination of β ‐gal DNA and further inhibited by additional IL ‐27 DNA administration or IL ‐12 family cytokines. Nevertheless, a population of β ‐gal‐specific CD 8 + T cells did not increase, and production of anti‐ β ‐gal antibody was not enhanced by IL ‐27 DNA administration. Spleen cells from mice bearing IL ‐27‐expressing Colon 26 tumours showed greater YAC ‐1‐targeted cytotoxicity although CD 3 − / DX 5 + natural killer ( NK ) cell numbers remained unchanged. Recombinant IL ‐27 enhanced YAC ‐1‐targeted cytotoxicity of IL ‐2‐primed splenic NK cells and augmented a phosphorylation of signal transducer and activator of transcription 3 and an expression of perforin. These data collectively indicate that IL ‐27 DNA administration activates NK cells and augments vaccination effects of DNA encoding a tumour antigen through non‐adaptive immune responses.

We examined cytotoxicity of replication-competent type 5 adenoviruses (Ad5) in human pancreatic carcinoma cells with a p53-defective genotype. The replication-competent Ad5 of which E1A gene was activated by exogenous transcriptional regulatory sequences, derived from the midkine and survivin genes, achieved cytotoxicity to the pancreatic carcinoma. These cells were susceptible to replication-incompetent Ad5 expressing the wild-type p53 gene. We also produced the replication-competent Ad5 bearing the same exogenous regulatory sequences and the type 35 Ad-derived fiber-knob region, and showed that the cytotoxicity was comparable to that of the replication-competent Ad5 prototype. We then investigated possible combinatory effects of the fiber-modified replication-competent Ad and Ad5 expressing the wild-type p53 gene, both of which did not interfere respective infections. The combination produced synergistic cytotoxic effects with enhanced cleavages of caspase-3 and PARP molecules, and with increased sub-G1 fractions and annexin V-positive populations although the viral production of the replication-competent Ad was rather suppressed by expressed p53. Pancreatic cells infected with both Ad showed increase of p53 and decrease of MDM2 and p21 levels, compared with those infected with Ad expressing the p53 gene. These data collectively indicated that replication-competent Ad augmented susceptibility of pancreatic cells to apoptosis through upregulated p53 expression.

Zoledronic acid (ZOL), a nitrogen-containing bisphosphonate, produced anti-tumor effects through apoptosis induction or S-phase arrest depending on human mesothelioma cells tested. An addition of isoprenoid, geranylgeraniol but not farnesol, negated these ZOL-induced effects, indicating that the ZOL-mediated effects were attributable to depletion of geranylgeranyl pyrophosphates which were substrates for prenylation processes of small guanine-nucleotide-binding regulatory proteins (small G proteins). ZOL-treated cells decreased a ratio of membrane to cytoplasmic fractions in RhoA, Cdc42 and Rab6 but less significantly Rac1 proteins, indicating that these proteins were possible targets for ZOL-induced actions. We further analyzed which small G proteins were responsible for the three ZOL-induced effects, caspase-mediated apoptosis, S-phase arrest and morphological changes, using inhibitors for respective small G proteins and siRNA for Cdc42. ZOL-induced apoptosis is due to insufficient prenylation of Rab proteins because an inhibitor of geranlygeranyl transferase II that was specific for Rab family proteins prenylation, but not others inhibitors, activated the same apoptotic pathways that ZOL did. ZOL suppressed an endogenous topoisomerase II activity, which was associated with apoptosis and S-phase arrest in respective cells because we detected the same cell cycle changes in etoposide-treated cells. Inhibitors for geranlygeranyl transferase I and for RhoA produced morphological changes and disrupted actin fiber structures, both of which were similar to those by ZOL treatments. These data demonstrated that anti-tumor effects by ZOL were attributable to inhibited functions of respective small G proteins and topoisomerase II activity, and suggested that cellular factors were involved in the differential cell cycle changes.

We examined the combinatory antitumor effects of adenoviruses expressing human mda-7/IL-24 gene (Ad-mda-7) and chemotherapeutic agents on nine kinds of human esophageal carcinoma cells. All the carcinoma cells expressed the melanoma differentiation-associated gene-7/interleukin-24 (MDA-7/IL-24) receptor complexes, IL-20R2 and either IL-20R1 or IL-22R1, and were susceptible to Ad-mda-7, whereas fibroblasts were positive only for IL-20R2 gene and resistant to Ad-mda-7-mediated cytotoxicity. Sensitivity of these esophageal carcinoma cells to Ad-mda-7 was however lower than that to Ad expressing the wild-type p53 gene. We thereby investigated a possible combination of Ad-mda-7 and anticancer agents and found that Ad-mda-7 with 5-fluorouracil (5-FU), cisplatin, mitomycin C or etoposide produced greater cytotoxic effects than those by Ad-mda-7 or the agent alone. Half-maximal inhibitory concentration values of the agents in respective cells were decreased by the combination with Ad-mda-7. Cell cycle analyses showed that Ad-mda-7 and 5-FU increased G2/M-phase and S-phase populations, respectively, and the combination augmented sub-G1 populations. Ad-mda-7-treated cells showed cleavages of caspase-8, -9 and -3 and poly (ADP-ribose) polymerase, but the cleavage levels were not different from those of the combination-treated cells. Ad-mda-7 treatments upregulated Akt phosphorylation but suppressed IκB-α levels, whereas 5-FU treatments induced phosphorylation of p53 and extracellular signal-regulated protein kinases 1 and 2. Molecular changes caused by the combination were similar to those by Ad-mda-7 treatments, but the Ad-mda-7-mediated upregulation of Akt phosphorylation decreased with the combination. These data collectively suggest that Ad-mda-7 induced apoptosis despite Akt activation and that the combinatory antitumor effects with 5-FU were produced partly by downregulating the Ad-mda-7-induced Akt activation.

Pancreatic cancer is one of the intractable diseases and an effective therapeutic strategy is required to improve the prognosis. We examined possible antitumor effects of adenoviruses expressing melanoma differentiation-associated gene-7/interleukin-24 (Admda-7) and a heat-shock protein 90 (Hsp90) inhibitor to human pancreatic carcinoma cells. Ad-mda-7 and an Hsp90 inhibitor, geldanamycin (GA), produced cytotoxic effects, and a combinatory use of Ad-mda-7 and GA further achieved synergistic effects. Administration of N-acetyl-L-cysteine, an inhibitor of reactive oxygen species, eliminated Ad-mda-7- and GA-mediated cytotoxicity. Ad-mda-7 augmented phosphorylated AKT levels but GA did not influence the phosphorylation. GA-treated cells showed cleavage of poly-(ADP-ribose) polymerase but not caspase-3, and upregulated Hsp70 and LC3A/B II levels, whereas Ad-mda-7-treated cells did not. GA treatments augmented ubiquitination and markedly increased melanoma differentiation-associated gene-7 (MDA-7) expression levels. These findings suggest that Ad-mda-7-mediated cytotoxicity is dependent on reactive oxygen species but independent of apoptosis or autophagy, and that GA-mediated cytotoxicity was linked with caspase-independent apoptosis and/or autophagy. A mechanism underlying the combinatory effects of Ad-mda-7 and GA remained complex and the synergism is attributable to multiple factors including increased MDA-7 protein stability by GA.

Background: Brain metastases (BM) are a common in patients with lung cancer. Although whole-brain radiation therapy (WBRT) is the standard therapy, it may have a risk of decline in cognitive function of patients. In this study, we evaluated the efficacy of gefitinib alone without radiation therapy for the treatment of patients with BM from lung adenocarcinoma.Materials and methods: Eligible patients had BM from lung adenocarcinoma with epidermal growth factor receptor (EGFR) mutations. Gefitinib was given at 250 mg orally once a day until tumor progression or unacceptable toxicity.Results: Forty-one patients were enrolled. The response rate was 87.8%. No patient experienced grade >= 4 toxicity. The median progression-free survival time was 14.5 months (95% CI, 10.2-18.3 months), and the median overall survival time was 21.9 months (95% CI, 18.5-30.3 months). In compared with L858R, exon 19 deletion was associated with better outcome of patients after treatment with gefitinib in both progression-free (p = 0.003) and overall survival (p = 0.025).Conclusion: Favorable response of BM to gefitinib even without irradiation was demonstrated. Exon 19 deletion was both a predictive and prognostic marker of patients with BM treated by gefitinib. (C) 2013 Elsevier Ireland Ltd. All rights reserved.

Background: Chemokines and chemokine receptors not only have significant roles in cancer metastasis and tumorigenesis but also act as antitumour agents. The interaction between the Crk-like adaptor protein (CrkL), which is encoded by the CRKL gene, and non-receptor tyrosine kinase c-ABL is reported to transform many cells into malignant cells. We examined the effects of CC chemokine receptor 7 (CCR7), CCR7 ligands and CrkL and c-ABL in lung adenocarcinoma.Methods: One hundred and twenty patients with lung adenocarcinoma were included in this historical cohort analysis. We examined CCR7 and CCR7 ligands and CrkL and c-ABL mRNA expressions in surgically resected lung adenocarcinoma specimens and evaluated their contribution to prognosis, and the relationship with epidermal growth factor receptor (EGFR) and TP53 mutations.Results: High CCR7 mRNA expressions indicated better prognoses than those of the groups with low CCR7 mRNA expressions (P = 0.007, HR = 2.00, 95% CI of ratio: 1.22 -3.31). In lung adenocarcinoma, CrkL and c-ABL mRNAs were related to CCR7 mRNA expression (P<0.0001). CrkL and c-ABL mRNA expressions were influenced by EGFR mutations. A high expression of CCL19 was a good prognostic factor of lung adenocarcinoma.Conclusion: We propose that CCR7 and CCL19 are clinically good prognostic factors and that CCR7 is strongly related to CrkL and c-ABL kinase mRNA expression in lung adenocarcinoma.