Combination of a p53-activating CP-31398 and an MDM2 or a FAK inhibitor produces growth suppressive effects in mesothelioma with wild-type p53 genotype.

A majority of mesothelioma had the wild-type p53 genotype but was defective of p53 functions primarily due to a genetic defect in INK4A/ARF region. We examined a growth suppressive activity of CP-31398 which was developed to restore the p53 functions irrespective of the genotype in mesothelioma with wild-type or mutated p53. CP-31398 up-regulated p53 levels in cells with wild-type p53 genotype but induced cell growth suppression in a p53-independent manner. In contrasts, nutlin-3a, an MDM2 inhibitor, increased p53 and p21 levels in mesothelioma with the wild-type p53 genotype and produced growth suppressive effects. We investigated a combinatory effect of CP-31398 and nutlin-2a and found the combination produced synergistic growth inhibition in mesothelioma with the wild-type p53 but not with mutated p53. Western blot analysis showed that the combination increased p53 and the phosphorylation levels greater than treatments with the single agent, augmented cleavages of PARP and caspase-3, and decreased phosphorylated FAK levels. Combination of CP-31398 and defactinib, a FAK inhibitor, also achieved synergistic inhibitory effects and increased p53 with FAK dephosphorylation levels greater than the single treatment. These data indicated that a p53-activating CP-31398 achieved growth inhibitory effects in combination with a MDM2 or a FAK inhibitor and suggested a possible reciprocal pathway between p53 elevation and FAK inactivation.

Japanese subgroup analysis of a phase III study of S-1 versus docetaxel in non-small cell lung cancer patients after platinum-based treatment: EAST-LC.

INTRODUCTION: The East Asia S-1 Trial in Lung Cancer (EAST-LC) was a randomized phase III study conducted in East Asia that demonstrated the non-inferiority of S-1 to docetaxel in previously treated patients with advanced non-small cell lung cancer (NSCLC). Here, we reported the results of the Japanese subgroup treated with docetaxel 60 mg/m2, the standard dosage in Japan. PATIENTS AND METHODS: Patients were randomized 1:1 to receive either S-1 or docetaxel. The primary endpoint was overall survival (OS); the secondary endpoints included progression-free survival (PFS), response rate (RR), quality of life (QOL), and safety. RESULTS: Patient characteristics in the Japanese subgroup (n = 724) were similar to those in the overall EAST-LC population. Median OS was 13.4 months in the S-1 group and 12.6 months in the docetaxel group. In pemetrexed-pretreated patients, OS with S-1 was similar to that with docetaxel. Median PFS was 2.9 and 3.0 months in the S-1 and docetaxel groups, respectively. RR was 9.4% and 10.3% in the S-1 and docetaxel groups, respectively. The QOL of patients treated with S-1 was better compared with that of patients treated with docetaxel. Decreased appetite and diarrhea were more common in the S-1 group, whereas the frequency of neutropenia and febrile neutropenia was markedly higher in the docetaxel group. CONCLUSIONS: This Japanese subgroup analysis showed that S-1 had similar efficacy to docetaxel in patients with previously treated advanced NSCLC. These results are similar to those of the overall EAST-LC population.

Augmented expression of cardiac ankyrin repeat protein is induced by pemetrexed and a possible marker for the pemetrexed resistance in mesothelioma cells.

BACKGROUND: Pemetrexed (PEM) is an anti-cancer agent targeting DNA and RNA synthesis, and clinically in use for mesothelioma and non-small cell lung carcinoma. A mechanism of resistance to PEM is associated with elevated activities of several enzymes involved in nucleic acid metabolism. METHODS: We established two kinds of PEM-resistant mesothelioma cells which did not show any increase of the relevant enzyme activities. We screened genes enhanced in the PEM-resistant cells with a microarray analysis and confirmed the expression levels with Western blot analysis. A possible involvement of the candidates in the PEM-resistance was examined with a WST assay after knocking down the expression with si-RNA. We also analyzed a mechanism of the up-regulated expression with agents influencing AMP-activated protein kinase (AMPK) and p53. RESULTS: We found that expression of cardiac ankyrin repeat protein (CARP) was elevated in the PEM-resistant cells with a microarray and Western blot analysis. Down-regulation of CARP expression with si-RNA did not however influence the PEM resistance. Parent and PEM-resistant cells treated with PEM increased expression of CARP, AMPK, p53 and histone H2AX. The CARP up-regulation was however irrelevant to the p53 genotypes and not induced by an AMPK activator. Augmented p53 levels with nutlin-3a, an inhibitor for p53 degradation, and DNA damages were not always associated with the enhanced CARP expression. CONCLUSIONS: These data collectively suggest that up-regulated CARP expression is a potential marker for development of PEM-resistance in mesothelioma and that the PEM-mediated enhanced expression is not directly linked with immediate cellular responses to PEM.

Cytotoxicity of replication-competent adenoviruses powered by an exogenous regulatory region is not linearly correlated with the viral infectivity/gene expression or with the E1A-activating ability but is associated with the p53 genotypes.

BACKGROUND: Replication-competent adenoviruses (Ad) produced cytotoxic effects on infected tumors and have been examined for the clinical applicability. A biomarkers to predict the cytotoxicity is valuable in a clinical setting. METHODS: We constructed type 5 Ad (Ad5) of which the expression of E1A gene was activated by a 5' regulatory sequences of survivin, midkine or cyclooxygenase-2, which were highly expressed in human tumors. We also produced the same replication-competent Ad of which the fiber-knob region was replaced by that of Ad35 (AdF35). The cytotoxicity was examined by a colorimetric assay with human tumor cell lines, 4 kinds of pancreatic, 9 esophageal carcinoma and 5 mesothelioma. Ad infectivity and Ad-mediated gene expression were examined with replication-incompetent Ad5 and AdF35 which expressed the green fluorescence protein gene. Expression of cellular receptors for Ad5 and AdF35 was also examined with flow cytometry. A transcriptional activity of the regulatory sequences was investigated with a luciferase assay in the tumor cells. We then investigated a possible correlation between Ad-mediated cytotoxicity and the infectivity/gene expression, the transcriptional activity or the p53 genotype. RESULTS: We found that the cytotoxicity was greater with AdF35 than with Ad5 vectors, but was not correlated with the Ad infectivity/gene expression irrespective of the fiber-knob region or the E1A-activating transcriptional activity. In contrast, replication-competent Ad produced greater cytotoxicity in p53 mutated than in wild-type esophageal carcinoma cells, suggesting a possible association between the cytotoxicity and the p53 genotype. CONCLUSIONS: Sensitivity to Ad-mediated cytotoxic activity was linked with the p53 genotype but was not lineally correlated with the infectivity/gene expression or the E1A expression.

Metformin produces growth inhibitory effects in combination with nutlin-3a on malignant mesothelioma through a cross-talk between mTOR and p53 pathways.

BACKGROUND: Mesothelioma is resistant to conventional treatments and is often defective in p53 pathways. We then examined anti-tumor effects of metformin, an agent for type 2 diabetes, and combinatory effects of metformin and nutlin-3a, an inhibitor for ubiquitin-mediated p53 degradation, on human mesothelioma. METHODS: We examined the effects with a colorimetric assay and cell cycle analyses, and investigated molecular events in cells treated with metformin and/or nutlin-3a with Western blot analyses. An involvement of p53 was tested with siRNA for p53. RESULTS: Metformin suppressed cell growth of 9 kinds of mesothelioma including immortalized cells of mesothelium origin irrespective of the p53 functional status, whereas susceptibility to nutlin-3a was partly dependent on the p53 genotype. We investigated combinatory effects of metformin and nutlin-3a on, nutlin-3a sensitive MSTO-211H and NCI-H28 cells and insensitive EHMES-10 cells, all of which had the wild-type p53 gene. Knockdown of p53 expression with the siRNA demonstrated that susceptibility of MSTO-211H and NCI-H28 cells to nutlin-3a was p53-dependent, whereas that of EHMES-10 cells was not. Nevertheless, all the cells treated with both agents produced additive or synergistic growth inhibitory effects. Cell cycle analyses also showed that the combination increased sub-G1 fractions greater than metformin or nutlin-3a alone in MSTO-211H and EHMES-10 cells. Western blot analyses showed that metformin inhibited downstream pathways of the mammalian target of rapamycin (mTOR) but did not activate the p53 pathways, whereas nutlin-3a phosphorylated p53 and suppressed mTOR pathways. Cleaved caspase-3 and conversion of LC3A/B were also detected but it was dependent on cells and treatments. The combination of both agents in MSTO-211H cells rather suppressed the p53 pathways that were activated by nutrin-3a treatments, whereas the combination rather augmented the p53 actions in NCI-H28 and EHMES-10 cells. CONCLUSION: These data collectively indicated a possible interactions between mTOR and p53 pathways, and the combinatory effects were attributable to differential mechanisms induced by a cross-talk between the pathways.

An image cytometric technique is a concise method to detect adenoviruses and host cell proteins and to monitor the infection and cellular responses induced.

BACKGROUND: Genetically modified adenoviruses (Ad) with preferential replications in tumor cells have been examined for a possible clinical applicability as an anti-cancer agent. A simple method to detect viral and cellular proteins is valuable to monitor the viral infections and to predict the Ad-mediated cytotoxicity. METHODS: We used type 5 Ad in which the expression of E1A gene was activated by 5'-regulatory sequences of genes that were augmented in the expression in human tumors. The Ad were further modified to have the fiber-knob region replaced with that derived from type 35 Ad. We infected human mesothelioma cells with the fiber-replaced Ad, and sequentially examined cytotoxic processes together with an expression level of the viral E1A, hexon, and cellular cleaved caspase-3 with image cytometric and Western blot analyses. RESULTS: The replication-competent Ad produced cytotoxicity on mesothelioma cells. The infected cells expressed E1A and hexon 24 h after the infection and then showed cleavage of caspase-3, all of which were detected with image cytometry and Western blot analysis. Image cytometry furthermore demonstrated that increased Ad doses did not enhance an expression level of E1A and hexon in an individual cell and that caspase-3-cleaved cells were found more frequently in hexon-positive cells than in E1A-positive cells. Image cytometry thus detected these molecular changes in a sensitive manner and at a single cell level. We also showed that an image cytometric technique detected expression changes of other host cell proteins, cyclin-E and phosphorylated histone H3 at a single cell level. CONCLUSIONS: Image cytometry is a concise procedure to detect expression changes of Ad and host cell proteins at a single cell level, and is useful to analyze molecular events after the infection.

Retrospective analysis of lung cancer patients treated with supportive care alone.

BACKGROUND: It is not uncommon for patients with lung cancer to receive supportive care alone. However, the clinical characteristics of these patients have not been fully studied. We conducted a retrospective study to identify the clinical characteristics of definitive lung cancer patients treated with supportive care alone. METHODS: We retrospectively analyzed the percentage of and reasons for definitive lung cancer patients treated with supportive care alone at a regional cancer center. We also investigated the histological diagnostic approaches, palliative therapy types, primary treatment locations after hospital consultation, and places of death. RESULTS: A total of 1,223 patients were histologically diagnosed as having lung cancer between 2011 and 2014. Of these, 160 (13%) patients were treated with supportive care alone. The primary reason for treatment with supportive care alone was a poor performance status (PS) in almost half of the patients. Overall, 40% of the patients received supportive care at home, and 17% were admitted to a palliative care unit (PCU). Death occurred at home for 17% of the patients and in the PCU for 42% of the patients. CONCLUSION: This study revealed that 13% of histologically proven lung cancer patients were treated with supportive care alone, mostly because of a poor PS. Only 40% of these patients received home care, suggesting the need for a more accessible home care system for patients and their families.

Combination of a third generation bisphosphonate and replication-competent adenoviruses augments the cytotoxicity on mesothelioma.

BACKGROUND: Approximately 80 % of mesothelioma specimens have the wild-type p53 gene, whereas they contain homozygous deletions in the INK4A/ARF locus that encodes p14 (ARF) and the 16 (INK4A) genes. Consequently, the majority of mesothelioma is defective of the p53 pathways. We examined whether zoledronic acid (ZOL), a third generation bisphosphonate, and adenoviruses with a deletion of the E1B-55kD gene (Ad-delE1B55), which augments p53 levels in the infected tumors, could produce combinatory anti-tumor effects on human mesothelioma cells bearing the wild-type p53 gene. METHODS: Cytotoxicity of ZOL and Ad-delE1B55 was assessed with a WST assay. Cell cycle changes were tested with flow cytometry. Expression levels of relevant molecules were examined with western blot analysis to investigate a possible mechanism of cytotoxicity. Furthermore, the expressions of Ad receptors on target cells and infectivity were estimated with flow cytometry. Viral replication was assayed with the tissue culture infection dose method. RESULTS: A combinatory use of ZOL and Ad-delE1B55 suppressed cell growth and increased sub-G1 or S-phase populations compared with a single agent, depending on cells tested. The combinatory treatment up-regulated p53 levels and subsequently enhanced the cleavage of caspase-3, 8, 9 and poly (ADP-ribose) polymerase, but expression of molecules involved in autophagy pathways were inconsistent. ZOL-treated cells also increased Ad infectivity with a dose-dependent manner and augmented Ad replication although the expression levels of integrin molecules, one of the Ad receptors, were down-regulated. CONCLUSIONS: These findings indicated that ZOL and Ad-delE1B55 achieved combinatory anti-tumor effects through augmented apoptotic pathways or increased viral replication.

Replication-competent adenoviruses with the type 35-derived fiber-knob region achieve reactive oxygen species-dependent cytotoxicity and produce greater toxicity than those with the type 5-derived region in pancreatic carcinoma.

Pancreatic carcinoma is relatively resistant to chemotherapy and cell death induced by replication of adenoviruses (Ad) can be one of the therapeutic options. Transduction efficacy of conventional type 5 Ad (Ad5) is however low and the cytotoxic mechanism by replication-competent Ad was not well understood. We constructed replication-competent Ad5 of which the E1A promoter region was replaced with a transcriptional regulatory region of the midkine, the survivin or the cyclooxygenase-2 gene, all of which were expressed at a high level in human tumors. We also prepared replication-competent Ad5 that were activated with the same region but had the type 35 Ad-derived fiber-knob region (AdF35) to convert the major cellular receptor for Ad infection from the coxsackie adenovirus receptor to CD46 molecules. Replication-competent AdF35 that were activated with the exogenous region produced cytotoxic effects on human pancreatic carcinoma cells greater than the corresponding Ad5 bearing with the same regulatory region. Cells infected with the AdF35 showed cytopathic effects and increased sub-G1 fractions. Caspase-9, less significantly caspase-8 and poly (ADP-ribose) polymerase, but not caspase-3 was cleaved and expression of molecules involved in autophagy and caspase-independent cell death pathways remained unchanged. Nevertheless, H2A histone family member X molecules were phosphorylated, and N-acetyl-L-cystein, an inhibitor for reactive oxygen species, suppressed the AdF35-mediated cytotoxicity. These data indicated a novel mechanism of Ad-mediated cell death and suggest a possible clinical application of the fiber-knob modified Ad.

A combinatory use of adenoviruses expressing melanoma differentiation-associated gene-7 and replication-competent adenoviruses produces synergistic effects on pancreatic carcinoma cells.

Type 5 adenoviruses expressing mda-7 gene (Ad-mda-7) induced cell death in various kinds of human tumors, but pancreatic carcinoma cells were relatively resistant to Ad-mda-7-mediated cytotoxicity. We then examined whether infection of Ad-mda-7 together with replication-competent Ad produced combinatory cytotoxic effects. We prepared replication-competent Ad, defective of the E1B55kDa gene or activated by a transcriptional regulatory region of the midkine or the survivin gene of which the expression was up-regulated in human tumors. Type 5 Ad bearing the exogenous regulatory region were further modified by replacing the fiber-knob region with that of type 35 Ad. Pancreatic carcinoma cells were infected with replication-incompetent Ad-mda-7 and the replication-competent Ad. Combinatory effects were examined with the CalcuSyn software and cell cycle analyses. Ad-mda-7 and the replication-competent Ad achieved cytotoxicity to pancreatic carcinoma. A combinatory use of Ad-mda-7 and either Ad defective of the E1B55kDa gene or Ad activated by the regulatory region produced synergistic cytotoxic effects. Cell cycle analyses demonstrated that the combination increased sub-G1 populations. These data collectively suggest that expression of MDA-7 augments cytotoxicity of replication-competent Ad and achieves adjuvant effects on Ad-mediated cell death.

Cytotoxic effects of replication-competent adenoviruses on human esophageal carcinoma are enhanced by forced p53 expression.

BACKGROUND: Improvement of transduction and augmentation of cytotoxicity are crucial for adenoviruses (Ad)-mediated gene therapy for cancer. Down-regulated expression of type 5 Ad (Ad5) receptors on human tumors hampered Ad-mediated transduction. Furthermore, a role of the p53 pathways in cytotoxicity mediated by replication-competent Ad remained uncharacterized. METHODS: We constructed replication-competent Ad5 of which the E1 region genes were activated by a transcriptional regulatory region of the midkine or the survivin gene, which is expressed preferentially in human tumors. We also prepared replication-competent Ad5 which were regulated by the same region but had a fiber-knob region derived from serotype 35 (AdF35). We examined the cytotoxicity of these Ad and a possible combinatory use of the replication-competent AdF35 and Ad5 expressing the wild-type p53 gene (Ad5/p53) in esophageal carcinoma cells. Expression levels of molecules involved in cell death, anti-tumor effects in vivo and production of viral progenies were also investigated. RESULTS: Replication-competent AdF35 in general achieved greater cytotoxic effects to esophageal carcinoma cells than the corresponding replication-competent Ad5. Infection with the AdF35 induced cleavages of caspases and increased sub-G1 fractions, but did not activate the autophagy pathway. Transduction with Ad5/p53 in combination with the replication-competent AdF35 further enhanced the cytotoxicity in a synergistic manner. We also demonstrated the combinatory effects in an animal model. Transduction with Ad5/p53 however suppressed production of replication-competent AdF35 progenies, but the combination augmented Ad5/p53-mediated p53 expression levels and the downstream pathways. CONCLUSIONS: Combination of replication-competent AdF35 and Ad5/p53 achieved synergistic cytotoxicity due to enhanced p53-mediated apoptotic pathways.

Phase II study of carboplatin, docetaxel and bevacizumab for chemotherapy-naïve patients with advanced non-squamous non-small cell lung cancer.

PURPOSE: To evaluate a 3-drug combination of carboplatin, docetaxel and bevacizumab as a front-line chemotherapy for patients with advanced non-squamous non-small cell carcinoma (NSCLC), a single arm phase II study was conducted. METHODS: Patients with stage IIIB/IV or postoperative recurrent non-squamous NSCLC were treated with carboplatin (targeted area under the curve of 6 mg h/L), docetaxel (60 mg/m(2)), and bevacizumab (15 mg/kg) on day 1, repeated every 3 weeks for 4 to 6 cycles, followed by maintenance with bevacizumab every 3 weeks until disease progression or occurrence of predefined toxicity. The planned patient number was 40, and the primary endpoint was progression free survival (PFS) as assessed by independent reviewers. RESULTS: One patient refused the treatment after enrollment; thus, 39 patients were treated and analyzed. The 3-drug therapy was delivered for a median of 4 cycles, and 54 % of the patients proceeded to the maintenance therapy for a median of 4 cycles. The overall response rate was 74.4 % (29/39), with a 95 % confidence interval (CI) of 60.0 to 88.7 %. The median PFS and overall survival (OS) were 6.2 months (95 % CI, 4.8-8.5 months) and 22.4 months (95 % CI, 11.3-26.2 months), respectively. Toxicities of grade 3 or higher included neutropenia in 71.8 %, febrile neutropenia in 23.1 %, and hypertension in 38.5 % of the patients, but they were transient and manageable. CONCLUSION: The primary endpoint was met. The regimen yielded promising results with an excellent overall response rate, PFS, and OS for chemotherapy-naïve patients with advanced non-squamous NSCLC. Further studies are warranted.

Frequency of brain metastases in non-small-cell lung cancer, and their association with epidermal growth factor receptor mutations.

BACKGROUND: The brain is a frequent site of metastases from non-small-cell lung cancer (NSCLC). We analyzed the frequency of brain metastases (BMs) from NSCLC in the era of magnetic resonance images, and evaluated the correlation between epidermal growth factor receptor (EGFR) mutations and BMs among East Asian patients. METHODS: Frequency, number, and size of BMs, and survival of 1,127 NSCLC patients were retrospectively reviewed. Mutation status of EGFR was evaluated in all cases, and its association with BMs was statistically evaluated. RESULTS: EGFR mutations were found for 331 cases (29.4 %). BM was the cause of primary symptoms for 52 patients (4.6 %), and found before initiation of treatment for 102 other patients (9.1 %); In addition to these 154 patients, 107 patients (9.5 %) developed BMs, giving a total of 261 patients (23.2 %) who developed BMs from 1,127 with NSCLC. BM frequency was higher among EGFR-mutated cases (31.4 %) than EGFR-wild cases (19.7 %; odds ratio: 1.86; 95 % confidence interval (CI) 1.39-2.49; P < 0.001). BMs from EGFR-mutated NSCLC were small, but often became disseminated. EGFR mutations accounted for 39.9 % of BMs, but patient survival after BMs was significantly longer for EGFR-mutated cases than for EGFR-wild cases (hazard ratio: 2.23; 95 % CI 1.62-3.10; P < 0.001). CONCLUSIONS: Patients with EGFR-mutated NSCLC were more likely to develop BMs, but apparently also survived longer after BMs.

The significance and robustness of a plasma free amino acid (PFAA) profile-based multiplex function for detecting lung cancer.

BACKGROUND: We have recently reported on the changes in plasma free amino acid (PFAA) profiles in lung cancer patients and the efficacy of a PFAA-based, multivariate discrimination index for the early detection of lung cancer. In this study, we aimed to verify the usefulness and robustness of PFAA profiling for detecting lung cancer using new test samples. METHODS: Plasma samples were collected from 171 lung cancer patients and 3849 controls without apparent cancer. PFAA levels were measured by high-performance liquid chromatography (HPLC)-electrospray ionization (ESI)-mass spectrometry (MS). RESULTS: High reproducibility was observed for both the change in the PFAA profiles in the lung cancer patients and the discriminating performance for lung cancer patients compared to previously reported results. Furthermore, multivariate discriminating functions obtained in previous studies clearly distinguished the lung cancer patients from the controls based on the area under the receiver-operator characteristics curve (AUC of ROC = 0.731 ~ 0.806), strongly suggesting the robustness of the methodology for clinical use. Moreover, the results suggested that the combinatorial use of this classifier and tumor markers improves the clinical performance of tumor markers. CONCLUSIONS: These findings suggest that PFAA profiling, which involves a relatively simple plasma assay and imposes a low physical burden on subjects, has great potential for improving early detection of lung cancer.

Real-World Data Analysis of Pembrolizumab Monotherapy for NSCLC Using Japanese Postmarketing All-Case Surveillance Data.

Introduction: Pembrolizumab is a programmed death-ligand 1 inhibitor that was initially indicated for monotherapy in patients with advanced lung cancer. The Japanese Lung Cancer Society conducted an observational study on pembrolizumab using confirmative data obtained through postmarketing all-case surveillance (PMACS), which was performed by a pharmaceutical company under the Japanese law in 2017. Methods: This multicenter observational study was conducted by the Japanese Lung Cancer Society using PMACS data with the newly created central registration system regarding patients with NSCLC who received pembrolizumab monotherapy between February 1, 2017 and June 30, 2017; a new database was created by adding the clinical information regarding prognosis for 3 years after therapy to the existing data collected by PMACS. Results: A total of 300 patients from 43 facilities were enrolled in this study. The median overall survival and progression-free survival after pembrolizumab initiation were 558 and 188 days, respectively. Moreover, the 1- and 3-year survival rates were 58.9% and 33.7%, respectively. Results of multivariate analysis revealed performance status (p < 0.0001), histology (p = 0.0118), previous chemotherapy (p = 0.0007), programmed death-ligand 1 expression status (p = 0.0195), and previous steroid use (p = 0.0460) as significant factors that affected overall survival. The toxicity profile was similar to that previously reported. Conclusions: In this first attempt to use PMACS data, we successfully collected clinical information and found the real-world efficacy and safety of pembrolizumab.

Comparison of 21-gauge and 22-gauge aspiration needle during endobronchial ultrasound-guided transbronchial needle aspiration.

BACKGROUND AND OBJECTIVE: Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) has typically been performed using the 22 gauge (G) dedicated TBNA needle. Recently a new 21G TBNA needle has been introduced. The efficacy of using a larger gauge biopsy needle during EBUS-TBNA has not been reported. The purpose of this study was to compare the diagnostic yield and utility of 21G and 22G needles during EBUS-TBNA. METHODS: EBUS-TBNA was performed using both 21G and 22G needles. Cytological and histological findings were recorded for each samples obtained by an independent cytologist and pathologist. The cellularity and blood contamination were evaluated in the cytological samples. The quality of the histological core was evaluated by the amount of blood clots versus the actual tissue. Each factor was compared within two slides from the two different size needles. The diagnostic yield and the differences of the cytology and histology were analysed. RESULTS: The evaluation of 45 lesions by EBUS-TBNA revealed that tumour cells were equally detected by both 21G and 22G needles. Two patients of adenocarcinoma were histologically diagnosed only by the 21G needle. Although histological structure was better preserved in five lesions collected by the 21G needle, there was more blood contamination with the 21G needle (P < 0.0001). CONCLUSIONS: There were no differences in the diagnostic yield between the 21G and 22G needles during EBUS-TBNA. The preserved histological structure of the samples obtained by the 21G needle may be useful for the diagnosis of mediastinal and hilar adenopathy of unknown aetiology which may be a challenge with the 22G needle.

An MDM2 inhibitor achieves synergistic cytotoxic effects with adenoviruses lacking E1B55kDa gene on mesothelioma with the wild-type p53 through augmenting NFI expression.

A majority of mesothelioma specimens were defective of p14 and p16 expression due to deletion of the INK4A/ARF region, and the p53 pathway was consequently inactivated by elevated MDM2 functions which facilitated p53 degradaton. We investigated a role of p53 elevation by MDM2 inhibitors, nutlin-3a and RG7112, in cytotoxicity of replication-competent adenoviruses (Ad) lacking the p53-binding E1B55kDa gene (Ad-delE1B). We found that a growth inhibition by p53-activating Ad-delE1B was irrelevant to p53 expression in the infected cells, but combination of Ad-delE1B and the MDM2 inhibitor produced synergistic inhibitory effects on mesothelioma with the wild-type but not mutated p53 genotype. The combination augmented p53 phosphorylation, activated apoptotic but not autophagic pathway, and enhanced DNA damage signals through ATM-Chk2 phosphorylation. The MDM2 inhibitors facilitated production of the Ad progenies through augmented expression of nuclear factor I (NFI), one of the transcriptional factors involved in Ad replications. Knocking down of p53 with siRNA did not increase the progeny production or the NFI expression. We also demonstrated anti-tumor effects by the combination of Ad-delE1B and the MDM2 inhibitors in an orthotopic animal model. These data collectively indicated that upregulation of wild-type p53 expression contributed to cytotoxicity by E1B55kDa-defective replicative Ad through NFI induction and suggested that replication-competent Ad together with augmented p53 levels was a therapeutic strategy for p53 wild-type mesothelioma.

Clinical value of PET/CT with carbon-11 4DST in the evaluation of malignant and benign lung tumors.

OBJECTIVES: The aim of this study was to assess the clinical value of [11C]4DST uptake in patients with lung nodules, including benign and malignant tumors, and to assess the correlation between [11C]4DST uptake and proliferative activity of tumors in comparison with [18F]FDG uptake. METHODS: Twenty-six patients (22 males and 4 females, mean age of 65.5-year-old) were analyzed in this prospective study. Patients underwent [11C]4DST and [18F]FDG PET/CT imaging on the same day. Diagnosis of each lung nodule was confirmed by histopathological examination of tissue specimens at surgery, or during clinical follow-up after the PET/CT studies. To assess the utility of the semi-quantitative evaluation method, the SUVmax was calculated of [11C]4DST and [18F]FDG uptake by the lesion. Proliferative activities of each tumor as indicated by the immunohistochemical Ki-67 index was also estimated using surgical specimens of patients. Then the relationship between the SUVmax of both PET/CT and the Ki-67 index was examined. Furthermore, the relationship between the uptake of [11C]4DST or [18F]FDG and the histopathological findings, the clinical stage, and the clinical outcome of patients were also assessed. RESULTS: There was a positive linear relationship between the SUVmax of [11C]4DST images and the Ki-67 index (Correlation coefficients = 0.68). The SUVmax of [11C]4DST in the 26 lung nodules were 1.65 ± 0.40 for benign lesions, 3.09 ± 0.83 for adenocarcinomas (P < 0.001 between benign and adenocarcinoma), and 2.92 ± 0.58 for SqCCs (P < 0.001 between benign and SqCC). Whereas, the SUVmax of [18F]FDG were 2.38 ± 2.27 for benign lesions, 6.63 ± 4.24 for adenocarcinomas (n.s.), and 7.52 ± 2.84 for SqCCs (n.s.). The relationship between TNM tumor stage and the SUVmax of [11C]4DST were 2.54 ± 0.37 for T1, 3.48 ± 0.57 for T2, and 4.17 ± 0.72 for T3 (P < 0.005 between T1 and T2, and P < 0.001 between T1 and T3). In comparison with the TNM pathological stage, SUVmax of [11C]4DST were 2.63 ± 0.49 for stage I, 3.36 ± 0.23 for stage II, 3.40 ± 1.12 for stage III, and 4.65 for stage IV (P < 0.05 between stages I and II). In comparison of the clinical outcome, the SUVmax of [11C]4DST were 2.72 ± 0.56 for the no recurrence (No Rec.) group, 3.10 ± 0.33 for the recurrence-free with adjuvant chemotherapy after the surgery (the No Rec. Adjv. CTx. group) and 4.66 ± 0.02 for the recurrence group (Rec. group) (P < 0.001 between the No Rec and Rec. groups, and P < 0.005 between the No Rec. Adjv. CTx. and Rec. groups). CONCLUSIONS: PET/CT with [11C]4DST is as feasible for imaging of lung tumors as [18F]FDG PET/CT. For diagnosing lung tumors, [11C]4DST PET is useful in distinguishing benign nodules from malignancies. [11C]4DST uptake in lung carcinomas is correlated with the proliferative activity of tumors, indicating a promising noninvasive PET imaging of DNA synthesis in malignant lung tumors.

A Case of Long-Term Survival after Checkpoint Inhibitor Pneumonitis in a Patient with Squamous Cell Lung Cancer.

The management of grade 1 checkpoint inhibitor pneumonitis (CIP) is to withhold immune checkpoint inhibitors; however, the natural history of this condition is unknown. We herein report the case of a woman with squamous cell lung cancer who was a long-term survivor after CIP. After 4 rounds of treatment with nivolumab, a chest CT revealed a reticular pattern and ground-glass attenuation with shrinkage of the primary nodule. Nivolumab treatment was withheld without the administration of steroids. Although she remained asymptomatic, subsequent images revealed an increasing interstitial shadow until 2 months after the stop of nivolumab treatment. Thereafter, the interstitial shadow began to improve spontaneously without steroid treatment. Moreover, although the patient has not received additional therapy, disease control of lung cancer has been obtained within a follow-up period of more than 3 years. Although the exacerbation of CIP may appear on images for several months, asymptomatic cases can be followed without the administration of steroids. If the tumor had already responded prior to the onset of CIP, a favorable long-term prognosis can be expected.

AMPK activation induced in pemetrexed-treated cells is associated with development of drug resistance independently of target enzyme expression.

Pemetrexed (PEM) inhibits DNA and RNA synthesis and is currently one of the first-line agents for mesothelioma. PEM suppresses the activities of several enzymes involved in purine and pyrimidine synthesis, and elevated activity of these enzymes in tumors is often linked with resistance to PEM. The agent also stimulates AMP-activated protein kinase (AMPK) and consequently influences the mammalian target of rapamycin complex 1 (mTORC1) pathways. Nevertheless, it remains unclear whether PEM resistance is linked to the AMPK or mTORC1 pathways. Here, we established two independent PEM-resistant mesothelioma cell lines in which expression of the PEM-target enzymes was not elevated, and found that levels of phosphorylated AMPK and p70S6K and, to a lesser extent, levels of phosphorylated AKT and p53, were increased in these cells as compared with the respective parent cells. PEM stimulation also augmented phosphorylation of AMPK, p70S6K, AKT and p53 in most cases. An AMPK activator increased phosphorylation and PEM resistance in parental cells, and the inhibitor decreased the resistance of PEM-resistant cells. In contrast, inhibitors for p70S6K and AKT did not influence PEM resistance; furthermore, increased levels of endogenous p53 did not affect PEM sensitivity. These data collectively indicate that constitutive activation of AMPK is associated with PEM resistance, and that this is unconnected with elevated DNA and RNA synthesis.