Atsushi Hayata

Peroxynitrite augments fibroblast-mediated tissue remodeling via myofibroblast differentiation

Irreversible airflow limitation in asthma is associated with airway remodeling in which the differentiation of fibroblasts to myofibroblasts plays a pivotal role. In asthmatic airways, excessive production of reactive nitrogen species (RNS) has been observed. The aim of this study is to evaluate whether peroxynitrite, one of the RNS, can affect the differentiation of fibroblasts to myofibroblasts. Human fetal lung fibroblasts were treated with various concentrations of authentic peroxynitrite or a peroxynitrite donor 3-morpholinosydnonimine hydrochloride (SIN-1), and the expressions of alpha-smooth muscle actin (alpha-SMA) and desmin, markers of myofibroblast differentiation, were evaluated. The releases of transforming growth factor-beta(1) (TGF-beta(1)) and ECM proteins including fibronectin and collagen I were assessed. To clarify the mechanism in this differentiation, the effect of anti-TGF-beta antibody or NF-kappaB inhibitors on the alpha-SMA expression and ECM production was assessed. Peroxynitrite and SIN-1 significantly augmented the alpha-SMA expression compared with control in a concentration-dependent manner (P < 0.01 and P < 0.05, respectively). Peroxynitrite significantly increased desmin and TGF-beta(1) production (P < 0.01). Peroxynitrite enhanced the translocation of NF-kappaB into the nucleus confirmed by immunocytostaining and immunoblotting. Peroxynitrite-augmented alpha-SMA expression was blocked by NF-kappaB inhibitors, MG132 and caffeic acid phenethyl ester (CAPE), and anti-TGF-beta antibody. CAPE completely inhibited the peroxynitrite-augmented TGF-beta(1) release. The production of fibronectin and collagen I was significantly increased by peroxynitrite (P < 0.01) and inhibited by anti-TGF-beta antibody. These results suggest that RNS can affect the differentiation to myofibroblasts and excessive ECM production via a NF-kappaB-TGF-beta(1)-dependent pathway.

Progression of Irreversible Airflow Limitation in Asthma: Correlation with Severe Exacerbations

BACKGROUND Severe exacerbations of asthma are periods of excess functional and pathological changes in the airways that have been proposed to induce airway remodeling. OBJECTIVE The objective of this study was to explore whether severe exacerbations are correlated with the decline in post-bronchodilator forced expiratory volume in 1 second (FEV1) and loss of bronchodilator reversibility (BDR). METHODS We examined the changes in FEV1 and BDR in 140 nonsmoking patients with well-controlled asthma at baseline and correlated these changes with the frequency of severe asthma exacerbations. RESULTS A 3-year follow-up assessment was completed in 128 patients. A total of 28 (21.9%) patients experienced at least 1 severe exacerbation with a mean rate of 0.16 year(-1). The exacerbation rate was significantly correlated with an annual rate of decline in FEV1 (ρ = 0.49, P < .0001). Both patients with 1 exacerbation and those with 2 or more exacerbations had greater declines in FEV1 than patients with no exacerbations (no exacerbation, 13.6 mL/year; 1 exacerbation, 41.3 mL/year; 2 or more exacerbations, 58.3 mL/year; P < .01 and P < .0001, respectively). The changes in BDR from baseline to the end of the study in patients who did or did not experience an exacerbation were -1.2% and 0.1%, respectively (P < .0005). The changes in BDR were significantly correlated with the annual rates of change in FEV1 (r = 0.40, P < .0001). CONCLUSION The occurrence of severe exacerbations of asthma is correlated with the progression of irreversible airflow limitation over time. This suggests that asthma exacerbations could have the long-term adverse consequences of structural and functional changes in the airways.

Correlation between immune-related adverse events and efficacy in non-small cell lung cancer treated with nivolumab

OBJECTIVES Patients treated with nivolumab often experience its unique adverse events, called immune-related adverse events (irAEs). Regarding the mechanisms of immune-checkpoint inhibitors (ICIs), the occurrence of irAEs may also reflect antitumor responses. Here, we report the clinical correlation between irAEs and efficacy in NSCLC patients treated with nivolumab. MATERIALS AND METHODS Between December 2015 and February 2017, 38 advanced NSCLC patients were treated in our institution. All the patients were enrolled in our single-institutional, prospective, observational cohort study (UMIN000024414). IrAEs were defined as having a potential immunological basis that required more frequent monitoring and potential intervention. We divided the patients into two groups (irAEs group or no-irAEs group) and evaluated the objective response rate (ORR) and progression-free survival (PFS). RESULTS The median age of the patients was 68.5 years (range 49-86 years); male/female ratio was 28/10; squamous/non-squamous cell carcinoma cases were 10/28; performance status was 0-1/2/3, 7/26/5. Among the overall population, ORR was 23.7% and median PFS was 91days. At the data cutoff, 14 irAEs were observed. The most common irAE was interstitial pneumonia (n=5). Other irAEs were hypothyroidism (n=4), hyperthyroidism, hypopituitarism, liver dysfunction, rash, and elevated thyroid stimulating hormone levels (n=1, each). Patients with irAEs had significantly higher ORRs compared with no-irAE patients (63.6% versus 7.4%, p <0.01). Similarly, the PFS among irAE patients was longer (median: not reached [95% confidence interval {CI}: 91days to not applicable]) than no-irAE patients (median 49days [95% CI: 36-127days], hazard ratio [HR] 0.10 [95% CI: 0.02-0.37, p<0.001]). Landmark analysis of patients who achieved PFS ≥60days demonstrated similar tendencies, but this was not significant (HR 0.28 [95% CI: 0.04-1.46], p=0.13). CONCLUSION There was a correlation between irAE and efficacy in NSCLC patients treated with nivolumab.

Development of an automated size-based filtration system for isolation of circulating tumor cells in lung cancer patients

Circulating tumor cells (CTCs), defined as tumor cells circulating in the peripheral blood of patients with solid tumors, are relatively rare. Diagnosis using CTCs is expected to help in the decision-making for precision cancer medicine. We have developed an automated microcavity array (MCA) system to detect CTCs based on the differences in size and deformability between tumor cells and normal blood cells. Herein, we evaluated the system using blood samples from non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) patients. To evaluate the recovery of CTCs, preclinical experiments were performed by spiking NSCLC cell lines (NCI-H820, A549, NCI-H23 and NCI-H441) into peripheral whole blood samples from healthy volunteers. The recovery rates were 70% or more in all cell lines. For clinical evaluation, 6 mL of peripheral blood was collected from 50 patients with advanced lung cancer and from 10 healthy donors. Cells recovered on the filter were stained. We defined CTCs as DAPI-positive, cytokeratin-positive, and CD45-negative cells under the fluorescence microscope. The 50 lung cancer patients had a median age of 72 years (range, 48–85 years); 76% had NSCLC and 20% had SCLC, and 14% were at stage III disease whereas 86% were at stage IV. One or more CTCs were detected in 80% of the lung cancer patients (median 2.5). A comparison of the CellSearch system with our MCA system, using the samples from NSCLC patients, confirmed the superiority of our system (median CTC count, 0 versus 11 for CellSearch versus MCA; p = 0.0001, n = 17). The study results suggest that our MCA system has good clinical potential for diagnosing CTCs in lung cancer.

Stratifying the risk of COPD exacerbation using the modified Medical Research Council scale: A multicenter cross-sectional CAP study

1016/j.resinv.2014.10.006 he Japanese Respiratory Society. Published by Else of Internal Medicine, Wakayama Medical Universit n; c National Hospital Organization Wakayama Hos raduate School of Medicine, Japan; f Kamei Respirator al, Japan; i NTT East Tohoku Hospital, Japan; j Hirosh spital, Japan; l Minami Tokushima Clinic, Japan; m Hir n; o Tohoku Rosai Hospital, Japan. uthor. Tel.: þ81 73 441 0619; fax: þ81 73 447 2201. kazmatsu@wakayama-med.ac.jp (K. Matsunaga) a-med.ac.jp (K. Akamatsu), tsuna@wakayama-m .pikara.ne.jp (T. Kamei), tsudat@k-you.or.jp (T. Ts i@east.ntt.co.jp (T. Takahashi), hozawa@vesta.ocn ikara.ne.jp (Y. Sakamoto), hrkjmsom@air.ocn.ne.j izusawa.iwate.jp (U. Katsumata), mmiura@tohok hoku.ac.jp (M. Ichinose). by physicians is limited [3], and there is a need for a simple assessment tool that can be used in everyday practice [4]. The modified Medical Research Council (mMRC) scale is a wellvalidated questionnaire that uses a simple grading system to quantify disability associated with breathlessness [5], and current guidelines advocate the use of this scale to assess

Stratifying a Risk for an Increased Variation of Airway Caliber among the Clinically Stable Asthma

BACKGROUND Recently, correlations of peak expiratory flow (PEF) variation have been shown to facilitate the prediction of later asthma symptoms and exacerbations. However, it has not been fully examined whether or not any patient characteristics are associated with the residual airway lability in treated asthmatics. The objective of this study is to examine a predictive marker for increased variation of PEF in patients with clinically stable asthma. METHODS We studied 297 asthmatic patients who were monitored for PEF twice a day. Asthma Control Questionnaire (ACQ), spirometry, and exhaled nitric oxide fraction (FENO) were measured. After the assessment of baseline values, PEF measuring was continued and associations between these clinical markers and later variation of PEF over a week (Min%Max) were investigated. RESULTS 17.5% of the subjects showed increased PEF variability (Min%Max < 80%). ACQ, forced expiratory volume in 1 s % of predicted (%FEV1), and FENO were identified as independent predictors of Min%Max < 80%. An ACQ ≥ 0.4 yielded 96% sensitivity and 59% specificity, a %FEV1 ≤ 85% yielded 62% sensitivity and 89% specificity, and a FENO ≥ 40 ppb yielded 75% sensitivity and 90% specificity for identifying the subjects with high variability in PEF. When we combine %FEV1 ≤ 85% and FENO ≥ 40 ppb, this index showed the highest specificity (98%) for increased PEF variability. CONCLUSIONS These results indicate that ACQ, %FEV1 and FENO can stratify the risk for increased variation in airway caliber among patients with stable asthma. This may help identify subjects in whom further monitoring of lung function fluctuations is indicated.

Abstract A056: Sequential tracking of PD-L1 expression on circulating tumor cells in NSCLC patients treated with nivolumab

Background: Nivolumab (anti-PD-1 antibody) has become a new standard treatment in pretreated, advanced non-small cell lung cancer (NSCLC). Although strong expression of PD-L1 on tumor tissue has predictive value, significance of its expression on circulating tumor cell (CTC) is unknown and its status can be changed during the treatment. Here, we conducted a serial evaluation of PD-1-expressing CTCs in NSCLC patients treated with nivolumab. Methods: Advanced NSCLC patients who receive nivolumab at Wakayama Medical University Hospital were enrolled in this prospective observational study (registered at UMIN (000024414)). Nivolumab was administered 3 mg/kg biweekly until progressive disease (PD) or unacceptable toxicity. Peripheral whole blood (3 mL) was collected in a EDTA collection tube (BD vacutainer) and processed within 3 hours for CTC evaluation at baseline, week 4, and week 8. CTCs were detected using microcavity array system (Hitachi Chemical Co.). PD-L1 expression was immunohistochemically examined on both tumor tissues and CTCs using anti-PD-L1 antibody, clone 28-8 (Abcam). Results: Thirty-eight patients were registered in this study between January 2016 and September 2016. Clinical characteristics of the patients were as follows: median age 68 (range, 49 to 86); male 73%; stage IV 100%; squamous/non-squamous, 30/65%. Regarding nivolumab treatment, overall response rate (ORR) was 22% (95% confidence interval (CI): 10-38%), and median progression-free survival (PFS) was 62 days (95%CI: 40-235 days). At baseline, CTCs were detected in all patients (median, 15; range, 1-90) and PD-L1-expressing CTCs were detected in 87% of patients. Tumor proportion score (TPS) of PD-L1 expression on CTCs varied from 6% to 100%. Matched tumor tissues were available from 14 patients and 7 showed the PD-L1 TPS ≥ 50%. PD-L1 status on CTCs was not correlated with that on tumor tissues both using proportional score and H score (Spearman’s correlation: r = 0.0007 and 0.08, respectively). On CTCs, patients with PD-L1 ≥ 50 have significantly higher disease control rate than those with below 50% (83.3% versus 36.4%, p Citation Format: Hiroaki Akamatsu, Yasuhiro Koh, Keita Mori, Kuninobu Kanai, Atsushi Hayata, Nahomi Tokudome, Masayuki Higuchi, HIsashige Kanbara, Keiichiro Akamatsu, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. Sequential tracking of PD-L1 expression on circulating tumor cells in NSCLC patients treated with nivolumab [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A056.

Abstract A057: Serial evaluation of multiple serum protein levels in non-small lung cancer patients treated with nivolumab

Background: Blockade of programmed death receptor-1 (PD-1) pathway is effective against various malignancies. Although PD-ligand 1 (PD-L1) expression on tumor tissue has been established as companion diagnostics in non-small cell lung cancer (NSCLC), additional biomarkers to enrich the patients likely to benefit from the therapy are critically needed. Here, we conducted a serial evaluation of multiple serum cytokines, growth factors, and angiogenesis factors relevant to immune checkpoint blockade in NSCLC patients treated with nivolumab. Patients and Methods: Advanced NSCLC patients after failure of at least one prior chemotherapy regimen received nivolumab monotherapy (3mg/kg, q2W) until progressive disease (PD) or unacceptable toxicity. Serum samples were collected in a serum separation tube (Venoject II autosep, TERUMO) at baseline and at week 4. Best response was classified into partial response (PR), stable disease (SD), or progressive disease (PD) according to RECIST v1.1. Using Luminex TM xMap TM technology, serum levels of 54 proteins consisting of cytokines, chemokines, growth factors, and angiogenesis factors were analyzed. All statistical analyses were carried out using JMP Pro software (ver. 13.0) and Mann-Whitney U test and Spearman9s test were performed accordingly. A p value Citation Format: Jun Oyanagi, Yasuhiro Koh, Hiroaki Akamatsu, Kuninobu Kanai, Atsushi Hayata, Nahomi Tokudome, keiichiro Akamatsu, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. Serial evaluation of multiple serum protein levels in non-small lung cancer patients treated with nivolumab [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A057.

O2-7-3Correlation between immune-related adverse events and efficacy in non-small cell lung cancer treated with nivolumab

events, nivolumab-induced pneumonitis, which infrequently develops but sometimes results in a fatal outcome, requires an early detection and prompt response. The purpose of this study was to understand the pathogenesis of nivolumab-induced pneumonitis, leading to avoiding its onset and increase in severity. Patients and Methods: The subjects were patients with malignant melanoma (n1⁄4 2) and non-small cell lung cancer (NSCLC) (n1⁄4 2), all of whom developed nivolumabinduced pneumonitis in Tokyo Medical University Hachioji Medical Center. We retrospectively analyzed clinicopathological characteristics of these patients from the medical records.

Applicability of the Japanese equation for estimating glomerular filtration rate in patients with advanced-stage thoracic cancer

BACKGROUND The 24-h creatinine clearance (24-h Ccr) and the Cockcroft-Gault equation (CG) are commonly used as markers of renal function in clinical practice. However, the utility of the Japanese equation for estimating glomerular filtration rate (eGFR) in cancer patients has not yet been evaluated. The aim of this cross-sectional study was to investigate the extent and correlating factors for differences between eGFR and both 24-h Ccr and CG in advanced-stage thoracic cancer patients. METHODS eGFR, 24-h Ccr, and CG were calculated in 90 patients with thoracic malignancies. We evaluated how these three parameters are affected by clinical factors, including age, body surface area, serum creatinine concentration, and body mass index. RESULTS eGFR and CG were significantly correlated with 24-h Ccr (r=0.64, p<0.001 and; r=0.67, p<0.001, respectively). However, the median value derived from eGFR was higher than the median 24-h Ccr and the CG value (74.0, 65.2, and 63.9mL/min, respectively). Age had a significant positive correlation with the differences between eGFR and both 24-h Ccr and CG value (r=0.30, p=0.005 and; r=0.47, p<0.001, respectively). The differences between eGFR and the other two parameters were significantly higher in older patients (age≥70 years) than in younger patients (age<70 years) (p=0.023, p<0.001, respectively). CONCLUSIONS eGFR is likely to overestimate the renal function of elderly cancer patients. A modified equation for evaluating the renal function of Japanese older patients might be needed.