Masato Katagiri

EGFR genetic heterogeneity of nonsmall cell lung cancers contributing to acquired gefitinib resistance

Gefitinib is dramatically effective for nonsmall cell lung cancers (NSCLCs) with activating mutations of the epidermal growth factor receptor (EGFR) gene, but these tumors eventually develop drug resistance, attributable to a secondary T790M mutation or acquired MET amplification in some relapsed tumors. We analyzed EGFR mutations in matched pre‐ and post‐therapeutic tumors of 6 gefitinib‐responding lung cancers. With conventional PCR‐based sequencing, classic mutations were detected in pretreatment samples of each case. The same mutations were readily confirmed in treated lesions of 4 cases, but were absent in those of Cases 1 and 2. Subsequent mutant‐enriched peptide‐nucleic‐acid‐mediated PCR clamping and subcloning assays detected the mutation in minor cells of treated lesions of Case 1, but still failed to detect a mutation in Case 2. We thus performed microdissection‐based cell cluster mutation analysis of pretreatment tumors, and found that 3, including the first 2, concurrently contained tumor cells with either mutant or wild‐type EGFR, although the latter was only a minor fraction. These findings suggest that some NSCLCs are genetically heterogeneous with regard to EGFR mutations; gefitinib‐sensitive mutants decrease or vanish while wild clones selectively survive with gefitinib treatment. In addition, T790M was detected in a small fraction of treated lesions of 3 cases, and MET amplification was revealed in 3 treated tumors of Case 2. Thus, our results suggest that multiple mechanisms underlie acquired gefitinib resistance, and selection on a background of EGFR genetic heterogeneity also contributes to acquisition of resistance in a proportion of NSCLCs.

Successful rechallenge with erlotinib in a patient with EGFR-mutant lung adenocarcinoma who developed gefitinib-related interstitial lung disease

Small-molecule tyrosine kinase inhibitors (TKIs) targeting the epidermal growth factor receptor (EGFR) pathways are used clinically for patients with non-small cell lung cancer (NSCLC). It is well established that somatic mutations in the kinase domain of the EGFR (Lynch et al. in N Engl J Med 350:2129–2139, 2004; Paez et al. in Science 304:1497–1500, 2004) are strongly associated with the tumor response and clinical outcomes in patients with NSCLC receiving EGFR-TKIs (Mitsudomi and Yatabe in Cancer Sci 98:1817–1824, 2007). Although the most common adverse events are skin rash and diarrhea, the most serious adverse effect reported is drug-related interstitial lung disease (ILD) (Inoue et al. in Lancet 361:137–139, 2003; Ando et al. in J Clin Oncol 24:2549–2556, 2006). The precise mechanism underlying the development of drug-related ILD remains unknown. Here, we describe a case of EGFR-mutant NSCLC who was rechallenged with the small-molecule EGFR antagonist erlotinib after developing gefitinib-related ILD.

Respiratory muscle electromyogram and mouth pressure during isometric contraction.

When extra-diaphragmatic muscles are activated progressively under approximately isometric conditions, we expect a corresponding increase in respiratory muscle output. Therefore, we examined relative recruitment shown as the latency to onset of EMG activity, and the relationship between mouth pressure and electromyogram activity of the neck accessory and transversus abdominis (TRANS) muscles during respiratory maneuvers against occlusion. Fine wire electrodes were inserted into the scalene (SCLN), sternocleidomastoid (STERNO), trapezius (TRAPZ) and TRANS in six awake, healthy subjects. Mouth pressure, raw and moving average EMG signals were recorded during gradual production of expiratory or inspiratory mouth pressure to maximum (MPmax) at FRC in the standing posture. Group mean linear regression lines of EMG activity versus mouth pressure were strongly significant for SCLN and TRANS, less for STERNO, and least for TRAPZ. The SCLN and STERNO showed EMG activities with low, and TRAPZ showed EMG activity only with high, mouth pressure. At 90% MPmax, TRAPZ was much less active compared with TRANS, SCLN, or STERNO. These results suggest that over a wide range of respiratory effort there is a significant difference in the relationship between mouth pressure and EMG activity in the accessory muscles, with differential recruitment of individual respiratory muscles.

Impact of PD-L1 Expression in Patients with Surgically Resected Non-Small-Cell Lung Cancer

Background: Immunotherapy can become a crucial therapeutic option to improve the prognosis of patients with non-small-cell lung cancer (NSCLC). Here, we evaluated the impact of programmed cell death ligand-1 (PD-L1) expression in surgically resected NSCLCs. Methods: We estimated PD-L1 expression in 229 consecutive NSCLC specimens using rabbit polyclonal antibodies to human PD-L1 in a SP263 immunohistochemical assay and evaluated PD-L1 expression for potential associations with clinicopathological parameters and survival time. Results: PD-L1 expression was significantly higher in tumors from men or current smokers. Squamous cell carcinoma histology was independently associated with high PD-L1 expression according to multivariate analysis (p = 0.015). The 5-year survival rate of patients was 70%, and the difference in the 5-year survival rate according to PD-L1 expression was not statistically significant (high expression group [67%] vs. low expression group [68%]); however, the squamous cell carcinoma group exhibited significantly lower 5-year survival rates as compared to the non-squamous cell carcinoma group (53 and 71%, respectively; p = 0.026). Conclusion: Here, we revealed high PD-L1 expression and poor prognosis observed in patients with surgically resected squamous NSCLC as compared with non-squamous NSCLC. Our results support the identification of patient subsets that most likely respond to anti-PD-1 therapy as the first step in precision medicine.

Effect of gefitinib on warfarin antithrombotic activity.

BackgroundDespite the literature indicating adverse interactions between warfarin and cytotoxic agents, whether such an interaction occurs when warfarin and gefitinib are used concomitantly is unknown. We analyzed the prevalence of the concomitant use of warfarin and gefitinib, and the incidence of prothrombin time-international normalized ratio (PT-INR) alterations or adverse interactions in concomitant users of warfarin and gefitinib.MethodsWe conducted a retrospective study of patients with non-small cell lung cancer treated at the Kitasato University Hospital who received concomitant warfarin and gefitinib between September 2002 and January 2007. Medical information, including the indication for warfarin use, warfarin dosing and dosing changes, and exposure to gefitinib were collected from computerized databases and medical records.ResultsTwelve (4.1%) of 296 patients treated with gefi— tinib received warfarin. PT-INR elevation occurred in 6 patients (50.0%). Two (16.7%) of the 12 patients had liver metastases. Liver dysfunction was associated with PT-INR elevation (P = 0.0100).ConclusionAs there is a possibility of PT-INR abnormalities occurring during the concomitant use of gefitinib and warfarin, clinicians should be aware of this interaction. Because of the potentially severe consequences of this interaction, close monitoring of PT-INR and warfarin dose adjustment are recommended for patients receiving warfarin and gefitinib, especially during the first 2 weeks in the beginning of warfarin therapy.

Angiotensin-converting enzyme inhibition curbs tyrosine nitration of mitochondrial proteins in the renal cortex during the early stage of diabetes mellitus in rats

Experiments were performed to evaluate the hypothesis that ACE (angiotensin-converting enzyme) inhibition (enalapril) suppresses 3-NT (3-nitrotyrosine) production in the renal cortex during the early stage of Type 1 DM (diabetes mellitus) in the rat. Enalapril was administered chronically for 2 weeks to subsets of STZ (streptozotocin)-induced DM and vehicle-treated sham rats. O2− (superoxide anion) and NOx (nitrate+nitrite) levels were measured in the media bathing renal cortical slices after 90 min incubation in vitro. SOD (superoxide dismutase) activity and 3-NT content were measured in the renal cortex homogenate. Renal cortical nitrated protein was identified by proteomic analysis. Renal cortical production of O2− and 3-NT was increased in DM rats; however, enalapril suppressed these changes. DM rats also exhibited elevated renal cortical NOx production and SOD activity, and these changes were magnified by enalapril treatment. 2-DE (two-dimensional gel electrophoresis)-based Western blotting revealed more than 20 spots with positive 3-NT immunoreactivity in the renal cortex of DM rats. Enalapril treatment blunted the DM-induced increase in tyrosine nitration of three proteins ACO2, GDH1 and MMSDH (aconitase 2, glutamate dehydrogenase 1 and methylmalonate-semialdehyde dehydrogenase), each of which resides in mitochondria. These data are consistent with enalapril preventing DM-induced tyrosine nitration of mitochondrial proteins by a mechanism involving suppression of oxidant production and enhancement of antioxidant capacity, including SOD activation.

Aminophylline increases parasternal intercostal muscle activity during hypoxia in humans.

To clarify the mechanism of action of aminophylline on the hypoxic ventilatory response in humans, we analyzed the effects of aminophylline on respiratory neural output. To evaluate the respiratory neural output, we analyzed the electromyogram (EMG) of the parasternal intercostal muscle, one of the major inspiratory muscles, in eight healthy subjects. Both before and during aminophylline administration, measurements of ventilatory parameters with EMG recordings were conducted in room air, mild hypoxia (F(I)(o)(2) 0.15), and severe hypoxia (F(I)(o)(2) 0.11). Before administering aminophylline, hypoxic stimulation elicited ventilatory augmentation in a hypoxia-intensity dependent manner. Administration of aminophylline caused significant increases in ventilation (V (I)), tidal volume (V(T)), respiratory frequency (f(R)), and the respiration-related phasic moving averaged EMG amplitude (tidal EMG), at corresponding levels of hypoxia compared to before aminophylline. Augmentation patterns of hypoxia-induced increases in V(T) and tidal EMG showed close similarity. These results indicate that augmentation of hypoxic ventilatory response by aminophylline is mainly mediated by an increase in the respiratory neural drive in healthy humans.

Effect of salbutamol on respiratory muscle function and ventilation in awake canines.

The effect of the beta-agonist bronchodilator salbutamol on respiratory muscles and ventilation is uncertain. The presence of beta2 receptors on skeletal muscles and increased diaphragm contractility in vitro with salbutamol predict a significant effect that has not been confirmed, in vivo in non-fatigued diaphragm or in clinical studies using standard bronchodilator dosages. Therefore, we infused salbutamol at a higher dosage (23.3 microg/min) used clinically for treatment of respiratory emergencies, while measuring directly the length, shortening and EMG activation of costal and crural diaphragm, parasternal intercostal and transversus abdominis muscles, in 10 awake canines. At this salbutamol dosage, ventilation and tidal volume increased significantly during both resting and CO2-stimulated breathing. Salbutamol elicited significant increases in respiratory muscle shortening with much smaller increases in EMG activity, so the proportionally greater muscle shortening per unit EMG showed increased muscle contractility. The effects of salbutamol were not extinguished by inspiratory flow resistance or fluid challenge but were reversed specifically by the beta-blocker, propranolol. This study demonstrates that, in sufficient intravenous dosage, the beta-agonist salbutamol elicits increased ventilation and a beta2 receptor-mediated increase in contractility of respiratory muscles.

Impact of Smoking History on the Efficacy of Gefitinib in Patients with Non-Small Cell Lung Cancer Harboring Activating Epidermal Growth Factor Receptor Mutations.

Background: Gefitinib treatment has come to be recognized as the standard therapy for patients with non-small cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutations. However, resistance to gefitinib has been observed in certain subpopulations of these patients. The purpose of this study was to evaluate the impact of smoking status on the efficacy of gefitinib in patients with NSCLC harboring EGFR mutations. Methods: The records of NSCLC patients harboring EGFR mutations who were treated with gefitinib at Kitasato University Hospital were retrospectively reviewed, and the treatment outcomes were evaluated. Results: In 153 patients with NSCLC harboring EGFR mutations, the overall response rate and progression-free survival (PFS) were 66.7% and 9.0 months, respectively. PFS differed significantly among the current smokers and never-smokers/former light smokers (10.7 vs. 5.4 months, p = 0.0002), and the response rate was significantly higher in the never-smokers/former light smokers than in the current smokers (72.3 vs. 55.8%, p = 0.04). Multivariate analysis identified smoking status as an independent predictor of PFS. Conclusion: The clinical data obtained in this study provide a valuable rationale for considering smoking status as a predictor of the efficacy of gefitinib in patients with NSCLC harboring activating EGFR mutations.

Aminophylline increases respiratory muscle activity during hypercapnia in humans

BACKGROUND Theophylline is an old drug traditionally used as a bronchodilator, although it was recently shown to possess anti-inflammatory properties, enhance the actions of corticosteroid actions, and stimulate the respiratory neuronal network. Theophylline has been recognized as an important drug for not only asthma but also corticosteroid-insensitive chronic obstructive pulmonary disease (COPD). To clarify the role of theophylline in hypercapnic ventilatory responses in humans, we analyzed the effects of aminophylline administered at the usual clinical therapeutic doses on ventilation and augmentation of respiratory muscle contractility in room air and under 3 conditions of hypercapnia. STUDY DESIGN We performed electromyography (EMG) of the parasternal intercostal muscle (PARA) and transversus abdominis muscle (TA) in 7 healthy subjects and recorded both ventilatory parameters and EMG data in room air and under 3 conditions of hypercapnia before (control) and during aminophylline administration. RESULTS Before aminophylline administration (control), hypercapnic stimulation elicited ventilatory augmentation in a hypercapnia intensity-dependent manner. Ventilatory parameters (tidal volume, frequency of respiration, and minute ventilation) showed significant increases from lower PaCO2 levels during aminophylline administration when compared with the corresponding values before aminophylline administration. EMG activity of both PARA and TA increased significantly at each level of hypercapnia, and those augmentations were shown from lower PaCO2 levels during aminophylline administration. CONCLUSION Aminophylline administered at the usual clinical therapeutic dose increases ventilation and EMG activity of both inspiratory and expiratory muscles during hypercapnia in healthy humans.