Daisuke Minami

Synergistic Effect of Olaparib with Combination of Cisplatin on PTEN-Deficient Lung Cancer Cells

PARP enzyme plays a key role in the cellular machinery responsible for DNA damage repair. PTEN is a tumor-suppressor gene deactivating PI3K downstream of EGFR signaling. We hypothesize that PTEN-deficient lung cancer cells suppressed DNA damage signaling and that the absence of PTEN can sensitize these cells to a concurrent treatment of a DNA-damaging agent (cisplatin) and a PARP inhibitor (olaparib). To investigate the effect of olaparib and cisplatin on PTEN-deficient lung tumors, two EGFR-mutant (deletion in exon19) non–small cell lung cancer (NSCLC) cell lines, PC-9 (PTEN wild-type) and H1650 (PTEN loss), were used. We transfected intact PTEN gene into H1650 cells (H1650PTEN+) and knocked down PTEN expression in the PC-9 cells (PC-9PTEN−) using short hairpin RNA (shRNA). Combination of cisplatin with olaparib showed a synergistic effect in vitro according to the combination index in H1650 cells. Restoration of PTEN in the H1650 cells decreased sensitivity to the combination. Ablation of PTEN in PC-9 cells increased sensitivity to olaparib and cisplatin. We also examined the effectiveness of cisplatin and olaparib in a xenograft model using H1650 and PC-9PTEN− cells. The combination of cisplatin with olaparib was more effective than each agent individually. This effect was not observed in a xenograft model using H1650PTEN+ and PC-9 cells. Mechanistic investigations revealed that PTEN deficiency caused reductions in nuclear RAD51 and RPA focus formation and phosphorylated Chk1 and Mre11. Thus, genetic inactivation of PTEN led to the suppression of DNA repair. Mol Cancer Res; 11(2); 140–8. ©2012 AACR.

Afatinib Prolongs Survival Compared with Gefitinib in an Epidermal Growth Factor Receptor-Driven Lung Cancer Model

An irreversible ErbB family blocker is expected to inhibit tumors with activating epidermal growth factor receptor (EGFR) mutations more strongly than reversible EGFR tyrosine kinase inhibitors and to overcome acquired resistance to the T790M secondary mutation. Eleven-week-old transgenic mice with Egfr exon 19 deletion mutation were treated with afatinib, gefitinib, or vehicle for 4 weeks. All mice were sacrificed at 15 weeks of age, and the number of superficial left lung tumors with a long axis exceeding 1 mm was counted. The afatinib-treated group had significantly fewer tumors than the vehicle group (P < 0.01) and tended to have fewer tumors than the gefitinib-treated group (P = 0.06). Pathologically, gefitinib-treated mice had clearer, more nodular tumors than afatinib-treated mice. Immunoblotting showed that afatinib suppressed not only pEGFR but also pHER2, and induced apoptosis for longer periods than gefitinib. Subsequently, when each drug was administered 5 days per week until death, afatinib significantly enhanced mouse survival compared with gefitinib (median survival time: 456 days vs. 376.5 days; log-rank test, P < 0.01). Finally, the combination of afatinib with bevacizumab was found to be superior to either drug alone in exon 19 deletion/T790M and L858R/T790M xenograft tumors. Overall, afatinib was more potent than gefitinib in tumors harboring an exon 19 deletion mutation, and the combination of afatinib with bevacizumab efficiently suppressed tumors harboring the T790M secondary mutation. Mol Cancer Ther; 12(5); 589–97. ©2013 AACR.

Impact of physical size on gefitinib efficacy in patients with non-small cell lung cancer harboring EGFR mutations.

Gefitinib is an essential drug for the treatment of non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) gene mutations. The approved dosage is 250 mg/body/day without adjustment for physical size such as body surface area (BSA), and the impact of physical size on the efficacy of gefitinib has not been evaluated. Here, we sought to clarify this issue using a retrospective cohort. We reviewed the medical records of patients with consecutive advanced NSCLC harboring EGFR mutations who underwent gefitinib monotherapy at Okayama University Hospital. In total, 101 patients were included in this study, and the median BSA in this cohort was 1.5 m(2). The median progression-free survival (PFS) of the patients with higher BSA (≥1.5 m(2)) was significantly worse than that of those with lower BSA (< 1.5 m(2)) (10.4 vs. 18.0 months; p = 0.019, log-rank test). Multivariate analysis also showed a significant impact of BSA on PFS (hazards ratio, 2.34; 95% confidence interval, 1.78-2.89; p = 0.002). By contrast, no significant association between BSA and PFS was observed in those undergoing cytotoxic chemotherapy (4.0 vs. 5.1 months; p = 0.989, log-rank test), suggesting that BSA is a predictive, rather than a prognostic, marker for gefitinib therapy in EGFR-mutated NSCLC. In conclusion, BSA affected PFS in patients with EGFR-mutated NSCLC who underwent gefitinib monotherapy, suggesting the need for appraisal of BSA-based dose adjustment, even for this molecular target-based therapy.

Vandetanib is effective in EGFR-mutant lung cancer cells with PTEN deficiency.

The effectiveness of vandetanib, an agent that targets RET, VEGFR and EGFR signaling, against EGFR-mutant lung cancer cells with PTEN loss was investigated. Two EGFR mutant non-small cell lung cancer (NSCLC) cell lines, PC-9 (PTEN wild type) and NCI-H1650 (PTEN null), were used. We transfected an intact PTEN gene into H1650 cells and knocked down PTEN expression in PC-9 cells using shRNA. The effectiveness of gefitinib and vandetanib was assessed using a xenograft model. While PC-9 cells were more resistant to vandetanib than gefitinib, H1650 cells were more sensitive to vandetanib than gefitinib. Both gefitinib and vandetanib suppressed the activation of EGFR and MAPK in H1650 cells, although phosphorylated AKT levels were not affected. In an H1650 cell xenograft model, vandetanib was also more effective than gefitinib. Although PTEN-transfected H1650 cells did not show restoration of sensitivity to gefitinib in vitro, the xenograft tumors responded to gefitinib and vandetanib. Knockdown of PTEN in PC-9 cells caused resistance to gefitinib. In conclusion, vandetanib might be effective in NSCLC with EGFR mutations that lack PTEN expression. The contribution of PTEN absence to vandetanib activity in NSCLC cells harboring EGFR mutations should be further examined.

Rapid on-site evaluation with BIOEVALUATOR(®) during endobronchial ultrasound-guided transbronchial needle aspiration for diagnosing pulmonary and mediastinal diseases.

AIM: Rapid on-site evaluation (ROSE) is used widely during endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA). BIOEVALUATOR® is a device used for determining whether the tissues obtained by EBUS-TBNA are appropriate for a pathological diagnosis. This study describes our experience with ROSE using BIOEVALUATOR® during EBUS-TBNA for diagnosing pulmonary and mediastinal diseases. MATERIALS AND METHODS: We retrospectively evaluated the results of 35 patients who underwent EBUS-TBNA with BIOEVALUATOR® between December 2011 and February 2013. For the diagnosis, the tissue areas were appearing white and red through BIOEVALUATOR® are considered to be appropriate and inappropriate, respectively. We examined their medical records to obtain information concerning the examination of BIOEVALUATOR® results of the patients materials (white/red), the diagnosis yield, site and size of lymph nodes and number of needle passes. RESULTS: The median longest diameter of 40 lymph nodes (21 #7, 13 #4R, 4 #4L and 2 #11) from 35 patients was 27.9 (range 12.4-50.6) mm and the median number of needle passes was 2 (range 1-5). The definitive diagnosis was made by EBUS-TBNA in 28 of 35 patients, by thoracotomy in one patient and BIOEVALUATOR® results were white and lymphocytes were seen in the rest six patients. The BIOEVALUATOR® results of other patients without accurate diagnosis were left indefinitive. Finally, the six patients were judged as having benign lymphadenopathy because the lymph node size on computed tomography decreased or remained stable after for at least 8 months. CONCLUSIONS: Checking aspirated samples using BIOEVALUATOR® appears useful for determining their adequacy for pathological diagnosis.

Endobronchial ultrasound-guided transbronchial biopsy with or without a guide sheath for diagnosis of lung cancer.

BACKGROUND Endobronchial ultrasound-guided transbronchial biopsy with a guide sheath (EBUS-GS) is widely used for diagnosing lung cancers; however, the diagnostic yield varies widely. This study aimed to assess the efficiency of EBUS-GS. METHODS We retrospectively evaluated the results of 110 patients who underwent transbronchial biopsy (TBB) for diagnosis of peripheral lung cancer. Bronchoscopy with and without EBUS-GS was performed in 60 (group A) and 50 patients (group B), respectively; their medical records were examined, and results from the two groups were compared by using the unpaired Student t-test. RESULTS The diagnostic sensitivity for lung cancer was 83.3% in group A and 68% in group B (P=0.066) while using at least one of the following procedures: TBB, cytological brushing, and bronchial washing. The diagnostic sensitivity for lesions ≥20mm was 86.4% in group A and 76.7% in group B (P=0.263). Moreover, the diagnostic sensitivity for lesions 10-20mm was 60% in group A and 14.2% in group B (P=0.0004); the diagnostic sensitivity with TBB alone was 63.3% in group A and 44% in group B (P=0.043). The diagnostic sensitivity with TBB alone for lesions ≥20mm was 70.2% in group A and 44.8% in group B (P=0.051). Moreover, the diagnostic sensitivity for lesions 10-20mm in size was 45% in group A and 14.2% in group B with TBB alone (P=0.115). CONCLUSION EBUS-GS with TBB, brushing, and bronchial washing is effective in diagnosing lung cancers sized <20mm.

Usefulness of Endobronchial Ultrasound-guided Transbronchial Needle Aspiration in Distinguishing Sarcoidosis from Recurrent Cancer in Patients with Lymphadenopathy after Surgery

OBJECTIVE Endobronchial ultrasound-guided transbronchial needle aspiration is a new minimally invasive test for investigating mediastinal and hilar lymphadenopathy. It is sometimes difficult to distinguish between a recurrent malignant lymph node and lymphadenopathy due to sarcoidosis in patients who develop lymphadenopathy after surgery for a malignant tumor. METHODS Between December 2009 and October 2012, we performed endobronchial ultrasound-guided transbronchial needle aspiration in 13 selected patients with a suspected recurrence in the mediastinum and/or hilum of the lung after surgical resection of a malignant tumor. We examined their medical records to obtain information on the diagnosis, the sizes of lymph nodes, the number of needle passes and other complications. RESULTS Definitive diagnoses were made using endobronchial ultrasound-guided transbronchial needle aspiration in 10 patients (three lung adenocarcinomas, one prostate carcinoma, one renal cell carcinoma, one neuroendocrine tumor and four sarcoidosis). Pathological specimens showing non-caseating granulomas led to the diagnosis of sarcoidosis in four patients; their previous malignancies had been papillary adenocarcinoma of the thyroid, carcinoma of the gingiva, thymoma and bladder cancer, but no recurrences were observed. The median of the longest diameter in 15 lymph nodes was 22 mm (range 13-35), and the median number of needle passes was two times (range 1-5) without severe complications. CONCLUSIONS Endobronchial ultrasound-guided transbronchial needle aspiration might be useful in differentiating between benign lymphadenopathy, including sarcoidosis, and cancer recurrence in patients with mediastinal or hilar lymphadenopathy after surgical resection of a malignant tumor.

Triplet therapy with afatinib, cetuximab, and bevacizumab induces deep remission in lung cancer cells harboring EGFR T790M in vivo

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have changed the treatment strategy for EGFR‐mutant lung cancers; however, resistance usually occurs due to a secondary mutation, T790M, in EGFR. Combination therapy using afatinib and cetuximab has had good results in lung tumors harboring EGFRT790M mutations in clinical trials. The effect of bevacizumab, an antivascular endothelial growth factor (VEGF) antibody, combined with EGFR‐TKIs has also been investigated. We hypothesized that the dose of afatinib and cetuximab could be reduced by combination with bevacizumab and that the triplet therapy may result in better tumor inhibition with tolerable toxicity. Using a xenograft mouse model with H1975‐harboring EGFRL858R+T790M and RPC‐9‐harboring EGFR19DEL+T790M, we tested the efficacy of the triplet therapy with a modified dose of afatinib, cetuximab, and bevacizumab, and compared this therapy to single and double therapies. Triplet therapy with afatinib, cetuximab, and bevacizumab induced pathological complete remission in xenograft tumors with H1975 and RPC‐9 cells versus tumors treated with single or double therapies. We saw no body weight loss in the mice. The triple therapy induced a significant reduction in CD31‐positive vascular endothelial cells and increased cleaved caspase‐3‐positive cells in the tumors. This suggests that one mechanism underlying the deep remission could be suppression of neovascularization and induction of apoptosis by intensive inhibition of driver oncoproteins and VEGF. These results highlight the potential of afatinib, cetuximab, and bevacizumab to induce deep remission in tumors harboring EGFRT790M mutations. Therefore, clinical trials of this combination therapy are warranted.

Phase II Study of the EGFR-TKI Rechallenge With Afatinib in Patients With Advanced NSCLC Harboring Sensitive EGFR Mutation Without T790M: Okayama Lung Cancer Study Group Trial OLCSG 1403

Abstract Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) as first‐line therapy for patients with EGFR‐mutated non–small‐cell lung cancer (NSCLC) have shown a significantly better objective response rate and progression‐free survival than platinum doublet therapy. However, acquired resistance often occurs within 12 months. One of the potential strategies for treating acquired resistance in NSCLC is the readministration of EGFR‐TKIs, a strategy that has mainly been evaluated using gefitinib or erlotinib. The aim of the present study is to investigate the efficacy and safety of EGFR‐TKI readministration with afatinib in patients with advanced NSCLC harboring activating EGFR mutations without T790M. The primary endpoint is progression‐free survival. The secondary endpoints include the objective response rate, disease control rate, overall survival, toxicity, and quality of life. A total of 12 patients will be enrolled in this trial.

Downregulation of TBXAS1 in an iron‐induced malignant mesothelioma model

Malignant mesothelioma is an aggressive and therapy‐resistant neoplasm arising from mesothelial cells. Evidence suggests that the major pathology associated with asbestos‐induced mesothelioma is local iron overload. In the present study, we induced iron‐induced mesothelioma in rats based on previous reports. Ten Wistar rats were given ferric saccharate and nitrilotriacetate i.p. for 5 days a week. Five of the ten rats exhibited widespread mesotheliomas in the peritoneum and tunica vaginalis. The tumor cells showed positive immunostaining for calretinin, wilms tumor‐1, podoplanin and the oxidative DNA marker 8‐hydroxy‐2′‐deoxyguanosine. In three of the five rats with mesothelioma, array‐based comparative genomic hybridization analysis identified a common chromosomal deletion mapped to the chromosomal 4q31 locus, which encompasses the TBXAS1 gene. Downregulation of the TBXAS1 gene was confirmed using quantitative PCR. TBXAS1 gene expression was also reduced in three of four human malignant pleural mesothelioma cell lines compared with normal bronchial epithelial cells. Immunohistochemistry revealed that TBXAS1 expression was weakly positive and positive in five and three out of eight human malignant mesothelioma samples, respectively. In conclusion, TBXAS1 gene expression was downregulated in rats with iron‐induced mesothelioma. The relationship between iron overload and TBXAS1 downregulation should be pursued further.