Yoshitaka Yagi

High-dose crizotinib for brain metastases refractory to standard-dose crizotinib.

Journal of Thoracic Oncology ® • Volume 8, Number 9, September 2013 CASE REPORT A 41-year-old man, a former smoker (20 pack-years), was referred to Kyoto University Hospital, Kyoto, Japan, because of an abnormal chest shadow detected at an annual medical checkup. Positron emission tomography revealed multiple metastases to both the liver and bone. A biopsy of the pulmonary nodule demonstrated adenocarcinoma. Combination chemotherapy with pemetrexed and carboplatin was started, but the disease progressed after six cycles of chemotherapy. He then received multilines of chemotherapy, including gemcitabine, bevacizumab, erlotinib, TS-1, and docetaxel. Two years after the start of treatment, he developed multiple brain metastases and received whole-brain radiation therapy. At that time, molecular testing for echinoderm microtubule-associated protein like 4 (EML4)anaplastic lymphoma kinase (ALK) had just become commercially available in Japan, and his tumor demonstrated ALK rearrangement (Fig. 1). Then crizotinib was started at a dose of 250 mg twice daily. Abdominal computed tomography showed a marked decrease in the size of liver metastases, and magnetic resonance imaging of the brain demonstrated improvement of brain metastases; however, regrowth of brain metastases was detected 8 months after the start of crizotinib. Extracranial disease, including liver metastases, was under control with crizotinib. We therefore decided to increase the dose of crizotinib after a thorough discussion. At first, the dose was escalated to 750 mg/d. The dose was then escalated to 1000 mg/d after we confirmed that no new toxicities had occurred. Brain magnetic resonance imaging reassessed 2 weeks after the start of dose escalation demonstrated the striking improvement of multiple brain metastases; however, his brain diseases rapidly progressed after 1 month from the dose escalation (Fig. 2). After the dose escalation, he developed bradycardia without accompanying any symptoms. Pulse rate before and after the dose escalation were 50/minute and 39/minute, respectively. DISCUSSION Although it was reported that crizotinib is effective for brain metastases, its penetration into cerebrospinal fluid (CSF) is considered to be poor. Costa et al. measured the concentration of crizotinib in both CSF and plasma and reported that the CSF-to-plasma ratio of crizotinib was only 0.0026. Because of this relatively low drug exposure, the brain may be a site susceptible to progression in patients with ALK rearrangement and treated with crizotinib. A similar scenario has been observed also in patients with epidermal growth factor receptor (EGFR) mutation and treated with EGFR tyrosine kinase inhibitors, and the efficacy of high-dose gefitinib was reported in patients with EGFR mutation who developed brain metastases during treatment with a standard dose of gefitinib. Recently, Gandhi et al. reported a patient who was effectively treated with high-dose pemetrexed and high-dose crizotinib in the same situation; however, the escalated dose of crizotinib was only 100 mg/d, and the efficacy mainly seemed to be the result of pemetrexed. Therefore, we believe that this is the first case in which high-dose crizotinib was used alone for refractory brain metastases that developed during treatment with the standard dose of crizotinib. However, in this case, response duration was quite short and CSF concentration was not measured. In addition, 1000 mg/d of crizotinib is above the maximum tolerated dose. High-dose crizotinib cannot be recommended in routine clinical practice because safety data and efficacy are lacking in a prospective cohort. Further research is required in this setting.

Significance of c-MET overexpression in cytotoxic anticancer drug-resistant small-cell lung cancer cells

The c‐MET receptor tyrosine kinase is the receptor for hepatocyte growth factor. Recently, activation of the c‐MET/hepatocyte growth factor signaling pathway was associated with poor prognosis in various solid tumors and was one of the mechanisms of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitor, gefitinib. But the link between c‐MET activation and the cytotoxic anticancer drug has not been fully examined. Here, we found that the enhanced expression and activation of c‐MET in cytotoxic anticancer agent‐resistant small‐cell lung cancer cells. Downregulation of c‐MET expression by siRNA against the c‐MET gene or inhibition of c‐MET activation by SU11274, a c‐MET inhibitor, in the resistant cells altered resistance to the cytotoxic anticancer agent. These results indicated that c‐MET overexpression might play an important role in acquired resistance to cytotoxic anticancer drugs. Furthermore, the number of c‐MET gene loci was increased in the resistant cells compared to the parental cells. In conclusion, increased c‐Met expression through an increase in the number of c‐MET gene loci is one of the mechanisms of acquired resistance to cytotoxic anticancer drugs. Our results add a new strategy, the targeting of c‐MET, for overcoming resistance to cytotoxic agents in small‐cell lung cancer.

Clinical Impact of Single Nucleotide Polymorphism in PD-L1 on Response to Nivolumab for Advanced Non-Small-Cell Lung Cancer Patients

This study was intended to determine the efficacy of nivolumab, we evaluated treatment response with respect to PD-1/PD-L1 SNPs among patients with NSCLC. A total of 50 patients with NSCLC were treated with nivolumab and were also evaluated for PD-1/PD-L1 single nucleotide polymorphisms (SNPs) from plasma DNA. We investigated the association among PD-1/PD-L1 SNPs, objective response rate (ORR) and progression-free survival (PFS). Two of seven SNPs studied showed association with ORR and PFS, with maximum evidence at the marker rs2282055. The ORR was 25%, 15%, and 0% for the G/G, G/T and T/T genotypes of PD-L1 rs2282055, respectively. The G allele of PD-L1 rs2282055 was significantly associated with better clinical response compared with the T allele (P = 0.0339 [Cochran-Armitage trend test]). The median PFS time was 2.6 months (95% confidence interval [CI], 1.8 months to 4.3 months) for the G/G and G/T genotypes and 1.8 months (95% confidence interval [CI], 0.4 months to 2.2 months) for the T/T genotype (P = 0.0163). Moreover, the C/C and C/G genotypes of PD-L1 rs4143815 were significantly associated with better ORR and PFS in NSCLC patients treated with nivolumab. These results suggest that rs2282055 and rs4143815 may be a biomarker for the efficacy of nivolumab.

Successful treatment with carboplatin and nanoparticle albumin-bound paclitaxel in a patient with pulmonary spindle cell carcinoma

Introduction Pulmonary spindle cell carcinoma (SpCC) is a rare subtype of non-small-cell lung cancer (NSCLC) and, in general, is chemoresistance. Case A sixty-five year-old male patient with metastatic pulmonary SpCC was initially treated with cisplatin and docetaxel, but his disease progressed. Then, he received a combination chemotherapy with carboplatin and nab-PTX followed by maintenanced chemotherapy with nab-PTX. Fluorodeoxyglucose (FDG) positron-emission CT revealed a substantial decrease of FDG accumulation in the primary tumor, and the response continued for more than 7 months. Discussion Preclinical models suggested that nab-PTX may reach the tumor microenvironment more efficiently than solvent-based paclitaxel (sb-PTX) and be preferentially taken up by cancer cells. Considering that there is no effective treatment for patients with pulmonary SpCC, nab-PTX may merit further investigation in patients with pulmonary SpCC.

Clinical impact of high serum hepatocyte growth factor in advanced non-small cell lung cancer

Activation of c-MET through hepatocyte growth factor (HGF) increases tumorigenesis, induces resistance, and is associated with poor prognosis in various solid tumors. However, the clinical value of serum HGF (sHGF) in patients with advanced non-small cell lung cancer (NSCLC), especially those receiving cytotoxic chemotherapy, remains unknown. Here, we show that sHGF may be useful to predict tumor response and progression-free survival (PFS) in patients with advanced NSCLC. A total of 81 patients with NSCLC were investigated. sHGF levels were evaluated using ELISA at 4 time-points: at pre-treatment, at response-evaluation (1–2 months after treatment initiation), at the best tumor response, and at disease progression. As a control biomarker, CEA was also evaluated in lung adenocarcinoma. Positive-sHGF at response-evaluation predicted poor PFS compared with Negative-sHGF in both first-line (median, 153.5 vs. 288.0; P < 0.05) and second-line treatment (87.0 vs. 219.5; P = 0.01). In 55 patients that received cytotoxic chemotherapy, multiple Cox proportional hazards models showed significant independent associations between poor PFS and Positive-sHGF at response-evaluation (hazard ratio, 4.24; 95% CI, 2.05 to 9.46; P < 0.01). Lung adenocarcinoma subgroup analysis showed that in patients receiving second cytotoxic chemotherapy, there were no significant differences in PFS between patients with low-CEA compared with those with high-CEA, but Positive-sHGF at pre-treatment or at response-evaluation predicted poor PFS (35.0 vs. 132.0; P < 0.01, 50.0 vs. 215.0; P < 0.01, respectively). These findings give a rationale for future research investigating the merit of sHGF as a potential clinical biomarker to evaluate HGF/c-MET activity, which would be useful to indicate administration of c-MET inhibitors.Activation of c-MET through hepatocyte growth factor (HGF) increases tumorigenesis, induces resistance, and is associated with poor prognosis in various solid tumors. However, the clinical value of serum HGF (sHGF) in patients with advanced non-small cell lung cancer (NSCLC), especially those receiving cytotoxic chemotherapy, remains unknown. Here, we show that sHGF may be useful to predict tumor response and progression-free survival (PFS) in patients with advanced NSCLC. A total of 81 patients with NSCLC were investigated. sHGF levels were evaluated using ELISA at 4 time-points: at pre-treatment, at response-evaluation (1-2 months after treatment initiation), at the best tumor response, and at disease progression. As a control biomarker, CEA was also evaluated in lung adenocarcinoma. Positive-sHGF at response-evaluation predicted poor PFS compared with Negative-sHGF in both first-line (median, 153.5 vs. 288.0; P < 0.05) and second-line treatment (87.0 vs. 219.5; P = 0.01). In 55 patients that received cytotoxic chemotherapy, multiple Cox proportional hazards models showed significant independent associations between poor PFS and Positive-sHGF at response-evaluation (hazard ratio, 4.24; 95% CI, 2.05 to 9.46; P < 0.01). Lung adenocarcinoma subgroup analysis showed that in patients receiving second cytotoxic chemotherapy, there were no significant differences in PFS between patients with low-CEA compared with those with high-CEA, but Positive-sHGF at pre-treatment or at response-evaluation predicted poor PFS (35.0 vs. 132.0; P < 0.01, 50.0 vs. 215.0; P < 0.01, respectively). These findings give a rationale for future research investigating the merit of sHGF as a potential clinical biomarker to evaluate HGF/c-MET activity, which would be useful to indicate administration of c-MET inhibitors.

Response to chemotherapy with carboplatin plus albumin‑bound paclitaxel in a patient with lymphoepithelioma‑like thymic carcinoma: A case report

Thymic carcinoma is a rare neoplasm with a poor outcome due to its aggressive characteristics. For patients who are not operable, radiation therapy and/or palliative chemotherapy are indicated. However, no optimal chemotherapy regimen has been established. The present study reports the case of a 22-year-old man with advanced lymphoepithelioma-like thymic carcinoma refractory to conventional chemotherapy with carboplatin plus solvent-based paclitaxel (sb-PAC) treatment. The patient was subsequently treated with carboplatin plus nanoparticle albumin-bound paclitaxel (nab-PAC). The treatment resulted in a partial response following three cycles of chemotherapy. Since only grade 3 neutropenia, but no other severe adverse effects, was observed, no dose reduction was required. To the best of our knowledge, the current study is the first to present the response to chemotherapy with carboplatin plus nab-PAC in a patient with lymphoepithelioma-like thymic carcinoma. Considering that no standard treatment has been established in thymic carcinoma, nab-PAC may merit further investigation in this rare, but aggressive disease.

Abstract 5045: Overexpression of ABC transporters through HGF/c-MET activation results in anti-cancer drug resistance in SCLC

Upregulation of hepatocyte growth factor (HGF)/c-MET pathway causes drug resistance to anti-cancer agents such as epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors in non-small lung cancer (NSCLC) with EGFR mutation and cytotoxic agents in small cell lung cancer (SCLC), but the roles and mechanisms of c-MET in drug-resistant cancer cells are not clarified. We studied to reveal the role of c-MET overexpression in the resistant lung cancer cell lines and to demonstrate whether MET-inhibition could restore drug sensitivity in anti-cancer drug resistant cell lines via ABC transporters down-regulation. In this study we used the 7-ethyl-10-hydroxycamptothesin (SN-38)-resistant cell line (PC-6/SN-38) that was derived from the human SCLC cell line PC-6. We compared the expression levels of the purposed genes by quantitative real-time PCR (qRT-PCR) and western blotting (WB). MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assay was used to measure cell viability of the resistant cells compared with the parental cells. Inhibition of c-MET activation was performed by c-MET inhibitors, PHA665752 and Crizotinib or small interfering RNA against c-MET. We found MET inhibitors (4 or 8μM) reduced cell growth and restored drug sensitivity (2μM) of resistant cells, PC-6/SN38, compared to parental cells. To reveal the mechanisms of c-MET in drug resistance we examined ATP-binding cassette (ABC) transporters expression in PC-6/SN-38 compared to the parental cells by qRT-PCR, and found the mRNA level of ABCG2, which effluxes SN-38 as substrate, in PC-6/SN-38 cells was extremely increased and another ABC transporters that do not efflux SN-38 were not. Recently we reported PC-6/SN-38 cells had c-MET overexpression, and c-MET inhibition in resistant cells such PC-6/SN-38 resulted in restoration drug resistance. Based on this report we examined c-MET inhibition by c-MET inhibitor and small interfering RNA against c-MET in PC-6/SN38, and qRT-PCR and WB were performed to investigate alteration of ABCG2 expression, and found both two c-MET inhibition reduced ABCG2 expression. In addition, to demonstrate c-MET upregulation in resistant cells is addicted to the ligand HGF, PC-6 cells were incubated with HGF treated for 3 weeks and made PC-6/HGF, then continued to incubate with HGF and SN-38 was added two times in a week. Although c-MET expression is equal in PC-6 and PC-6/HGF, after SN-38 treatment c-MET was overexpressed. Furthermore, serum HGF level in SCLC patients correlated with clinical course. We demonstrated upregulation of ABC transporters via HGF/c-MET signaling activation might be one of the mechanisms of acquired resistance to cytotoxic anticancer agents in lung cancer cells. Decreased ABC transporters expression through inhibition of c-MET activation might overcome drug resistance to cytotoxic agents. Citation Format: Yoshitaka Yagi, Hiroaki Ozasa, Takahiro Tsuji, Yuichi Sakamori, Takeshi Nomizo, Hiroki Nagai, Young Hak Kim, Ken Maeno, Tetsuya Oguri, Michiaki Mishima. Overexpression of ABC transporters through HGF/c-MET activation results in anti-cancer drug resistance in SCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5045.

Abstract 2946: Increased ALDH7A1 expression enhances the resistance to the anticancer drugs and colony formation in lung cancer cell lines

Background: Aldehyde dehydrogenase (ALDH) forms a superfamily of enzymes that catalyze conversion of aldehydes into carboxylic acids, and they are categorized into 19 families in human. Increased ALDH activity was reported to be a potential marker of cancer stem cell (CSC) in various solid tumors including lung cancer. Furthermore, High ALDH expression has been shown to be involved in drug resistance to conventional cytotoxic drugs. Recent studies revealed that ALDH7A1 is highly expressed in prostate cancer and associated with recurrence in patients with surgically resected non-small-cell lung cancer. However, there is not a fundamental link between ALDH7A1 expression and malignant phenotype, including drug resistance and colony formation. Material and methods: Anticancer drug resistant cell lines were established from lung cancer cell lines (PC-6, DMS53, PC-14, PC-9, NCI-H23 and NCI-H2228 cells) by exposure to various anticancer drug, including 7-ethyl-10-hydroxycamptothesin (SN-38), gemcitabine (GEM), cisplatin (CDDP), etoposide, paclitaxel (TXL), pemetrexed, amrubicin, erlotinib and crizotinib. Protein expression was determined by western blotting and gene expression was examined by RT-PCR. ALDH enzyme activity was measured by ALDEFLOUR TM assay. Proliferation was measured using MTS assay. ALDH7A1encoding plasmid was stably transfected into PC-14 cell. These transfected cells displayed sphere formation on ultralow binding plates and survived for more than 3 weeks. Results: We found that the levels of ALDH7A1 expression were higher in various anticancer drug-resistant lung cancer cell lines compared with the parental cells. ALDH enzyme activity was about 4-9 fold higher in SN-38, TXL, CDDP and GEM resistant cell lines (PC6/SN38, PC6/TXL, PC14/CDDP and PC14/GEM) than the parents cells. Up-regulation of ALDH7A1 expression by using a forced expression vector in PC14 cell altered cytotoxicity to SN-38 and TXL. Furthermore, overexpression of ALDH7A1in PC-14 cells enhanced sphere formation relative to the cells transfected with control vector. Conclusion: These results suggested that increased ALDH7A1 expression in lung cancer cell lines might be one of the mechanisms of acquired resistance to anticancer agents and enhance colony formation. Citation Format: Yuichi Sakamori, Hiroaki Ozasa, Eiji Kunii, Yoshitaka Yagi, Takahiro Tsuji, Takeshi Nomizo, Hiroki Nagai, Young Hak Kim, Ken Maeno, Tetsuya Oguri, Michiaki Mishima. Increased ALDH7A1 expression enhances the resistance to the anticancer drugs and colony formation in lung cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2946.

Abstract 2941: Increased expression of ABCB1/MDR1 could be associated with alectinib resistance in ALK-rearranged lung cancer cells

Alectinib, a second-generation anaplastic lymphoma kinase (ALK) inhibitor, has shown promising activity in ALK-rearranged non-small cell lung cancer (NSCLC). The mechanisms of drug resistance to alectinib have been still unclear. To explore whether overexpression of ATP-binding cassette (ABC) transporters might provide a potential mechanism of alectinib resistance in lung cancer, we established alectinib-resistant cell lines from NCI-H2228 and primary ALK-rearranged cells and evaluated the expression of ABC transporters. We established ALK-rearranged NSCLC cell lines (KTOR 1, 2, and 3) from tumor cells in pleural effusion obtained from 3 patients with alectinib naive ALK-rearranged NSCLC. In the 2 of 3 patients, tumor cells in pleural effusion were also obtained when disease progression was observed during alectinib treatment (KTOR1-RE and KTOR2-RE). Expression profiling of ABC transporters using quantitative real-time polymerase chain reaction (qRT-PCR) revealed that the expression of ABCB1 was significantly increased in KTOR1-RE and KTOR2-RE compared with the parental cells (4.6 and 10.8 folds), but not other ABC transporters. Then, alectinib-resistant NCI-H2228 cell lines (H2228-AR) were established by exposing the parental cells to stepwise-increasing alectinib up to 5μM for 10 months. We found that the ABCB1/MDR1 expression in H2228-AR cells were significantly increased (2.09-16.6 folds) compared with the parental cells using qRT-PCR and immunoblotting. Then, we examined the alternation of alectinib cytotoxicity by inhibition of ABCB1. Knockdown of ABCB1 expression using small interfering RNA and inhibition of ABCB1 by verapamil enhanced alectinib cytotoxicity in H2228-AR cells. Next, the levels of ABCB1 expression after short-term alectinib exposure were examined in NCI-H2228 and the three alectinib naive ALK-rearranged NSCLC primary cell lines (KTOR 1, 2, and 3). In all the four ALK-rearranged cell lines, exposure to 100 nM alectinib for 72 hours induced increased expression of ABCB1 compared with control medium. These data suggested that increased expression of ABCB1/MDR1 might be, in part, responsible acquired resistance and sensitivity to alectinib in ALK-rearranged NSCLC cells. Citation Format: Takahiro Tsuji, Hiroaki Ozasa, Yuichi Sakamori, Takashi Nomizo, Yoshitaka Yagi, Hiroki Nagai, Young Hak Kim, Michiaki Mishima. Increased expression of ABCB1/MDR1 could be associated with alectinib resistance in ALK-rearranged lung cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2941.

Long survival of a small-cell lung cancer patient who received maintenance chemotherapy with irinotecan.

Lung cancer is the leading cause of cancer-related death worldwide. Small-cell lung cancer (SCLC) accounts for approximately 15% of all lung cancers. It is characterized by rapid tumor growth and early metastasis to multiple organs. Response to initial chemotherapy is generally good; however, the majority of patients develop recurrence and the prognosis of such patients is reportedly 2-4 months. Evolution of the treatment for SCLC has stagnated, and cisplatin + etoposide has been the standard chemotherapy for decades. Meanwhile, the combination of cisplatin + irinotecan has demonstrated equivalent efficacy to cisplatin + etoposide. Recently, maintenance chemotherapy has been extensively investigated in non-small-cell lung cancer (NSCLC), and is currently recommended as a standard treatment in clinical guidelines. On the contrary, a maintenance strategy has not been established for SCLC. Here, we describe an SCLC patient who received maintenance chemotherapy with irinotecan for more than 2 years after induction chemotherapy with cisplatin + irinotecan, and survived long term with no recurrence.