Tomoki Tamura

Abstract 1368: The impact of bevacizumab on combination low-dose afatinib and cetuximab therapy in lung cancer cells harboring activated EGFR mutations

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background. The combination of afatinib and cetuximab induced an unprecedented response in lung tumors harboring EGFR T790M in clinical trials (Cancer Discov. 2014). However, up to 20% of patients discontinued treatment due to adverse events, including skin rashes and diarrhea, and the median progression-free survival of the combination therapy was 4.7 months. There is clearly still room for improvement in the therapy. Bevacizumab, a key drug in lung cancer treatment, is thought to have a wide variety of effects, including anti-vascularization, normalization of tumor vasculature, and improved drug delivery. Thus, we hypothesized that the dosage of afatinib and cetuximab could be reduced by combining with bevacizumab, and that the triple therapy might produce better tumor inhibition with tolerable toxicity. Methods. Two lung cancer cell lines, H1975, with EGFR L858R and T790M, and RPC-9, with EGFR exon 19 deletion and T790M, were injected subcutaneously into nude mice as xenograft models. Mice were divided into four groups 10 days after tumor cell inoculation (vehicle, afatinib, afatinib + cetuximab, afatinib + cetuximab + bevcacizumab). Compared with previous reports (afatinib 25 mg/kg, 5 times/week, cetuximab 1 mg/mouse, every 3 days; J. Clin. Invest. 2009), the low dose of afatinib (10 mg/kg, 5 times/week; i.e., 60% less drug) or cetuximab (0.1 mg/mouse, once/week; i.e., 90% less drug) were administered to the mice for 1 month. Bevacizumab was injected at 2 mg/kg twice/week. Following the therapies, the mice were observed for 1 month without treatment. Results. Surprisingly, the triple therapy induced a pathological complete response (pCR) in H1975 and RPC-9 cell xenograft tumors; in contrast, the tumors treated with single or double therapy were inhibited only partially. Furthermore pCR was maintained during the observation period in the triple therapy group. There was no body weight loss in any group. Frozen tumors were obtained from RPC-9 xenograft models after 1 week treatment with each regimen. Expression levels of pEGFR, pAKT, and pERK were decreased in the triple therapy group. CD31-positive blood vessels and Ki-67-positive cells in tumors with triple therapy were reduced significantly versus the tumors in other groups. Cleaved caspase-3 expression in the tumors with triple therapy was positive in a higher number of tumor cells than in the other groups. Conclusions. We demonstrated that a low-dose of afatinib and cetuximab combined with bevacizumab induced pCR in EGFR T790M mutated cell xenograft tumors with no obvious adverse events. We suggest that the suppression of neovascularization and induction of apoptosis may play important roles in the triple therapy. Note: This abstract was not presented at the meeting. Citation Format: Kenichiro Kudo, Kadoaki Ohashi, Eiki Ichihara, Daisuke Minami, Hisao Kubo, Akiko Sato, Yuka Kato, Hideko Isozaki, Hiroe Kayatani, Tomoki Tamura, Mitsune Tanimoto, Katsuyuki Kiura. The impact of bevacizumab on combination low-dose afatinib and cetuximab therapy in lung cancer cells harboring activated EGFR mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1368. doi:10.1158/1538-7445.AM2015-1368

Abstract 3164: A comprehensive analysis of autopsied specimens and patient-derived cell lines in ALK-positive lung cancers with rapid acquired resistance to alectinib

Background Alectinib, a highly selective anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor (TKI), demonstrated a stunning disease control rate (95.7%) and unprecedented median progression-free survival (not reached: 95% CI: 20.3-) (Lancet Oncology 2013, ASCO 2016, Abstract #9008). However, acquired resistance to alectinib is inevitable; some ALK-positive lung tumors rapidly acquire alectinib resistance. Therefore, we sought to investigate the mechanism of the rapid resistance to alectinib using clinical samples and patient-derived ALK-positive cell lines. Materials and Methods Autopsied samples (primary lung tumors and metastatic tumors from the esophagus, liver, and bilateral kidney) were obtained from a 51-year-old male with advanced ALK-positive lung cancer. The lung tumors acquired resistance to first-line treatment of alectinib in three months, and subsequent treatment with ceritinib or crizotinib showed only a moderate and temporary effect. We established novel ALK-positive cell lines; ABC-14 was established from a pleural effusion after alectinib treatment, and ABC-17 was established from patient-derived xenograft (PDX) tumors of autopsied liver metastases. Western blot analysis, immunostaining, a receptor tyrosine kinase assay, fluorescence in situ hybridization (FISH), and next-generation sequencing were performed. Results ALK overexpression was confirmed in all autopsied samples by immunostaining. PCR revealed that ABC-14 harbored the EML-4/ALK fusion variant 3. An in vitro cell proliferation assay showed that ABC-14 exhibited resistance to alectinib (IC 50 > 1 µM), but was sensitive to crizotinib (IC 50 = 98 nM). The receptor tyrosine kinase assay revealed the activation of MET and EGFR in ABC-14. Quantitative RT-PCR and FISH confirmed MET gene amplification. Quantitative RT-PCR also indicated the overexpression of an EGFR ligand, EGF, TGF-α but no EGFR mRNA overexpression. The combination of crizotinib (dual ALK/MET-TKI) and an EGFR-TKI, gefitinib, showed an additive inhibitory effect on cell growth compared with each drug alone in vitro. ABC-17 showed resistance to both alectinib and crizotinib; consistently showed no activation of MET and no MET gene amplification. Interestingly, ABC-17 showed metastatic ability in the lung in NOG mouse PDX models. Conclusion The mechanism of rapid resistance to alectinib may be complicated and heterogeneous. Crizotinib combined with gefitinib, which inhibits the ALK, EGFR, and MET pathways, may represent one potent strategy against alectinib resistance. Further next-generation sequencing of clinically relevant samples should provide deeper insights into its resistance. (This work was supported by KAKEN 16K19454 and KAKEN 15H04830.) Citation Format: Go Makimoto, Kadoaki Ohashi, Kazuya Nishii, Shuta Tomida, Hiroe Kayatani, Tomoki Tamura, Hisao Higo, Kiichiro Ninomiya, Takashi Ninomiya, Toshio Kubo, Eiki Ichihara, Akiko Sato, Katsuyuki Hotta, Masahiro Tabata, Katsuyuki Kiura. A comprehensive analysis of autopsied specimens and patient-derived cell lines in ALK-positive lung cancers with rapid acquired resistance to alectinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3164. doi:10.1158/1538-7445.AM2017-3164

Efficacy of multimodal treatment for leptomeningeal metastases in a lung cancer harboring an EGFR mutation

For lung cancer patients with epidermal growth factor receptor (EGFR) mutations, the advent of EGFR tyrosine kinase inhibitors (TKIs) has prolonged survival rates. Even though disease sites have been well controlled by EGFR-TKIs, some patients develop carcinomatous meningitis, which reduces their quality of life drastically. Although multidisciplinary approaches have improved patient survival and quality of life, the outcomes are not yet satisfactory. We report the case of a 54-year-old Japanese woman diagnosed with leptomeningeal metastases (LM) from a lung adenocarcinoma harboring an EGFR exon 21 L858R point mutation. She was treated with gefitinib for 2 months, and symptoms of LM emerged during the treatment period. Although the treatment was switched to erlotinib, disturbance of consciousness worsened because of progressive hydrocephalus. Because all extracranial lesions remained responsive to treatment, and the exon 20 T790M point mutation was not detected in cerebrospinal fluid, we placed a ventriculoperitoneal shunt. The patient’s disturbed consciousness improved dramatically after the shunt was placed; however, the optic and auditory nerve impairments due to direct invasion of LM lesions into nerve canals persisted. Administration of bevacizumab subsequent to whole-brain radiotherapy reduced the cranial nerve impairment, and the patient survived for 10 months. In conclusion, a combination of erlotinib and ventriculoperitoneal shunt was effective for hydrocephalus, and the immediate administration of additional therapies, including bevacizumab and radiation therapy, was useful in a patient suffering from LM.

Abstract 250: Interleukin-6 as potential predictive marker for therapeutic effect of Gefitinib in patients with advanced non-small-cell lung cancer harboring EGFR mutations

Background: Although epidermal growth factor receptor (EGFR) -tyrosine kinase inhibitors (TKIs) are the key drug in patients with EGFR-mutant (mt) Non-small-cell Lung Cancer (NSCLC), some of them can not respond well to its therapy. An overexpression of Interleukin (IL)-6 in tumor cells is postulated as a potential mechanism for such resistance or low sensitivity to EGFR-TKI in the preclinical models (PNAS 2010). Here, we evaluated clinically if tumor IL-6 level can be predictive for the effect of EGFR-TKI therapy, and evaluated the expression of IL-6 in EGFR-mt NSCLC cell-lines, and the effect of EGFR-TKI. Methods: A total of 52 pts with advanced EGFR-mt NSCLC who had received gefitinib (G) were retrospectively assessed. The protein expression of IL-6 in the tumor cells was immunostained. Each specimen was assessed independently by 2 physicians (TT and YK) and 2 pathologists (KI and TT), and judged as positive if ≥ 50% of 100 tumor cells were stained positively (BJC 1999). Serum IL-6 level was measured by CLEIA in 11 (21%) of 52 pts. Next we checked 13 EGFR-mt NSCLC cell lines. Enzyme linked immunosorbent assay (ELISA) and real time PCR (RT-PCR) of cell culture supernate were peformed. Results: Patients demographics were as follows: 24 men; median age, 66 yrs; PS 0-1, 48; stage IV, 22; Ad, 49; exon19, 29. Of these, 24 (46%) and 28 (54%) were defined as IL-6-postitive (group P) and IL-6-negative (group N), respectively. Group P had worse PFS (75% v 92% at 6m; p The IL-6 level in cell culture supernatant was ranged from 1.46 to 1219.75 pg/ml (median: 85.84 pg/ml). 2 of 13 cell lines showed especially high IL-6 level; IL-6 level of 11-18 was 1090.91 pg/ml and H1650 was 1219.75 pg/ml. RT-PCR also showed the high expression level of IL-6 mRNA in 11-18 and H1650 compared with other cell lines. 11-18 and H1650 showed low sensitivity to G therapy. Conclusions: Pts in group P benefited less from G therapy. In vitro experiment suggested that secretion of IL-6 may confer the less sensitivity to EGFR-TKIs. These data suggest that the expression of IL-6 lead to early acquisition of resistance to EGFR-TKIs in EGFR-mt tumors. Citation Format: Tomoki Tamura, Katsuyuki Hotta, Yuka Kato, Takehiro Tanaka, Kouichi Ichimura, Kadoaki Oohashi, Takashi Ninomiya, Toshio Kubo, Eiki Ichihara, Mitsune Tanimoto, Katsuyuki Kiura. Interleukin-6 as potential predictive marker for therapeutic effect of Gefitinib in patients with advanced non-small-cell lung cancer harboring EGFR mutations. [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 250.

Abstract 2103: Activating alternative receptor tyrosine kinases induced alectinib-resistance in ALK rearranged non-small cell lung cancer cells

Background The second-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor (TKI), alectinib, demonstrated high response rate, long response duration and a favorable toxic profile in patients with ALK-rearranged advanced non-small cell lung cancer in a phase II study (Lancet Oncol 14:590-8, 2013). However, even this promising drug is predicted to develop acquired resistance. Therefore, we investigated the mechanisms of resistance using two alectinib-resistant cell lines. Methods We established alectinib-resistant cell lines, H2228/CHR and ABC-11/CHR, from H2228 (EML4-ALK fusion genes variant 3a/b E6) and ABC-11 (EML4-ALK fusion genes variant 3b E6) respectively, by continuous exposure to alectinib. They were characterized using MTT assay, Western blotting, receptor tyrosine kinase array, ELISA, FISH, RT-PCR, and xenograft models. Results H2228/CHR and ABC-11/CHR cells were 117- and 40-fold more resistant than the parental lines, respectively, and maintained downstream AKT and ERK phosphorylation even in the presence of 10 μM alectinib. There were no ALK secondary mutations in those resistant cell lines. H2228/CHR lost the EML4-ALK fusion gene, and exhibited increased activation of insulin-like growth factor-1 receptor (IGF-1R) and human epidermal growth factor receptor 3 (HER3) with overexpression of the HER3 ligand neuregulin 1. Accordingly, pharmacologic inhibition of IGF-1R and HER3 signaling overcame the resistance. In ABC-11/CHR, MET was activated by stimulated hepatocyte growth factor (HGF) autocrine signaling. We found HGF gene translocation underlying the HGF autocrine system. Anti-HGF antibody suppressed the MET activation and combined treatment with alectinib and anti-HGF antibody or a MET inhibitor suppressed downstream signaling in ABC-11/CHR cells. Finally, crizotinib, which targets both ALK and MET, most effectively inhibited the growth of ABC-11/CHR both in vitro and in vivo. Conclusions We identified novel alectinib resistance mechanisms caused by the activation of alternative tyrosine kinase receptors. Our findings provide new insights into constructing a therapeutic strategy for ALK-positive lung cancer. Citation Format: Hideko Isozaki, Eiki Ichihara, Masayuki Yasugi, Nagio Takigawa, Kadoaki Ohashi, Toshio Kubo, Takashi Ninomiya, Nobuaki Ochi, Daisuke Minami, Kenichiro Kudo, Yuka Kato, Hiroe Kayatani, Tomoki Tamura, Kiichiro Ninomiya, Toshio Higo, Tsuyoshi Makimoto, Akiko Sato, Katsuyuki Hotta, Kunio Matsumoto, Toshiaki Sendo, Mitsune Tanimoto, Katsuyuki Kiura. Activating alternative receptor tyrosine kinases induced alectinib-resistance in ALK rearranged non-small cell 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 2103.

Differences in the toxicity and efficacy in phase I studies between cytotoxic and molecular-targeted therapies

13527 Background: In recent phase I trials, many molecular-targeted agents have been studied, but the differences in the toxicity and efficacy between cytotoxic and targeted agents have not yet been evaluated. Methods: We reviewed phase I trials published between 1999 and 2004 collected by a Medline search. Molecular-targeted agents, defined as agents designed and developed to inhibit specific molecules involved in the growth, invasion or metastasis of cancer cells, were compared with cytotoxic agents in terms of the toxicity and efficacy. Results: A total of 200 studies were collected, of which 8 were excluded because these did not evaluate the agents for their antitumor effect. The numbers of patients per number of studies evaluating molecular-targeted and cytotoxic treatments were 1369/44 and 4634/148, respectively. As for the molecular-targeted agents evaluated in the 44 studies, the agents employed were small molecules in 33, antisense agents in 5, and others in 6 studies. The molecular target was th...