Takahiro Ebata

Drug resistance originating from a TGF-β/FGF-2-driven epithelial-to-mesenchymal transition and its reversion in human lung adenocarcinoma cell lines harboring an EGFR mutation

Epithelial-to-mesenchymal transition (EMT) is a malignant cancer phenotype characterized by augmented invasion and metastasis, chemoresistance, and escape from host-immunity. This study sought to identify efficient methods for inducing EMT reversion, to evaluate alterations in chemosensitivity and immune-protectiveness, and to elucidate the underlying mechanisms. In this study, the human lung adenocarcinoma cell lines PC-9 and HCC-827, harboring an EGFR mutation, were treated with TGF-β and FGF-2 to induce EMT. The phenotypic alterations were evaluated by RT-PCR, fluorescent immunohistochemistry, cell-mobility, and flow cytometry. Chemosensitivity to gefitinib and cisplatin was evaluated using an MTT assay and apoptosis. Immune-protectiveness was evaluated by PD-L1 expression. A combination of TGF-β and FGF-2 efficiently induced EMT in both cell lines: through Smad3 pathway in PC-9, and through Smad3, MEK/Erk, and mTOR pathways in HCC-827. The mTOR inhibitor PP242, metformin, and DMSO reverted EMT to different extent and through different pathways, depending on the cell lines. EMT induction reduced the sensitivity to gefitinib in both cell lines and to cisplatin in HCC-827, and it increased PD-L1 expression in both cell lines. EMT reversion using each of the 3 agents partly restored chemosensitivity and suppressed PD-L1 expression. Thus, chemoresistance and increased PD-L1 expression caused by EMT can be successfully reverted by EMT-reverting agents.

Retrospective analysis of unknown primary cancers with malignant pleural effusion at initial diagnosis

Malignant pleural effusion (MPE) can occur during the progression of various cancers. However, factors, such as the incidence of MPE associated with different types of cancers and its potential for diagnosing previously undetected cancers, are unknown. Moreover, MPE may accompany potentially curable cancers or those with a favorable survival prognosis with adequate treatment. The present study determined the types of cancers accompanied by MPE at initial diagnosis and investigated appropriate related methods for diagnosing previously unknown cancers.

Safety of a short hydration method for cisplatin administration in comparison with a conventional method—a retrospective study

OBJECTIVE Cisplatin is administered in combination with massive hydration to avoid renal toxicity, making its administration difficult in an outpatient setting. Although a short hydration protocol for cisplatin has been recently developed, its safety is not fully understood. METHODS Consecutive patients with lung or other cancer and an Eastern Cooperative Oncology Group performance status of 0-2 who were receiving chemotherapy containing cisplatin at a dose of ≥60 mg/m(2) in a single administration were evaluated. Seventy-four patients were treated with a short hydration protocol consisting of 1750-2250 ml of hydration with mannitol and magnesium supplementation over a period of 3.75-4.75 h on Day 1. Sixty-nine patients were treated with a conventional hydration protocol consisting of 2100-2600 ml of hydration over 6.5-7.5 h on Day 1 with pre- and post-hydration on Days 0, 2 and 3. Toxicity was then compared between the two groups. RESULTS An elevated serum creatinine level ≥grade 1 was significantly less frequent in the group receiving the short hydration protocol than in the group receiving conventional hydration. Other toxicities were similar between the two groups. Consequently, the completion rate for the planned treatment in the short hydration group (73.0%, 54/74) was significantly higher than that in the conventional hydration group (53.6%, 37/69). CONCLUSIONS Short hydration is safe, making cisplatin-containing chemotherapy easier to perform.

Efficacy and Safety of Pazopanib for Recurrent or Metastatic Solitary Fibrous Tumor

Objective: To investigate the efficacy and safety of pazopanib for recurrent or metastatic solitary fibrous tumor (SFT) in first- and second-line settings. Methods: Patients histologically diagnosed with SFT at our hospital who received pazopanib monotherapy for inoperable disease between January 2013 and November 2016 were eligible. We retrospectively investigated treatment outcomes according to the treatment lines and assessed adverse events. Results: Nine patients were eligible. The median age was 67 years (range 42–81), and 6 patients (66.7%) were male. Four patients (50%) received pazopanib as second-line treatment. According to the RECIST and Choi criteria, the respective response rates were 0 and 50%, while the respective disease control rates were 88.9 and 75%. The median progression-free survival (PFS) was 6.2 months (95% confidence interval 3.2–8.8). Treatment line and high frequency of mitosis were not predictive of PFS (p = 0.67, 0.92). Two patients (22.2%) experienced elevated liver enzymes of grade 3 or higher. Conclusion: Pazopanib is an effective treatment option for recurrent or metastatic SFT in first- and second-line settings. Liver injury is a major adverse event and adequate treatment withdrawal and dose reduction should be considered when necessary.

Pirfenidone may revert the epithelial-to-mesenchymal transition in human lung adenocarcinoma

The epithelial-to-mesenchymal transition (EMT) in cancer is associated with invasion, metastasis and chemoresistance. Recent studies have revealed the increased expression of programmed death-ligand 1 (PD-L1) in cells undergoing EMT. The underlying mechanism of EMT involves transforming growth factor-β (TGF-β) and fibroblast growth factor-2 (FGF-2). Pirfenidone and the known EMT-suppressor nintedanib suppress pulmonary fibrosis partially through suppression of TGF-β. The present study aimed to determine whether pirfenidone has the potential to induce EMT-reversion, using nintedanib as a reference. The human lung adenocarcinoma cell lines A-549, HCC-827, and PC-9 were treated with TGF-β and FGF-2 to induce EMT. The EMT-induced cells were further treated with pirfenidone or nintedanib. Phenotypic alterations associated with EMT were assessed by examining the following: i) The expression levels of E-cadherin, vimentin, fibronectin and slug, using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and fluorescent immunohistochemistry; ii) cell motility via wound-healing assays; and iii) the expression of PD-L1 using RT-qPCR. The combination of TGF-β and FGF-2 successfully induced EMT in all three cell lines, characterized by a significant reduction in E-cadherin expression in the A-549 and HCC-827 cells, increased expression levels of vimentin, fibronectin, slug and PD-L1, and increased cell motility in all three cell lines. Pirfenidone and nintedanib reverted all of these phenotypes, with the exception of unaltered E-cadherin expression in all three cell lines, and inconsistent expression of vimentin in the HCC-827 and PC-9 cells. Thus, pirfenidone and nintedanib have the ability to induce EMT-reversion in human lung adenocarcinoma.

Amrubicin Monotherapy for Patients with Platinum-Pretreated Non-Gastrointestinal Non-Pancreatic Extrapulmonary Neuroendocrine Carcinoma

Objective: The aim of this study was to investigate the clinical usefulness of amrubicin therapy for patients with non-gastrointestinal (GI) non-pancreatic extrapulmonary neuroendocrine carcinoma (EP-NEC). Methods: The medical records of patients from the 2 participating institutions were retrospectively reviewed. The eligibility criteria were: patients with non-GI non-pancreatic EP-NEC who received amrubicin monotherapy after platinum-based chemotherapy. Patients in whom the platinum-free interval (interval between the last day of platinum administration and the first subsequent documentation of disease progression) was 90 days or longer were classified into the platinum-sensitive group. Results: The study was conducted in a total of 13 patients identified as eligible. The response rate was 45.4% (5/11). The median progression-free survival and overall survival were 6.0 and 10.6 months, respectively. A platinum-free interval of ≥90 days was identified as a significant predictor of a longer progression-free survival time. Grade 3 or 4 neutropenia was observed in 61.5% (8/13) of the patients. One patient died of treatment-related febrile neutropenia. Conclusions: Amrubicin monotherapy as second-line chemotherapy after failure of first-line platinum-based chemotherapy showed good efficacy in patients with non-GI non-pancreatic EP-NEC. Neutropenia was encountered as the most serious adverse event.

Potential Activity of Amrubicin as a Salvage Therapy for Merkel Cell Carcinoma

Merkel cell carcinoma (MCC) is a rare neuroendocrine carcinoma of the skin with an aggressive clinical course. Although anthracycline- and platinum-based regimens are empirically used as first-line treatments for metastatic or unresectable cases, no salvage therapy has been established. A 73-year-old man with platinum-refractory recurrent MCC was treated with amrubicin. The symptoms improved soon, and a partial response was achieved. A total of nine cycles of amrubicin were administered in nine months with manageable adverse events until disease progression was finally observed. The present findings suggest the potential of amrubicin monotherapy as a second-line therapy for patients with advanced/recurrent MCC.

Nivolumab-induced Acute Fibrinous and Organizing Pneumonia (AFOP)

Although nivolumab is known to cause immune-related interstitial lung diseases (ILD), the detailed characteristics of ILD are still not fully understood. A 68-year-old man was treated with nivolumab because of unresectable sinonasal melanoma, he achieved a complete response soon after the initiation of the therapy and a complete response was thereafter maintained for 30 weeks until the patient experienced dyspnea of subacute onset. CT images revealed patchy infiltrates and ground-glass opacifications. The bronchoalveolar lavage fluid (BALF) contained elevated percentages of lymphocytes (53%) and neutrophils (30%). A transbronchial lung biopsy revealed intraalveolar fibrin balls without hyaline membranes, which was considered to be consistent with the pattern of acute fibrinous and organizing pneumonia (AFOP). This is the first report of AFOP induced by nivolumab.

Abstract 239: Alteration of drug sensitivity according to the induction and reversion of epithelial-to-mesenchymal transition (EMT) in human lung adenocarcinoma cell lines harboring an EGFR mutation

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Introduction: EMT phenotype is known to relate to drug resistance and immune tolerance of cancer. However, mechanism underling these phenomena, including the mechanism of the induction and reversion of EMT, are not fully understood. Methods: EGFR mutated human lung adenocarcinoma cell lines, HCC-827 and PC-9, were treated with TGF-s and/or FGF-2 to induce EMT. EMT was reversed by treating the induced EMT cells with either of PP242 (mTOR inhibitor), metformin and DMSO. The phenotypic alterations according to the induction and reversion of EMT were accessed by a wound healing assay for mobility, flow cytometry for cell cycle, MTT and apoptosis assays for drug sensitivity to cisplatin and gefitinib, and RT-PCR and fluorescence IHC for PD-L1 expression. Results: The treatment with combination of TGF-s/FGF-2 showed an efficient increase in expressions of mesenchymal markers and slug in both cell lines, and a decrease in E-cadherin expression in HCC827. In immunoblotting analyses, the Smad3 pathway in PC-9, and the Smad3, MEK/Erk and mTOR pathways in HCC-827 were involved in the induction of the EMT. The induced EMT was accompanied by enhanced cell motility and accumulation in the G0/G1 phases in both cell lines. The induced EMT also made the cells less sensitive to gefitinib in both cell lines, and to cisplatin in HCC-827. Increased PD-L1 expression was observed in both cell lines. Treatment of the induced EMT cells with PP242, metformin and DMSO reverted the altered expressions of epithelial and mesenchymal markers, cell motility and cell cycle. These agents reverted the EMT to different extents and through different pathways, depending on the cell lines. Reversion of the EMT using each of the 3 agents partly restored drug sensitivity, and suppressed PD-L1 expression. Conclusion: Inducing EMT by treatment with TGF-s/FGF-2 is an effective way to acquire drug resistance and up-regulation of PD-L1. Reversion of the EMT, therefore, has a potential to overcome drug resistance and immune tolerance of cancer. Citation Format: Ryota Kurimoto, Shunichiro Iwasawa, Takahiro Ebata, Tsukasa Ishiwata, Ikuo Sekine, Yuji Tada, Koichiro Tatsumi, Shuhei Koide, Atsushi Iwama, Yuichi Takiguchi. Alteration of drug sensitivity according to the induction and reversion of epithelial-to-mesenchymal transition (EMT) in human lung adenocarcinoma cell lines harboring an EGFR mutation. [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 239.