Ayumi Kuroda

Trametinib plus 4-Methylumbelliferone Exhibits Antitumor Effects by ERK Blockade and CD44 Downregulation and Affects PD-1 and PD-L1 in Malignant Pleural Mesothelioma

Introduction: Malignant pleural mesothelioma (MPM) is a highly aggressive malignancy in which the mitogen‐activated protein kinase pathway plays a critical role in the regulation of tumorigenesis. Hyaluronic acid (HA) is a major component of the extracellular matrix, and elevated HA levels with a concurrent increase in malignant properties are associated with MPM. Methods: We evaluated the effects of trametinib, a mitogen‐activated protein kinase (MEK) inhibitor, and 4‐methylumbelliferone (4‐MU), an HA synthesis inhibitor, alone and in combination on MPM cells in vitro and in vivo. We studied the effects of trametinib, 4‐MU, and their combination on MPM cells by using cell viability assays, Western blot analysis, and a mouse xenograft model. Results: Trametinib and 4‐MU exhibited antiproliferative activity in MPM cells. Trametinib blocked MEK‐dependent extracellular signal‐regulated kinase (ERK) phosphorylation and decreased CD44 expression in a concentration‐dependent manner. Trametinib inhibited the expression of Fra‐1 (the activator protein 1 [AP1] component), inhibited ERK phosphorylation, and decreased CD44 expression. 4‐MU inhibited ERK phosphorylation but not CD44 expression. In a mouse xenograft model, trametinib and 4‐MU alone suppressed tumor growth compared with a control. The combination had a greater inhibitory effect than either monotherapy. Immunohistochemical analysis showed that trametinib treatment alone significantly reduced expression of programmed cell death 1 ligand 1. Furthermore, the combination of trametinib and 4‐MU resulted in higher expression of programmed cell death 1 and programmed cell death 1 ligand 1 than did the 4‐MU treatment alone. Conclusions: Our results suggest that trametinib and 4‐MU are promising therapeutic agents in MPM and that further study of the combination is warranted.

Pleural thickness after neoadjuvant chemotherapy is a prognostic factor in malignant pleural mesothelioma

Objectives: Definitive diagnosis of the T‐component is sometimes challenging in malignant pleural mesothelioma (MPM). Pleural thickness has been reported to be a prognostic factor for MPM and is a potential T‐component. Methods: We conducted a historical cohort study of patients who underwent neoadjuvant chemotherapy (NAC) and curative‐intent surgery as a multimodal treatment for MPM from January 2007 to June 2016. The maximum measurement of pleural thickness among 3 levels and the sum at each level determined using axial computed tomography imaging before and after NAC were termed as “max” and “sum,” respectively. We assessed the association between pleural thickness and the primary and secondary end points of overall survival and recurrence‐free survival. Survival was analyzed using the Kaplan–Meier curve, log rank test, and multivariate Cox regression model. Results: We enrolled 105 patients. We excluded 1 because of missing data; thus, the sample size was 104. The median follow‐up period was 29.1 months with recurrence in 78 patients (70.3%) and death in 67 (60.4%). Max and sum ranged from pre (before NAC) values of 0 to 35 (median, 6.05) and 0 to 97 (median, 12.9) to post (after NAC) values of 0 to 30.8 (median, 4.25) and 0 to 67.0 (median, 9.25), respectively. Post values max and sum were associated with overall survival and recurrence‐free survival. Post sum values were associated with recurrence (adjusted hazard ratio, 2.59; 95% confidence interval, 1.42‐3.83) and death (adjusted hazard ratio, 2.13; 95% confidence interval, 1.16‐4.52), respectively. Conclusions: Pleural thickness after NAC was an independent prognostic factor in patients who underwent multimodal treatment.

The clinical value of circulating tumour cells (CTCs) in patients undergoing pulmonary metastasectomy for metastatic colorectal cancer

Background Circulating tumour cells (CTCs) are a potential surrogate for distant metastasis and are considered a useful clinical prognostic marker for metastatic colorectal cancer (mCRC). This prospective study evaluated the preoperative CTC count as a prognostic factor for pulmonary metastasectomy in mCRC patients. Methods Seventy-nine mCRC patients who underwent curative-intent pulmonary metastasectomy were included. Preoperatively, 7.5 mL of peripheral blood from each patient was quantitatively evaluated for CTCs with the CellSearch® system. The clinical significance of CTC count was evaluated according to Kaplan-Meier analyses and log-rank test. Multivariate analyses of the perioperative variables were performed. Results The distribution of CTC counts were as follows; 0 in 66 patients (83.5%), 1 in eight patients (10.1%), 2 in three patients (3.8%), and 3 and 6 in one patient (1.3%). The patients with multiple CTCs (CTC count ≥2) had significant shorter disease-free survival (DFS) (P=0.005, median DFS; 19.8 vs. 8.6 months) and overall survival (OS) (P=0.035, median DFS; not reached vs. 37.8 months), respectively. Multivariate analysis showed the patients with multiple CTCs had elevated risk of recurrence [hazard ratio (HR), 3.28; 95% confidence interval (CI), 1.24-8.67; P=0.017]. Conclusions The detected rate of CTCs was quite low in mCRC patients who underwent pulmonary metastasectomy. The patient with multiple CTCs had shorter DFS in this study. The larger prospective clinical study is needed to establish the meaning of CTC in mCRC candidate for pulmonary metastasectomy.

Abstract 2488: Stat3 inhibitor (BBI-608) with radiation therapy is promising in malignant pleural mesothelioma

Background: The prognosis of Malignant pleural mesothelioma (MPM) is very poor, the new drug for MPM is need. IL-6 in serum with MPM patients is high, IL-6/Stat3 pathway is activated. We investigated that Stat3 is the potential target for the treatment of MPM. Methods: Cell viability was assayed with Cell Counting Kit-8 (CCK-8: WST-8 Dojindo). MPM cells (NCI-H28, NCI-H226, NCI-H2052, NCI-H2452, MSTO-211H) were seeded into 96-wellplates. After the treatment with Stat3 inhibitor (BBI-608). Cell Counting Kit-8 solution was added to each well of the plate and measured the absorbance using a microplate reader. The levels of phosphorylated Stat3 (p-Stat3) were measured in cell lysates using an InstantOne ELISA assays (eBioscience). The translocated p-Stat3, c-Myc were analyzed by Confocal immunofluorescent. In vivo study, H226 cells were injected into the subcutaneous over the flank region of nude mice. Mice were randomly assigned into four groups (5 mice each group) 1) vehicle control 2) treated with BBI-608 3) Radiation 4) BBI608/RT. Tumor is measured twice per week. Results: BBI-608 inhibited MPM cell lines (NCI-H28, NCI-H226, NCI-H2052, NCI-H2452, MSTO-211H) viability in a dose-dependent manner. The level of p-Stat3 was decreased 90% by BBI-608 10μM treatment in H226. Untreated H226, p-stat3 was observed in cytoplasma and localized in the nucleus. As compared with untreated cells, p-stat3, c-Myc were decrease in cytoplasma and was not localized in the nucleus with BBI-608 treated cells. BBI-608 suppressed tumor growth (p Conclusion: In this study, we have shown that BBI-608 inhibited the proliferation of all MPM cell (epithelial, biphasic, sarcomatoid) lines and inhibited Stat3 phosphorylation and blocked the translocation to the nucleus. We also demonstrated that BBBI-608 completely suppressed tumor growth with radiation in mouse model. Furthermore, Stat3 inhibitor with Radiation Therapy (START) is promising in MPM therapy. Citation Format: Seiji Matsumoto, Hiroshi Doi, Tohoru Nakamichi, Ayumi Kuroda, Masaki Hashimoto, Teruhisa Takuwa, Nobuyuki Kondo, Seiki Hasegawa. Stat3 inhibitor (BBI-608) with radiation therapy is promising in malignant pleural mesothelioma. [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 2488.

Abstract 2581: Antitumor effect of Trametinib, a selective MEK inhibitor, in combination with 4-methylumbelliferone, a hyaluronic acid synthesis inhibitor, in Malignant pleural mesothelioma cell lines

Introduction: Malignant pleural mesothelioma (MPM) is a highly aggressive malignancy for which no targeted therapy exists. The mitogen-activated protein kinase (MAPK) pathway plays critical roles in the regulation of tumorigenesis in multiple solid tumors, including MPM. Trametinib, a selective MEK inhibitor, have a survival benefit in patients with V600 BRAF-mutant metastatic melanoma. The effect of trametinib on MPM cells has not been well studied. Hyaluronan (HA) is one of the major components of the extracellular matrix. MPM is in most cases associated with elevated amounts of HA, which has increased the malignant properties of MPM cells. The HA synthesis inhibitor 4-methylumbelliferone (4-MU) has antitumor effects in various malignant tumors, but its effect on MPM cells has not been well studied. Purpose: We evaluated the effects of trametinib, 4-MU and their combination on MPM cells in vitro and in vivo. Experimental Design: The effects of trametinib, 4-MU, and their combination on MPM cells were evaluated using cell viability assay, western blot analysis, and mouse xenograft model. Results: Trametinib exhibited an antiproliferative activity in all four MPM cell lines, NCI-H226, NCI-H2452, NCI-H2052, and MSTO-211H, with IC50 values ranging from 0.15 μM to 12.7 μM. Trametinib blocked the phosphorylation of ERK until 72 hours and decreased the expression of CD44 in a dose-dependent manner. In addition, the expression of CD44 was inhibited (48-72 hours) after the suppression of ERK phosphorylation by trametinib. 4-MU exhibited an antiproliferative activity in MPM cells. 4-MU inhibited ERK phosphorylation but not CD44 expression. In mouse xenograft model, trametinib and 4-MU suppressed tumor growth (trametinib vs. control, P Conclusions: Trametinib and 4-MU exhibited antitumor activities in MPM cell lines. Furthermore, their combination exhibited more potent antitumor activities. These results suggests that Trametinib and 4-MU are promising therapeutic agents in MPM, and these combination therapies may be effective for the treatment of MPM. Citation Format: Hiroyuki Cho, Seiji Matsumoto, Yoshiko Fujita, Ayumi Kuroda, Masaki Hashimoto, Teruhisa Takuwa, Toshi Menju, Makoto Sonobe, Nobuyuki Kondo, Hiroshi Date, Seiki Hasegawa. Antitumor effect of Trametinib, a selective MEK inhibitor, in combination with 4-methylumbelliferone, a hyaluronic acid synthesis inhibitor, in Malignant pleural mesothelioma cell lines. [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 2581. doi:10.1158/1538-7445.AM2015-2581

Abstract 742: Antitumor activity of Trametinib, a MEK1/2 inhibitor, in malignant pleural mesothelioma cells in vitro

Introducion: Malignant pleural mesothelioma (MPM) is a highly aggressive malignancy and there is no approved targeted therapy. The MAPK pathway plays critical roles in the regulation of cell proliferation, growth, differentiation, and survival in multiple solid tumors, including MPM. Trametinib, a selective MEK inhibitor, have a survival benefit in patients with V600 BRAF-mutant metastatic melanoma. FDA approved Trametinib for these patients in May 2013. The effect of Trametinib in MPM cells has not been well studied. Purpose: We examined the effects of Trametinib in MPM cells in vitro. Methods: To examine the effect of Trametinib on the proliferation of MPM cells, we performed cell proliferation assay using four MPM cell lines, NCI-H2452, NCI-H226, NCI-H2052 and MSTO-211H. To examine the effect of Trametinib on intracellular signaling, we performed Western blot analysis in NCI-H226 cell line. Results: Trametinib exhibited potent antiproliferative activity in all four MPM cell lines with IC50 values ranging from 0.5 μM to 44 μM. Trametinib blocked the phosphorylation of ERK until 72 hours and decreased the expression of CD44 in a dose-dependent manner. In addition, the expression of CD44 was inhibited (48-72 hours) after the suppression of ERK phosphorylation by Trametinib. Conclusions: Our results suggest that Trametinib is a promising therapeutic agent for MPM. Citation Format: Hiroyuki Cho, Seiji Matsumoto, Yoshiko Fujita, Ayumi Kuroda, Masaki Hashimoto, Teruhisa Takuwa, Toshi Menju, Makoto Sonobe, Nobuyuki Kondo, Hiroshi Date, Seiki Hasegawa. Antitumor activity of Trametinib, a MEK1/2 inhibitor, in malignant pleural mesothelioma cells in vitro. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 742. doi:10.1158/1538-7445.AM2014-742

1561PPOST-RECURRENCE ADDITIONAL CHEMOTHERAPY IS FEASIBLE AND EFFECTIVE IN PATIENTS UNDERGOING EXTRAPLEURAL PNEUMONECTOMY FOR MALIGNANT PLEURAL MESOTHELIOMA

ABSTRACT Aim: It is believed that additional chemotherapy is infrequently feasible in patients with recurrent malignant pleural mesothelioma (MPM) after extrapleural pneumonectomy (EPP) due to deteriorated cardiopulmonary reserve. We examined the feasibility and efficacy of additional chemotherapy in patients with recurrent MPM after EPP. Methods: A retrospective review was conducted on 56 consecutive patients who underwent bi-/tri-modality treatment with induction chemotherapy, EPP, and with/without radiation therapy during July 2004 and November 2013 at Hyogo College of Medicine. Results: Of 56 patients, 31 (M/F=25/6, right/left=11/20, p-stage I/II/III/IV=1/5/22/3, bi-/tri-modality=12/19) relapsed at median age of 60 years (37-70). Median time to recurrence after EPP was 8.9 months. Of 31 relapsed patients, 10 received best supportive care (BSC) alone, 4 started but discontinued chemotherapy and had BSC, and the remaining 17 (55%) completed>3 cycles of intravenous chemotherapy. Median survival time after recurrence was significantly longer in 17 patients who received additional chemotherapy than that in 14 patients who did not (20 months vs. 3.1 months, p=0.001). Overrall(n=56) Relapsed(n=31) BSC(n=14) additional chemo(n=17) M/F 48/8 12/2 14/3 Right/left 26/30 5/9 6/11 Age at recurrence 62(37-71) 63(37-70) 61(44-68) Time to rucurrence after EPP NA 5.2 mo 24 mo MST after recurrence NA 3.1 mo 20 mo MST after EPP 34 mo 9.4 mo 39 mo Conclusions: Additional systemic chemotherapy was successfully administered in approximately 60% of relapsed patients after bi-/trimodality treatment including EPP, which resulted in a longer survival in comparison with BSC alone. Disclosure: All authors have declared no conflicts of interest.

Abstract 3508: Zoledronate induce expansion of glypican-3 (GPC3) peptide specific cytotoxic T lLymphocytes sufficient for adoptive cancer immunotherapy

BACKGROUND Glypican-3 (GPC3) is an onco-fetal antigen which is overexpressed in human hepatocellular carcinoma (HCC), and is only expressed in the placenta and embryonic liver among normal tissues. Previously, we completed a phase I clinical trial of HLA-A2-restricted GPC3 144-152 (FVGEFFTDV) and A24-restricted GPC3 298-306 (EYILSLEEL) peptide vaccine in 33 patients with advanced HCC. This clinical trial of GPC3-derived peptide vaccine showed the vaccination to be safe, and indicates many immunological responses. To develop more efficient immunotherapy, we investigated large scale expansion of GPC3 peptide specific CTLs for adoptive immunotherapy. PURPOSE In this study, we investigated the efficiency of new method to induce expansion of GPC3 peptide specific CTLs. METHODS Treatment of human peripheral blood mononuclear cells (PBMCs) with zoledronate is a method that enables large-scale γδ T cell expansion. To induce expansion of GPC3 peptide specific CTLs and γδ T cells, the PBMCs of patients vaccinated with GPC3 peptide were cultured with GPC3 peptide and zoledronate for 14 days. We used CD8 + and CD8 − cells that isolated from cultured cells using CD8 microbeads at day 14 as a effector cells. GPC3 peptide specificity and cytotoxic activity of CTLs were analysed by Dextramer assay, cytotoxicity assay and IFN-γ ELISPOT assay. We used human liver cancer cell line SK-Hep-1 (GPC3 − , HLA-A*02:01/24:02) and a human GPC3 gene transfectant, SK-Hep-1 [[Unsupported Character - ⁄]] hGPC3 (GPC3 + , HLA-A*02:01/24:02) as target cells. T2 (HLA-A*02:01, TAP − ) was pulsed with GPC3 peptide or HIV peptide at room temperature for 1h. To evaluate antitumor activity in vivo, we inoculated SK-Hep-1/hGPC3 and SK-Hep-1/vec cells at the right flank of NOD/SCID mice. We injected the CD8 + , CD8 − or all cells as effector cells intravenously. RESULTS The expansion of Lymphocytes from a few PBMCs of vaccinated patients with advanced HCC using zoledronate yields cell numbers sufficient for adoptive transfer. The rate of increase of GPC3 specific CTLs was approximate 490 to 170,000-fold, whereas the rate of increase of total cell number was approximate 13 to 1,000-fold. These CD8 + cells including CTLs showed GPC3 specific cytotoxicity against SK-Hep-1/hGPC3 and T2 pulsed with GPC3 peptide, but not against SK-Hep-1/vec and T2 pulsed with HIV peptide, whereas CD8 − cells including γδ T cells showed cytotoxicity against SK-Hep-1/hGPC3 and SK-Hep-1/vec while did not show GPC3 specificity. Furthermore, adoptive cell transfer of CD8 + cells, CD8 − cells and total cells after expansion significantly inhibited tumor growth in NOD/SCID mouse model. CONCLUSION This study indicates that zoledronate is useful to large-scale expand antigen-specific CTLs and γδ T cells for adoptive immunotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3508. doi:1538-7445.AM2012-3508