Kawakami, Hisato

203 Background: D2 gastrectomy followed by adjuvant S-1 is one of the standard therapy for the patients (pts) with stage III gastric cancer (GC) in Japan; however, the outcome is not satisfactory. We examined the efficacy of perioperative capecitabine and oxaliplatin (CapeOx) in pts with clinical SS/SE N1-3 M0 GC. Methods: The eligibility criteria included histopathologically confirmed clinical T3(SS)/T4a(SE) N1-3 M0 GC according to the Japanese Classification of GC (JCGC; 3rd English Edition). Three cycles of neoadjuvant CapeOx (NAC; capecitabine, 2,000 mg/m2 for 14 days; oxaliplatin, 130 mg/m2 on day 1, every 3 weeks) were administered, followed by five cycles of adjuvant CapeOx after D2 gastrectomy. The primary endpoint was the pathological response rate (pRR) according to JCGC ( ≥Grade 1b). Results: Thirty-seven pts were enrolled from April 2016 to May 2017, and fully evaluated for efficacy and toxicity. Thirty-three pts (89.2%) completed the planned three cycles of NAC and underwent gastrectomy, with an R0 resection rate of 78.4% (n = 29) and a pRR of 54.1% (n = 20, p = .058; 90% confidence interval [CI], 39.4–68.2) were demonstrated. The relative dose intensity (RDI) of capecitabine and oxaliplatin were 90.5% and 91.9%, respectively. Among 27 pts who initiated AC, 21 (63.6%) completed the treatment, and the RDI of capecitabine and oxaliplatin were 80.9% and 65.1%, respectively. Grade 3–4 toxicities during NAC included neutropenia (8%), thrombocytopenia (8%), and anorexia (8%) and during AC included neutropenia (37%), diarrhea (4%), and anorexia (4%), but no treatment-related death was reported. The overall survival (OS) rate and relapse free survival (RFS) rate at 3 years was 83.8% (95% CI, 72.7-96.5%) and 73.0% (95% CI, 60.0-88.8%), respectively. Subgroup analyses according to residual tumor after surgery (R status) showed a 3-year OS and RFS rate of 86.2% (95% CI, 74.5-99.7%) and 75.7% (95% CI, 63.0-90.8%) for R0. Conclusions: Perioperative CapeOx showed good feasibility and favorable prognosis with sufficient pathological response, although statistical significance at .058 did not reach the commonly accepted cutoff of .05. The data obtained using this novel approach warrant further investigations. Clinical trial information: 000021641.

AbstractBackgroundHepatectomy is standard treatment for colorectal liver metastases; however, it is unclear whether liver metastases from other primary cancers should be resected or not. The Japanese Society of Hepato‐Biliary‐Pancreatic Surgery therefore created clinical practice guidelines for the management of metastatic liver tumors.MethodsEight primary diseases were selected based on the number of hepatectomies performed for each malignancy per year. Clinical questions were structured in the population, intervention, comparison, and outcomes (PICO) format. Systematic reviews were performed, and the strength of recommendations and the level of quality of evidence for each clinical question were discussed and determined. The Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach was used to assess evidence and make recommendations.ResultsThe eight primary sites were grouped into five categories based on suggested indications for hepatectomy and consensus of the guidelines committee. Fourteen clinical questions were devised, covering five topics: (1) diagnosis, (2) operative treatment, (3) ablation therapy, (4) the eight primary diseases, and (5) systemic therapies. The grade of recommendation was strong for one clinical question and weak for the other 13 clinical questions. The quality of the evidence was moderate for two questions, low for 10, and very low for two.A flowchart was made to summarize the outcomes of the guidelines for the indications of hepatectomy and systemic therapy.ConclusionsThese guidelines were developed to provide useful information based on evidence in the published literature for the clinical management of liver metastases, and they could be helpful for conducting future clinical trials to provide higher‐quality evidence.

106 Background: Single agent of panitumumab (Pmab) is expected to be well tolerated and to improve survival in first-line setting in patients (pts) who are not eligible for intensive chemotherapy, although the efficacy and safety of Pmab for chemotherapy-naïve frail or elderly Japanese pts with wild-type (wt) RAS unresectable colorectal cancer (CRC) have not been yet studied. Methods: We conducted a multi-center phase II study. Pts aged over 76 years, or over 65 who were considered unsuitable for intensive chemotherapy. Pmab 6 mg/kg was administered intravenously every 2 weeks. The primary endpoint was disease control rate (DCR), and secondary endpoints included progression-free survival (PFS), overall survival (OS), response rate (RR), time to treatment failure (TTF), and the incidence of grade 3 or 4 toxicities. Sample size was set to 36 with exact p-value of 0.05, a power of 0.90, the null of hypothesis of 45% and alternative hypothesis of 70% based on the Clopper-Pearson method. Results: A total 36 pts were enrolled in February 2017 to August 2018. Two pts were excluded; one was a lack of image examination at baseline, and the other was a lack of measurable lesion. The median age was 81 (67-88), with 29 pts (85%) being aged over 76 years. There were 33 (92%) pts with performance status (PS) 0/1, while two (6%) and one (3%) pts were PS 2/3, respectively. Twenty-eight pts (78%) had left-sided CRC, while eight pts had right-sided CRC. The RR was 50.0% (95%CI, 32.4-67.6) including three cases (8.8%) of complete response, and SD was 26.5%, yielding 76.5% of DCR (p < 0.001, 90% confidence interval [CI], 61.5-87.7). The RR with left sided tumor was 65% (95%CI, 44.3-82.8), while that pts with right-sided tumor was 0% (95%CI, 0.0-36.9)(p = 0.003). The major grade 3 or 4 nonhematologic toxicities were rash (n = 6, 17%), hypomagnesemia (n = 4, 11%), fatigue (n = 3, 8%), paronychia (n = 3, 6%), and hyponatremia (n = 3, 6%). The grade 3 hematologic toxicities was neutropenia (n = 1, 3%). Conclusions: Pmab monotherapy showed the favolable efficacy and feasibility in the frail or elderly pts with RAS wt, unresectable CRC. The survival analysis including OS, PFS and TTF is awaiting. Clinical trial information: UMIN000024528.

102 Background: This phase II study aimed to investigate the safety and efficacy of XP compared to SP in the first-line treatment of HER2 negative AGC. Methods: Patients were randomly assigned to receive either SP (S-1 at 40–60 mg twice daily for 21 days plus cisplatin at 60 mg/ m2 on day 8, every 5 weeks) or XP (capecitabine 1,000 mg/m2 twice daily for 14 days plus cisplatin 80 mg/m2 on day 1, every 3 weeks). Primary endpoint was response rate (RR), and secondary endpoints were progression-free survival (PFS), overall survival (OS), time to treatment failure (TTF), and adverse events. Results: 84 eligible patients were randomly assigned to receive SP ( N = 41) or XP ( N = 43). No statistical difference was observed in overall RR between the SP and XP groups [51.2% (95% CI, 35.1% to 67.1%) vs. 53.5% (95% CI, 37.7% to 68.8%), P = 1.000]. Despite not significant, however, SP vs. XP showed a trend toward better PFS [median, 5.9 months vs. 4.1 months; hazard ratio (HR), 0.763; 95% CI, 0.462 to 1.259; P = .284] and OS (median, 13.5 months vs. 10.0 months; HR, 0.776; 95% CI, 0.485 to 1.244; P = .290). This trend in the SP vs. XP comparison was more pronounced in TTF (median, 4.5 months vs. 3.1 months; HR, 0.651; 95% CI, 0.421 to 1.006; P = .052). Common grade 3 to 4 hematological toxicities were neutropenia and anemia (SP group, 23% and 23%; XP group, 35% and 28%). Grade 3-4 anorexia and hyponatremia were more frequently seen in the XP group (31% and 16%) compared to the SP group (13% and 5%). Treatment-related deaths occurred in one patient (2.3%) in the XP group. Conclusions: XP failed to demonstrate the superior efficacy over SP. Higher incidence of severe toxicities by XP suggests SP as the standard 1st line chemotherapy for HER2 negative AGC in Japan. Clinical trial information: UMIN000006755.

Abstract Background. Mutation of the BRAF(V600E) oncogene occurs in ~8% of human colorectal cancers (CRC) and its inhibition is associated with treatment resistance that is due, in part, to paradoxical re-activation of MEK/ERK signaling. Recently, pathway analysis identified cyclin-dependent kinase 1 (CDK1) upregulation in a subset of human BRAFV600E CRCs. We determined whether CDK1 antagonism can enhance the efficacy of MEK inhibition in BRAFV600E CRC cell lines. Methods. Manipulation of gene expression was achieved by siRNA and/or ectopic expression, or using isogenic cell pairs. Cells were treated with the CDK1 selective inhibitor RO-3306 or dinaciclib (CDK1,2,5,9 inhibitor) alone or combined with the MEK inhibitor cobimetinib. Apoptosis was quantified by Annexin V staining with flow cytometry; apoptotic signaling was analyzed by immunoblotting of caspase-8 phosphorylation and caspase cleavage. Long-term cell survival was evaluated by clonogenic assay. In vivo efficacy of the drug combination was determined in a murine flank xenograft model. Results. BRAFV600E CRC cell lines expressed CDK1 whose siRNA knockdown or pharmacological inhibition by RO-3306 or dinaciclib was shown to sensitize them to apoptosis (annexin V labeling). The combination of RO-3306 or dinaciclib with cobimetinib cooperatively enhanced apoptosis and reduced clonogenic survival compared to drug monotherapy. Cells isogenic or ectopic for BRAFV600E displayed apoptosis resistance to CDK inhibitors, as did cells with ectopic expression of constitutively active MEK. CDK1 inhibition by RO-3306 or dinaciclib induced a caspase-8-dependent apoptosis shown by caspase-8 restoration in deficient NB7 cells that enhanced dinaciclib-induced caspase-3 cleavage. Furthermore, CDK inhibitors suppressed procaspase-8 phosphorylation at S387 shown by drug withdrawal that restored phospho-S387 and increased cell mitosis. In a CRC xenograft model, dinaciclib plus cobimetinib produced significantly greater tumor growth inhibition in association with a caspase-dependent apoptosis than did either drug alone. Analysis of TCGA datasets revealed overexpression of CDK1 in human CRCs compared to normal colonic tissue. An inverse relationship existed between expression of CDK1 and the cell cycle inhibitor p16. In CRC cells, ectopic p16 was shown to downregulate CDK1 expression. In conclusion, our data establish CDK1 as a novel mediator of apoptosis resistance in BRAFV600E CRC cells lines whose combined targeting with CDK and MEK/ERK inhibitors may represent an effective therapeutic strategy. Citation Format: Peng Zhang, Hisato Kawakami, Weizhen Liu, Xiangyu Zeng, Klaus Stebhardt, Kaixiong Tao, Shengbing Huang, Frank A. Sinicrope. Targeting CDK1 and MEK/ERK overcomes apoptosis resistance in BRAFV600E human colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2336.

Abstract The BRAFV600E mutation occurs in approximately 8% of human colorectal cancers and is associated with therapeutic resistance that is due, in part, to reactivation of MEK/ERK signaling cascade. Recently, pathway analysis identified cyclin-dependent kinase 1 (CDK1) upregulation in a subset of human BRAFV600E colorectal cancers. Therefore, it was determined whether CDK1 antagonism enhances the efficacy of MEK inhibition in BRAFV600E colorectal cancer cells. BRAFV600E colorectal cancer cell lines expressing CDK1 were sensitized to apoptosis upon siRNA knockdown or small-molecule inhibition with RO-3306 (CDK1 inhibitor) or dinaciclib (CDK1, 2, 5, 9 inhibitors). Combination of RO-3306 or dinaciclib with cobimetinib (MEK inhibitor) cooperatively enhanced apoptosis and reduced clonogenic survival versus monotherapy. Cells isogenic or ectopic for BRAFV600E displayed resistance to CDK1 inhibitors, as did cells with ectopic expression of constitutively active MEK. CDK1 inhibitors induced a CASP8-dependent apoptosis shown by caspase-8 restoration in deficient NB7 cells that enhanced dinaciclib-induced CASP3 cleavage. CDK inhibitors suppressed pro-CASP8 phosphorylation at S387, as shown by drug withdrawal, which restored p-S387 and increased mitosis. In a colorectal cancer xenograft model, dinaciclib plus cobimetinib produced significantly greater tumor growth inhibition in association with a caspase-dependent apoptosis versus either drug alone. The Cancer Genome Atlas (TCGA) transcriptomic dataset revealed overexpression of CDK1 in human colorectal cancers versus normal colon. Together, these data establish CDK1 as a novel mediator of apoptosis resistance in BRAFV600E colorectal cancers whose combined targeting with a MEK/ERK inhibitor represents an effective therapeutic strategy. Implications: CDK1 is a novel mediator of apoptosis resistance in BRAFV600E colorectal cancers whose dual targeting with a MEK inhibitor may be therapeutically effective. Mol Cancer Res; 16(3); 378–89. ©2017 AACR.

Abstract BRAFV600E mutation occurs in ~10% of human colorectal cancers (CRC) where it is associated with treatment resistance and poor prognosis. Data from TCGA and a clinical trial cohort identified a distinct subset of BRAF mutant colon cancers with deregulation of the cell cycle and overexpression of CDK1 (Barras D, et al, Clin Cancer Res. 2016). We tested the hypothesis that CDK1 inhibition can enhance apoptosis in BRAFV600E CRC cells. Since BRAF mutant cells show p-ERK activation, combined inhibition of CDK1 and MEK/ERK was evaluated. Using isogenic colon cancer cells, BRAF mutant alleles were shown to confer resistance to the CDK1 inhibitor, R0-3306, in a gene dose-dependent manner that was associated with reduced cleavage of caspase-3 and downstream PARP, and decreased pH2AX (DNA fragmentation marker). Ectopic BRAFV600E or constitutively active MEK mutant also conferred resistance to R0-3306. CDK1 siRNA was shown to increase cobimetinib-induced apoptosis, including annexin V labeling, and increased pH2AX. Furthermore, ERK siRNA or cobimetinib treatment attenuated CDK1 protein expression and increased R0-3306-induced pH2Ax. Moreover, treatment with R0-3306 plus cobimetinib significantly enhanced a caspase-dependent apoptosis and markedly reduced colony formation vs either drug alone in two CRC cell lines. Caspase-3 cleavage by R0-3306 + cobimetinib was dependent upon caspase-8. Mechanistically, CDK1 inhibition by R0-3306 suppressed the phosphorylation of procaspase-8 at Ser-387 shown by R0-3306 withdrawal which restored p-C8-S387 coincident with expression of the mitotic marker, pH3S10. In conclusion, inhibition of CDK1 can significantly augment apoptosis induction by MEK/ERK inhibition in BRAFV600E CRC cells, suggesting a novel therapeutic strategy to overcome mutant BRAF-mediated resistance. Apoptosis induction by CDK1 inhibitor (R0-3306) ± MEK/ERK inhibitor cobimetinibTreatmentTreatmentTreatementTreatmentCRC cell linesDMSOR0-3306 (5 µM)cobimetinib (5 µM)combinationAnnexin V+ (%)RKO13.43 ± 1.4132.55 ± 2.9315.04 ± 0.2652.72 ± 3.16Annexin V+ (%)HT-2911.22 ± 1.8442.44 ± 2.7025.29 ± 0.1665.62 ± 4.11 Citation Format: Hisato Kawakami, Shengbing Huang, Frank A. Sinicrope. Targeting CDK1 and MEK/ERK overcome mutant BRAF-mediated apoptosis resistance in human colorectal cancer cells [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 2173. doi:10.1158/1538-7445.AM2017-2173

603 Background: Oncogenic BRAFV600E mutations activate MAP kinase signaling and are associated with treatment resistance and poor prognosis in patients with colorectal cancer (CRC). In BRAFV600E mutant CRCs, treatment failure may be related to BRAFV600E-mediated apoptosis resistance that occurs by an as yet undefined mechanism. Methods: BRAF isogenic RKO CRC cells and RKO, HT29, WiDr cell lines were treated with cobimetinib ± the small molecule MCL-1 inhibitor, A-1210477. Apoptosis was measured by Annexin V staining and caspase cleavage. Gene knockdown or overexpression was achieved by lentivirus; ERK siRNA was utilized. Competitive RT-PCR was performed for MCL-1 mRNA expression. HT-29 cells with control or MCL-1 shRNA were xenografted into SCID mice, treated with cobimetinib or vehicle, and tumor volume was measured. Results: We found that BRAFV600E can upregulate anti-apoptotic MCL-1 in a gene dose-dependent manner using CRC cell lines isogenic for BRAF. BRAFV600E-induced MCL-1 upregulation was confirmed by ectopic BRAFV600E expression that activated MEK/ERK signaling to phosphorylate (MCL-1Thr163) and stabilize MCL-1. Upregulation of MCL-1 was mediated by MEK/ERK shown by ERK siRNA that suppressed MCL-1. Stabilization of MCL-1 by phosphorylation was shown by a phosphorylation-mimicking mutant and an unphosphorylated MCL-1 mutant that decreased or increased MCL-1 protein turnover, respectively.MEK /ERK inhibition by cobimetinib suppressed MCL-1 expression/phosphorylation and induced pro-apoptotic BIM to a greater extent than did vemurafenib in BRAFV600E cell lines. MCL-1 knockdown vs control shRNA significantly enhanced cobimetinib-induced apoptosis in vitro and in HT29 colon cancer xenografts. A-1210477 also enhanced cobimetinib-induced apoptosis in vitrothat was due to disruption of the interaction of MCL-1 with pro-apoptotic BAK and BIM. Knockdown of BIM attenuated BAX, but not BAK, activation by cobimetinib plus A-1210477. Conclusions: BRAFV600E-mediated MEK/ERK activation can upregulate MCL-1 by phosphorylation/stabilization to confer apoptosis resistance that can be reversed by MCL-1 antagonism.

Abstract Oncogenic BRAFV600E mutations activate MAPK signaling and are associated with treatment resistance and poor prognosis in patients with colorectal cancer. In BRAFV600E-mutant colorectal cancers, treatment failure may be related to BRAFV600E-mediated apoptosis resistance that occurs by an as yet undefined mechanism. We found that BRAFV600E can upregulate anti-apoptotic MCL-1 in a gene dose-dependent manner using colorectal cancer cell lines isogenic for BRAF. BRAFV600E-induced MCL-1 upregulation was confirmed by ectopic BRAFV600E expression that activated MEK/ERK signaling to phosphorylate (MCL-1Thr163) and stabilize MCL-1. Upregulation of MCL-1 was mediated by MEK/ERK shown by the ability of ERK siRNA to suppress MCL-1. Stabilization of MCL-1 by phosphorylation was shown by a phosphorylation-mimicking mutant and an unphosphorylated MCL-1 mutant that decreased or increased MCL-1 protein turnover, respectively. MEK/ERK inhibition by cobimetinib suppressed MCL-1 expression/phosphorylation and induced proapoptotic BIM to a greater extent than did vemurafenib in BRAFV600E cell lines. MCL-1 knockdown versus control shRNA significantly enhanced cobimetinib-induced apoptosis in vitro and in HT29 colon cancer xenografts. The small-molecule MCL-1 inhibitor, A-1210477, also enhanced cobimetinib-induced apoptosis in vitro that was due to disruption of the interaction of MCL-1 with proapoptotic BAK and BIM. Knockdown of BIM attenuated BAX, but not BAK, activation by cobimetinib plus A-1210477. In summary, BRAFV600E-mediated MEK/ERK activation can upregulate MCL-1 by phosphorylation/stabilization to confer apoptosis resistance that can be reversed by MCL-1 antagonism combined with cobimetinib, suggesting a novel therapeutic strategy against BRAFV600E-mutant CRCs. Mol Cancer Ther; 15(12); 3015–27. ©2016 AACR.

Abstract Background HER2-targeted therapies have been shown clinical benefits in patients with HER2 positive breast and gastric cancers. However, resistance against such targeted therapies eventually develops in most cases. Overexpression of heregulin, a ligand for HER3, has been reported as one of the resistant mechanism against HER2-targeted therapies. Upregulation of heregulin activates HER3-PI3K-AKT signaling, leading to the resistance against HER2-targeted therapies. We investigated the effects of heregulin in cell lines and evaluate its clinical impacts in HER2 positive breast and gastric cancers. Materials and methods We utilized transfected or recombinant heregulin to investigate the effect of heregulin on proliferation and apoptosis of SK-BR-3 and NCI-N87 cell lines treated with lapatinib, trastuzumab, trastuzumab-emtansine and paclitaxel. Clinical data and specimens were obtained from patients with HER2 positive breast and gastric cancers, evaluating their heregulin value from both serum and tissues using ELISA and RT-PCR, respectively. Results Heregulin transfection and recombinant heregulin conferred resistance against treatment with lapatinib, trastuzumab, and weak resistance against trastuzumab-emtansine, but no resistance against paclitaxel in HER2 positive cell lines. Clinical data of heregulin among patients treated with HER2-targeted therapies revealed that patients with relatively high heregulin value tended to have worse prognosis. Conclusions Together these data, overexpression of heregulin may at least in part play a role in resistance against HER2-targeted therapies in HER2 positive cancer patients. Further investigation is warranted to elucidate the relevance of treatments targeting heregulin-HER3-PI3K signaling. Citation Format: Yoshikane Nonagase, Kimio Yonesaka, Satomi Watanabe, Koji Haratani, Takayuki Takahama, Naoki Takegawa, Hiroto Ueda, Hisato Kawakami, Hidetoshi Hayashi, Masayuki Takeda, Haruka Sakamoto, Takao Tamura, Kazuhiko Nakagawa, Junji Tsurutani. Heregulin-induced resistance against HER2-targeted therapies in HER2 positive breast and gastric cancer in vitro and in vivo. [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 2939.