Espinoza‐Delgado, Igor

4083 Background: Thymidylate synthase (TS) over-expression is associated with 5-FU resistance. Pre-clinical studies demonstrate that vorinostat down-regulates intra-tumor TS in a dose-dependent fashion and augments 5-FU antitumor activity in xenograft models. We conducted a phase I clinical trial of an intermittent schedule of QD x 3 vorinostat in combination with a fixed dose of fluorouracil (5-FU) and leucovorin (LV) in patients (pts) with refractory solid tumors. Methods: Vorinostat was escalated in a standard 3 x 3 design in combination with a fixed dose of 5-FU and LV (simplified de Gramont regimen, sFULV2). Vorinostat was given QD x 3 on an every-2-week cycle. sFULV2 started on day 2 of vorinostat and consisted of leucovorin 400 mg/m2 i.v. over 2 hrs followed by 5-FU 400 mg/m2 bolus and 5-FU 2400 mg/m2 over 46 hrs. Results: 24 pts were enrolled: Male/Female: 11/13; ECOG 0/1: 6/18; Age: median 60 (range 42–77) yrs. 21 pts had colorectal cancer (CRC), 1 had gastric, 1 had esophageal, and 1 had anal cancer. Vorinostat dose-levels (DL) were 600 mg, 800 mg, 1000 mg, 1200 mg, 1400 mg, 1700 mg, and 2000 mg. Dose-limiting toxicities (DLT), consisting of fatigue and hand-and-foot syndrome (H&F), were seen in 2 of 3 pts at the 2000 mg DL. None of the 6 pts at the 1700 mg DL had a DLT. Cycle 1 grade 3/4 toxicities consisted of thrombocytopenia, GI bleeding, fatigue, and H&F in 2 pts at the 2000 mg DL and a non-DLT G3 diarrhea (lasted <24 hrs) in 1 pt at the 1700 mg DL. Grade 2 nausea, fatigue, and anorexia were common; especially at DL ≥ 1700 mg. Antitumor activity was noted in pts with CRC despite prior refractoriness to 5-FU and failure to oxaliplatin, irinotecan, and cetuximab in all pts. 12/21 CRC pts had a confirmed SD (11) or PR (1). CRC pts had a median PFS of 4 months, a ≥ 6 months PFS rate of 43%, and a ≥ 8 months PFS rate of 33%. Conclusions: The maximum tolerated dose (MTD) of vorinostat in combination with sFULV2 is 1700 mg PO QD x 3 every 2 weeks. This combination is associated with considerable activity in pts with 5-FU-refractory CRC and warrants further investigation. An expanded MTD cohort is accruing to investigate 5-FU-vorinostat PK interaction and intra-tumor TS down-regulation. (This work was supported by a grant from CTEP and the ACS.) No significant financial relationships to disclose.

Abstract Abstract 2742 Poster Board II-718 Background: The BCR-ABL inhibitor imatinib has revolutionized the treatment of CML, and improves the efficacy of chemotherapy in Ph+ ALL. Acquired imatinib resistance (IR) is an emerging problem. Patients who develop IR have a dismal prognosis and novel therapeutic strategies are needed. Mechanisms of resistance include mutations and overexpression of BCR/ABL. In addition, BCR/ABL-independent pathways may contribute to resistance. Mounting evidence suggests that aberrant histone deacetylase (HDAC) expression plays a key role in acquired IR. HDAC inhibition has been shown to downregulate BCR/ABL expression via multiple mechanisms. The functional effects of HDAC inhibition include cell cycle arrest and induction of mitochondrial damage and caspase-dependent apoptosis, both in cells that are imatinib sensitive (IS) and IR. We hypothesized that HDAC inhibition will demonstrate synergistic activity in combination with imatinib in IR cells. To test this hypothesis, we employed entinostat (SNDX-275), a novel, class I HDAC isoform selective inhibitor currently being tested in multiple phase 2 clinical trials. We explored the use of SNDX-275 alone and in combination with imatinib and dasatinib in IR and IS p210BCR/ABL expressing cell lines. Methods: K562 (p210BCR/ABL expressing CML cell line), IR K562 cells (Weisberg E, et al. Blood. 2000;95:3498), Ba/F3 cells, Ba/F3 cells transfected with p210BCR/ABL and Ba/F3 cells transfected with the T315I mutant form of BCR/ABL were treated with increasing concentrations of SNDX-275 (0.2 to 1.6μM), imatinib (0.1 to 1.6 μM), and dasatinib (0.3 to 250 nM) for 72 hours. The cells were also treated with combinations of SNDX-275 plus imatinib and SNDX-275 plus dasatinib at the same range of concentrations for 72 hours. All experiments were done in triplicate. Drug-related effects on cell viability were determined by MTT cytotoxicity assays. CalcuSyn software was used to calculate the IC50 for each drug in each cell line and to calculate the combination index (CI) for cell lines treated with combinations of imatinib or dasatinib plus SNDX-275. A CI from 0.9 to 1.1 indicates that the two drugs have an additive effect, below 0.9 indicates synergy between the two drugs and above 1.1 indicates antagonism. Results: In the Ba/F3 p210BCR/ABL and Ba/F3 T315I cells, SNDX-275 alone had minimal activity up to 1 μM. In K562 and IR K562 cells SNDX-275 alone led to significant cytotoxicity with an IC50 of 0.662 μM and 0.495 μM respectively. In the IR cell lines, cotreatment with SNDX-275 enhanced the activity of both imatinib and dasatinib. For the IR K562 cells, imatinib plus SNDX275 was potently synergistic with a CI of 0.6234. Dasatinib plus SNDX275 treatment of IR K562 cells was additive with a CI of 1.1015 (figure 1). Similarly, in the Ba/F3 T315I cells imatinib plus SNDX275 resulted in synergistic activity (CI = 0.87) and additive activity when treated with dasatinib plus SNDX275 (CI = 0.97). On the contrary, cotreatment of non-IR cell lines with dasatinib or imatinib plus SNDX-275 did not result in synergistic activity and revealed evidence of antagonism. In the K562 cells, the combination of imatinib plus SNDX-275 was antagonistic with a CI of 1.308 (figure 1A). In the Ba/F3 p210BCR/ABL cells, the combination of dasatinib plus SNDX-275 was strikingly antagonistic with a CI of 2.9. Conclusions: BCR/ABL-independent mechanisms of resistance, including aberrant acetylation of histone and non-histone proteins secondary to the deregulation of HDACs, contribute to IR in patients with CML and Ph+ ALL. Our data support this hypothesis, as the HDAC inhibitor SNDX-275 enhances the activity of imatinib and, to a lesser extent, dasatinib in IR p210BCR/ABL expressing cell lines. Therefore, SNDX-275 may represent a novel therapeutic approach to patients with IR CML and Ph+ ALL, and a phase I clinical study is planned. In contrast, SNDX-275 may antagonize the activity of the tyrosine kinase inhibitors in nonresistant CML and Ph+ ALL. To our knowledge, this is the first report of antagonism between a HDAC inhibitor and tyrosine kinase inhibitors in CML. Further investigation is necessary to define the mechanism and clinical significance of this antagonism. Disclosures: No relevant conflicts of interest to declare.

Abstract Purpose: A phase I, dose-finding study of vorinostat in combination with temozolomide (TMZ) was conducted to determine the maximum tolerated dose (MTD), safety, and pharmacokinetics in patients with high-grade glioma (HGG). Experimental Design: This phase I, dose-finding, investigational study was conducted in two parts. Part 1 was a dose-escalation study of vorinostat in combination with TMZ 150 mg/m2/day for 5 days every 28 days. Part 2 was a dose-escalation study of vorinostat in combination with TMZ 150 mg/m2/day for 5 days of the first cycle and 200 mg/m2/day for 5 days of the subsequent 28-day cycles. Results: In part 1, the MTD of vorinostat administered on days 1 to 7 and 15 to 21 of every 28-day cycle, in combination with TMZ, was 500 mg daily. Dose-limiting toxicities (DLT) included grade 3 anorexia, grade 3 ALT, and grade 5 hemorrhage in the setting of grade 4 thrombocytopenia. In part 2, the MTD of vorinostat on days 1 to 7 and 15 to 21 of every 28-day cycle, combined with TMZ, was 400 mg daily. No DLTs were encountered, but vorinostat dosing could not be escalated further due to thrombocytopenia. The most common serious adverse events were fatigue, lymphopenia, thrombocytopenia, and thromboembolic events. There were no apparent pharmacokinetic interactions between vorinostat and TMZ. Vorinostat treatment resulted in hyperacetylation of histones H3 and H4 in peripheral mononuclear cells. Conclusion: Vorinostat in combination with temozolomide is well tolerated in patients with HGG. A phase I/II trial of vorinostat with radiotherapy and concomitant TMZ in newly diagnosed glioblastoma is underway. Clin Cancer Res; 18(21); 6032–9. ©2012 AACR.

Abstract Background: Epigenetic alterations in the genome, including abnormal DNA methylation and histone hypoacetylation, initiate and promote cancerous changes via several mechanisms, including inactivation of tumor suppressor genes. Preclinical investigations in breast cancer suggest that use of epigenetic modifiers results in re-expression of aberrantly silenced genes and proteins that represent important therapeutic targets (e.g. estrogen receptor alpha, ER). Combination therapy with a DNA methyltransferase inhibitor (DNMTI) and a histone deacetylase inhibitor (HDACI) has yielded superior ER reexpression and greater restoration of tamoxifen responsiveness than with HDACI alone. We hypothesize that clinically tolerable doses of the DNMTI 5-azacitidine (5-AZA) and the HDACI entinostat may not only effect changes in DNA methylation and gene expression, but also yield objective disease responses in women with advanced breast cancer. Trial design: This multicenter phase II study (NCT01349959) is enrolling patients with advanced human epidermal growth factor receptor 2 (HER2)-negative breast cancer with triple negative (ER/progesterone receptor [PR]/HER2−negative, Cohort A) or hormone-resistant (Cohort B) disease. Patients will receive 5-AZA 40 mg/m2 subcutaneously days 1–5 and 8–10 and entinostat 7 mg orally days 3 and 10 every 28 days. Because of the potential for re-expression of the ER with epigenetic agents, patients will be offered continuation of 5-AZA and entinostat at progression with the addition of hormonal therapy (investigator discretion). Mandatory tumor biopsies will be performed at baseline and after 8 weeks of therapy to evaluate correlative biomarkers. Eligibility Criteria: Eligible patients must be ≥ 18 years, have measurable locally advanced/metastatic triple-negative (at least one prior chemotherapy received adjuvant/metastatic setting) or hormone-resistant (must have received two prior hormonal agents and one prior chemotherapy) disease, adequate organ function and ECOG PS ≤ 2. Specific Aims: 1. Objective response rate (ORR) by RECIST 1.1 criteria. 2. Safety and tolerability 3. Progression-free survival, overall survival and clinical benefit rate. 4. Safety and toxicity data, feasibility and response rate where hormonal therapy is added to the combination under investigation at the time of progressive disease. 5. Pharmacokinetics, cytidine deaminase, changes from baseline of candidate gene methylation and expression in circulating deoxyribonucleic acid (DNA) and malignant tissue. Statistical Methods: Using a two-stage three-outcome design to assess the efficacy of the combination, a maximum of 30 patients (requiring 27 evaluable) will be accrued to each cohort unless undue toxicity is encountered for a maximum sample size of 60 patients. The study design tests the null hypothesis that the ORR is at most 5% against the alternative hypothesis that it is at least 20% with a type I error of 4% and power of 90%. Present and Targeted Accrual: This study has just opened to patient enrollment. We anticipate a rapid accrual of 60 patients within 1 year.br](Funding from Stand Up to Cancer and CTEP). Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr OT3-01-06.

Abstract Abstract #404 Background: The oral HDACi vorinostat (V) sensitizes breast cancer cells to tubulin polymerizing agents and anti-VEGF directed therapies in vitro. We sought to determine the safety and efficacy of V plus paclitaxel (P) and bevacizumab (B) as first-line therapy for MBC.&#x2028; Methods: Eligible patients (pts) with measurable disease, ECOG PS 0 or 1, and no prior chemotherapy for MBC, received P (90mg/m2) on days 1, 8, 15 and B (10mg/kg) on days 1,15 every 28 days, plus V 200 mg (N=3) or 300 mg (N=28) BID orally for 3 days given the day before, day of, and day after each P dose. Response was assessed after every 3 cycles (RECIST). The trial was designed to distinguish between a response rate of &lt; 40% vs. &gt; 60% (alpha=0.10, beta=0.10) using Simon's optimal 2-stage design. At least 12 responses were required in the first 28 evaluable pts in the 1st stage and 21 of 41 in the 2nd stage.&#x2028; Results: There were no dose-limiting toxicities among the first 6 pts treated at the 1st (200 mg BID; N=3) and 2nd (300 mg BID; N=3) dose levels. Among the 26 evaluable pts treated at the recommended phase II dose of 300mg bid in the 1st stage, 14 patients had a partial response (PR-54%; 90% confidence interval [C.I.] 36%-71%), 6 had stable disease (SD) at 3 months and remain on treatment, 2 had SD as the best response with progressive disease (PD) at 11.8 and 15 months, and 4 had PD as their best response. Additionally, 2 of 3 pts treated at the first V dose level had a PR, indicating PR in 16 of 29 overall (55%, 90% C.I 38%-71%). Only 11 of 29 evaluable pts (38%) have progressed after a median followup of 10.5 months (R-2.3-14.9). Two pts had tumor biopsies and peripheral blood mononuclear cells (PBMC) collected before and 4 hours after the 3rd V dose (300 mg BID) which showed increased acetylation of K69 lysine residue of the chaperone protein Hsp 90, upregulation of Hsp70, and downregulation of AKT by Western Blot, providing evidence of Hsp90 inhibition in tumor and PBMC.&#x2028; &#x2028; Gr 3-4 toxicities included neutropenia (26%), neuropathy (23%), fatigue (16%), thrombosis (10%), vomiting (6%), hypertension (3%), and diarrhea (3%).&#x2028; Conclusions: Our preliminary findings indicate that the HDACi V inhibits Hsp90 and downregulates AKT in breast cancer in vivo, and enhances the effectiveness of P plus B in MBC. Accrual is nearly complete and the final efficacy analysis will be presented. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 404.

AbstractPurpose: We conducted a phase I clinical trial to determine the maximum tolerated dose (MTD) of daily or twice daily vorinostat × 3 days when combined with fixed doses of 5-fluorouracil (FU) and leucovorin every 2 weeks.Experimental Design: Vorinostat doses were escalated in a standard 3 × 3 phase I design. FU/leucovorin was started on day 2 of vorinostat and consisted of leucovorin 400 mg/m2 i.v. over 2 hours followed by FU 400 mg/m2 i.v. bolus and 2,400 mg/m2 over 46 hours (sLV5FU2).Results: Forty-three patients were enrolled. Grade 3 fatigue, and hand and foot syndrome were the dose-limiting toxicities (DLT) at the 2,000 mg vorinostat once-daily dose level. Grade 3 fatigue and mucositis were DLTs at the 800 mg vorinostat twice-daily dose level. None of six patients at the 1,700 mg once daily or six patients at the 600 mg twice daily dose levels had a DLT; those dose levels represent the MTD. Twenty-one of 38 patients with FU-refractory colorectal cancer had stable disease, and one had a partial response. Vorinostat maximum serum concentrations at the MTD exceeded concentrations associated with thymidylate synthase downregulation in vitro. No pharmacokinetic interactions were noted between vorinostat and FU.Conclusions: The MTD of vorinostat in combination with sLV5FU2 is 1,700 mg orally once daily × 3 or 600 mg orally twice daily × 3 days every 2 weeks. Clinical activity in refractory colorectal cancer supports further clinical development of this combination. Clin Cancer Res; 16(14); 3786–94. ©2010 AACR.