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To identify molecular determinants of histone deacetylase inhibitor (HDI) resistance, we selected HuT78 cutaneous T-cell lymphoma (CTCL) cells with romidepsin in the presence of P-glycoprotein inhibitors to prevent transporter upregulation. Resistant sublines were 250- to 385-fold resistant to romidepsin and were resistant to apoptosis induced by apicidin, entinostat, panobinostat, belinostat, and vorinostat. A custom TaqMan array identified increased insulin receptor (INSR) gene expression; immunoblot analysis confirmed increased protein expression and a four- to eightfold increase in mitogen-activated protein kinase (MAPK) kinase (MEK) phosphorylation in resistant cells compared with parental cells. Resistant cells were exquisitely sensitive to MEK inhibitors, and apoptosis correlated with restoration of proapoptotic Bim. Romidepsin combined with MEK inhibitors yielded greater apoptosis in cells expressing mutant KRAS compared with romidepsin treatment alone. Gene expression analysis of samples obtained from patients with CTCL enrolled on the NCI1312 phase 2 study of romidepsin in T-cell lymphoma suggested perturbation of the MAPK pathway by romidepsin. Immunohistochemical analysis of Bim expression demonstrated decreased expression in some skin biopsies at disease progression. These findings implicate increased activation of MEK and decreased Bim expression as a resistance mechanism to HDIs, supporting combination of romidepsin with MEK inhibitors in clinical trials.

Background: Over-expression of Aurora A has been shown in many subtypes of non-Hodgkin lymphoma (NHL) and is a negative prognostic factor in mantle cell lymphoma (MCL), where expression correlates with tumor proliferative signature (Ki67), and may contribute to cell cycle dysregulation. Aurora A regulates chromosomal stability through effects on mitosis and cell cycle regulation. Bortezomib increases both pro-apoptotic proteins and cell cycle dependent kinase inhibitors. Thus, there is a strong scientific rationale to combine Aurora A kinase and proteasome inhibition. We evaluated the combination of alistertib, an oral Aurora A kinase inhibitor, with bortezomib and rituximab in a CTEP sponsored phase 1 study of patients with relapsed /refractory (RR) low grade NHL or MCL. Methods: Patients with RR low grade NHL or MCL, who had received at least one line of immuno-chemotherapy were treated according to a 3 + 3 design with 3 dose escalation cohorts (DL1, DL2, and DL3) to identify the recommended phase 2 dose (RP2D) with an expansion cohort at the RPTD. Patients received alisertib 30mg BID (DL1 and DL2) or 40mg BID (DL3) on days 1-7, bortezomib subcutaneous 1mg/m2 (DL1) or 1.3 mg/m2 (DL2 and DL3) on days 1, 8, and 15, and rituximab 375mg/m2 on day 1 for the first 8 cycles, and subsequently q 3 months. An expansion cohort was treated at DL3. Treatment cycles were 28 days. Dose Limiting Toxicity (DLT) was defined within the first cycle of therapy. Results: A total of 24 patients at 3 sites were enrolled between 06/2013 and 02/2018, including DL1: 3, DL2: 7; DL3: 14. Median age was 64 (range 46-87). Twelve patients (50%) were male. Nineteen patients had low grade NHL, and 5 patients had MCL. Performance status was 0-1 in 22 (92%) patients, and 2 in 2 (8%) patients. Dose Escalation and Safety: The recommended phase II dose (RP2D) was DL3, with the only DLT in DL3 (prolonged grade 4 neutropenia). The most common grade 3-4 AEs at all dose levels were neutropenia (34%), thrombocytopenia (13%), anemia (8%), and fatigue (8%); grade 2 alopecia occurred in 37% (Table 1). Other grade 4 AEs included hypertension & hypotension, respiratory failure, hyperkalemia and hyperuricemia (n=1 (4%) each); one patient died of GI hemorrhage deemed unrelated to study treatment (NSAID overdose). Grade 3 neutropenia and leukopenia was seen in 4 patients and thrombocytopenia in 1. There was one grade 3 AE each of: hypertension, pulmonary hypertension, pneumonitis, fatigue, diarrhea, infusion reaction, lung infection, and febrile neutropenia. Efficacy: A total of 18 patients were evaluable for response. Five patients were not evaluable for response for the following reasons: withdrawal of consent (3, 1 each DL1, 2, 3), non-compliance (1 DL2), and death unrelated to study drug (1, DL2). For patients who were treated with at least 3 cycles of therapy, and had a restaging CT scan, the median duration of therapy was 142 days, (range 29-590). Response was seen in 7/18 evaluable patients for an overall response rate (ORR) of 38%; 4 of 18 patients (22%) had a complete response (CR), and 4 of 18 patients (22%) had a partial response (PR). An additional 8/18 patients (44%) had stable disease for a clinical benefit rate of 88%. With a median follow-up of 30.9 months, the median PFS was 6.9 months (95%CI 4.1-28.0) and median OS not reached. OS at 3 years was 64.9% (95% CI 44.1-95.3%; Fig. 1). Median duration of response was 14.1 mo (95% CI 2.63-NR). Conclusion: Alisertib in combination with bortezomib and rituximab is a well-tolerated regimen with significant and durable activity in heavily pretreated low grade NHL and MCL. The RP2D is DL3 (R + bortezomib 1.3mg/m2 and 1.8 alisertib 40mg bid). A correlation of Aurora Kinase tumor expression with clinical outcome is planned. Disclosures Barta: Janssen: Membership on an entity's Board of Directors or advisory committees; Merck, Takeda, Celgene, Seattle Genetics, Bayer: Research Funding. Diefenbach:Merck: Consultancy, Research Funding; Genentech: Consultancy; Incyte: Research Funding; Millenium/Takeda: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Acerta: Research Funding; Seattle Genetics: Consultancy, Research Funding; Trillium: Research Funding; Denovo: Research Funding.

Depsipeptide (FK228) is a novel histone deacetylase inhibitor currently in clinical trials and the first to demonstrate clinical activity in patients. Responses have been observed in patients with T-cell lymphomas, despite prior treatment with multiple chemotherapeutic agents. To better understand the effects of histone deacetylase inhibitors on T-cell lymphoma, the human T-cell lymphoma cell line HUT78 was tested for sensitivity and molecular response to depsipeptide. Treatment with depsipeptide, as well as other histone deacetylase inhibitors, caused induction of histone acetylation, induction of p21 expression, and substantial apoptosis without significant cell cycle arrest. Treatment with the caspase inhibitor z-VAD-fmk significantly inhibited depsipeptide-induced apoptosis, enabling detection of cell cycle arrest. Treatment with depsipeptide increased expression of the interleukin-2 (IL-2) receptor, and combination with the IL-2 toxin conjugate denileukin diftitox resulted in more than additive toxicity. Cells selected for resistance to depsipeptide overexpressed the multidrug resistance pump, P-glycoprotein (Pgp). However, cells selected for resistance to depsipeptide in the presence of a Pgp inhibitor had a Pgp-independent mechanism of resistance. These studies confirm the activity of depsipeptide in a T-cell lymphoma model and suggest a general sensitivity of T-cell lymphoma to histone deacetylase inhibitors, an emerging new class of anticancer agents. (Blood. 2004;103:4636-4643)

Background: MLL partial tandem duplication (MLL PTD) occurs in approximately 5-8% of patients with acute myeloid leukemia (AML) and is associated with an adverse prognosis. Our group has published that the MLL wild type (WT) allele is epigenetically silenced in MLL PTD; we showed that re-expression of this gene can be induced with methyltransferase (DNMT) and/or histone deacetylase (HDAC) inhibitors. Further, re-expression of MLL WT following combined decitabine and HDAC inhibitor treatment sensitized MLL PTD myeloid leukemia cells to chemotherapy in vitro. We hypothesized that epigenetic silencing of the MLL WT contributes to MLL PTD-associated leukemogenesis and that its pharmacologic re-expression with DNMT and HDAC inhibitors would activate apoptotic mechanisms important for chemo-response in the clinic. We aimed to develop a regimen to be tested in this unique molecular subset of disease. Because of the relatively low frequency of MLL PTD AML, this dose finding study was conducted in relapsed/refractory (R/R) AML regardless of molecular subtype but was enriched for MLLPTD. Methods:In this phase 1 study, adults aged 18-59 years with R/R AML, ECOG 0-2, and preserved organ function were enrolled. Patients received decitabine 20mg/m2 daily on days 1-10 and vorinostat 400mg daily on days 5-10 for all dose levels. Dose-escalated cytarabine was given on days 12, 14, 16 (six doses) according to the following schedule: dose level (DL) 1, 1.5g/m2/q12hr; DL 2, 2g/m2/q12hr; DL 3, 2.5g/m2/q12hr; and DL 4, 3g/m2/q12hr. Standard method 3+3 phase I design was used with the primary objective to determine the maximum tolerated dose and define specific toxicities with this combination therapy, in order to ultimately develop a regimen for MLLPTD AML. Results:Seventeen adults with R/R AML and median age of 46 years (range, 21-59 years) enrolled. The median number of prior induction therapies was 2 (range, 1-4). European LeukemiaNet (ELN) genetic risk classification frequencies were: favorable (n=2), intermediate-I (n=3), intermediate-II (n=5), and adverse (n=7), respectively. Four patients had MLL PTD. A total of 6 patients were treated at DL 1 with no non-hematologic dose limiting toxicity (DLT). DL 1 was expanded after two patients experienced prolonged but uncomplicated myelosuppression. Since both patients achieved complete remission (CR) shortly after passing the day 42 DLT cut off for hematologic recovery, the protocol was amended to allow further time for count recovery (up to 56 days). Three patients each were treated on DL 2 and 3; 5 patients were treated on DL 4 with no other DLTs observed. Diarrhea, nausea, fatigue, febrile neutropenia, and elevated ALT were the most common toxicities (any grade, regardless of attribution), occurring in 41%, 29%, 29%, 35%, and 35% of patients, respectively, but none were DLTs. In regards to ≥ Grade 3 toxicities, febrile neutropenia and catheter-related infections were most common at 35% and 24%. Significant mucositis was not observed. DL 4 was the recommended phase 2 dose (RP2D). CR or CR with incomplete count recovery (CRi) was observed in 6/17 patients (35%, 5 with CR). Of 6 responders, all (n=4) patients with abnormal karyotype achieved cytogenetic remission. The median number of prior therapies for patients achieving CR/CRi was 2 (range, 1-3). Four patients subsequently received allogeneic transplantation. Of the four patients known to have MLL PTD mutations, two responded (1 with CR and 1 with CRi). It is interesting to note that the two patients with MLL PTD mutations who did not respond to treatment also had FLT3-ITD mutations, while this mutation was absent in the two responding patients who had MLLPTD. Conclusions: We successfully determined the RP2D for this novel treatment regimen. The regimen had modest toxicities beyond uncomplicated (though prolonged) myelosuppression, and we propose that the study provides a framework for larger efficacy studies for AML patients with the uncommon but biologically distinct molecular feature of MLLPTD. Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Number U01CA076576. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Also supported by P30 CA016058/CA/NCI. Disclosures No relevant conflicts of interest to declare.

Background: The histone deacetylase inhibitor (HDACi) romidepsin (Coiffier 2010) and other HDACi such as belinostat (O'Connor 2015) are management options for relapsed peripheral T-cell lymphoma (PTCL), and HDACi have also shown benefit for B-cell lymphomas (Younes 2012). The aurora A kinase (AURKA) inhibitor alisertib has shown promising results in aggressive lymphomas including Burkitt lymphoma (BL), diffuse large B cell lymphoma (DLBCL), and PTCL (Friedberg 2011, Barr 2014), and a registration trial in PTCL has recently completed enrollment. Given preclinical evidence for synergy when combining an AURKA inhibitor plus an HDACi (Zullo 2015, Muscal 2011, Kretzner 2008), we initiated a Phase I trial (NCT01897012, supported by CTEP-NCI) combining alisertib plus romidepsin in patients with multiple lymphoma subtypes. Methods: 12 patients have been enrolled to date, 3 at each of 4 escalation dose levels with patients treated with alisertib on days 1 to 7 and romidepsin on days 1 to 8 of 21 day cycles. Of the eligible histologies there was 1 patient with BL, 3 with DLBCL and FISH positive for translocations of MYC and BCL2 (double-hit lymphoma, DHL), 4 with DLBCL with other unfavorable features (FISH-positive for c-myc alone; 90% Ki-67 positive; high protein staining for MYC and BCL2, but not FISH-positive; and transformed from follicular lymphoma), 3 with PTCL, and 1 with composite PTCL and DLBCL (Fanale 2014). Tumor core biopsies were performed at baseline and at the end of 1 cycle of therapy, part of which was collected in RNAlater for gene expression profiling (GEP) using Illumina HT12v4.0 arrays. Whole peripheral blood was also collected at these and other time points in PAXgene tubes for future GEP. Results: Dose escalation and safety data have been previously presented (Fanale 2014). Responses to date are CR (PTCL, dose level 1), SD (PTCL, dose level 3, composite PTCL/DLBCL, dose level 4), PD (3 DHL, 1 HG DLBCL, 1 DLBCL with c-Myc, 1 PTCL). 4 of the patients with PD have died from continued refractory disease and 1 has been transitioned to hospice. The CR patient received 7 prior lines of treatment and had a duration of remission of 10 month. The PTCL patient with SD underwent an allogeneic stem cell transplant (SCT) but developed further PD 5 months after completion of SCT. Technically-satisfactory GEP results were obtained for 9 pairs of baseline (pre-Rx) and post-cycle 1 (post-Rx) tumor biopsies. Hierarchical clustering of highly-variant genes across all samples, including additional samples from 2 patients (pre-Rx only), showed appropriate grouping of samples according to B- or T-cell lineage. For all but one sample pair, pre-Rx and post-Rx samples from the same patient clustered as nearest neighbors, indicating that treatment had less effect on GEP data than did patient origin, and that "subtraction" (comparing post-Rx to pre-Rx samples) would be necessary for comparing treatment effects. To identify treatment-induced GEP changes associated with response to alisertib plus romidepsin, log2-transformed and subtracted data for the lone patient (with PTCL) who reached CR were compared to those for the other 8 pairs. There were 160 gene probes that were upregulated by log2 >=1.5 in the lone responder, but by >=0.75 in 2 or fewer of the other patients; similarly, 163 probes were downregulated by log2 5-fold induction of TMSB15A, repressed by TGF-beta, and BATF3, essential for CD8a+ dendritic cells in animal models of anti-tumor immune response; and >3-fold induction of TNFRSF18 (GITR), upregulated by T-cell activation. Conclusions: Based on toxicity and response profile thus far, an amendment is under review to modify dosing schema for alisertib and romidepsin, and once the MTD is reached a potential cohort expansion for PTCL patients is planned. GEP changes suggest that induction of an antitumor response may underlie clinical responses, and will be investigated further by other studies of tumor biopsies and whole-blood GEP. Disclosures No relevant conflicts of interest to declare.

Background MLN8237 is an oral inhibitor of aurora kinase A (AURKA) that causes mitotic spindle defects, mitotic delay, and apoptosis in lymphoma cell lines and mouse models. Human studies have shown promising responses in hematologic malignancies. Vorinostat is an oral HDAC inhibitor that is FDA-approved for cutaneous T-cell lymphoma, and is under study in other lymphomas. AURKA inhibitors in combination with vorinostat show synergistic pro-apoptotic effects in vitro in Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) cell lines (Kretzner 2011, Cancer Res 71:3912). Our phase I multicenter study assessed the safety and tolerability of MLN8237 combined with vorinostat in patients with lymphoid malignancies [[NCT01567709][1]] and determined the maximum tolerated dose (MTD) to be 20 mg twice daily (BID) of MLN8237 and 200 mg BID of vorinostat orally in an interrupted dosing schedule (Schedule II). We have recently completed accrual to an expanded cohort at MTD and report preliminary data on our secondary endpoints among patients treated on Schedule II of this study. Methods Eligible patients were ≥18 years old with relapsed or refractory (r/r) lymphoid malignancies (HL, B-NHL, T-NHL), measureable disease, ECOG performance status 0-2, neutrophils ≥1500/µL, platelets ≥100,000/µL, and adequate kidney and liver function. Secondary endpoints were toxicities, clinical response, pharmacokinetic (PK) analysis, and correlative studies. A 3+2 modified rolling-6 design was employed to determine the MTD. Enrollment was initiated on a continuous dosing schedule (Schedule I) that was poorly tolerated, with adverse events (AEs) on dose levels 1 and 2 leading to many dose delays primarily due to gastrointestinal intolerance and myelosuppression. The protocol was amended to the interrupted dosing Schedule II: MLN8237 escalated from 20 to 50 mg BID on days 1-3 and 8-10, and vorinostat given at 200 mg BID on days 1-5 and 8-12 of a 21-day cycle. Results We treated 25 patients (11 DLBCL, 7 HL, 3 FL, 2 MCL, 1 PTCL, 1 NK/T cell) on the interrupted dosing Schedule II. Median age was 59 years (range 26-78). Mediannumber of prior therapies was 4 (range 1-10); 9 patients (36%) underwent prior stem cell transplantation. See Table for treatment summary. MTD of the combination is 20 mg BID for MLN8237 and 200 mg BID for vorinostat on the interrupted schedule. The commonest (>5%) ≥ grade 3 drug-related AEs were neutropenia (52%), thrombocytopenia (44%), leukopenia (44%), anemia (28%), lymphopenia (24%), febrile neutropenia (12%), oral mucositis (8%), diarrhea (8%), and lung infection (8%). There were no study-related deaths. 4 patients stopped treatment due to AEs and 13 due to progressive disease (PD). 2 patients achieved complete remission (CR); both had DLBCL, and both halted therapy after completing 2 further cycles of treatment post-CR. They both remain in CR (18 months and 1 month at data lock). 1 patient had a partial response (PR), and 8 patients maintained stable disease (SD). PKs demonstrated a clearance of 230 L/h (sd=495) and 2.94 L/h (sd=1.57) for vorinostat and MLN8237, respectively. Archived baseline biopsies are being analyzed to determine AURKA expression. Six fresh paired tumor biopsies were obtained before and on-treatment in the expanded cohort at MTD for correlative studies. Conclusions MLN8237 when given in combination with vorinostat is safe and tolerable in an interrupted dosing schedule among heavily pre-treated patients with r/r lymphoid malignancies. The MTD for MLN8237 is 20 mg BID on days 1-3 and 8-10, combined with vorinostat at 200 mg BID on days 1-5 and 8-12, of 21 day cycles. The commonest AEs were hematologic and gastrointestinal. Promising responses were seen in several patients, especially those with DLBCL, which support phase 2 exploration of this therapy in patients with intermediate-high grade NHL. PK analysis suggests that combination therapy exposures are similar to single agent exposure. Correlative studies done in a 12-patient expanded cohort will be presented. [Trial supported in part by UM1CA186717] | Schedule II: MLN8237 (mg)/ Vorinostat (mg) | # of patients treated | # of cycles completed Median (range) | # of dose limiting toxicities (DLT) | DLT Description | Best response | | ------------------------------------------------- | --------------------- | -------------------------------------------- | ----------------------------------- | --------------------------------------------------------------------------------------------- | --------------------------------------------- | | Dose level 1 (30/200) | 7 | 3 (0-18) | 2 | 1 pt had grade 3 febrile neutropenia; 1 pt had grade 3 thrombocytopenia requiring transfusion | 1 CR, 3 SD, 2 PD, 1 N/A | | Dose level -1 (20/200) | 18 | 2 (0-14) | | | 1 CR, 1 PR, 5 SD, 8 PD, 3 too early to assess | Table 1. Disclosures Siddiqi: Kite pharma: Other: attended advisory board meeting; Seattle Genetics: Speakers Bureau; Pharmacyclics/Jannsen: Speakers Bureau. Off Label Use: Vorinostat is only FDA-approved for CTCL but in this study it is being used in conjunction with MLN8237 (not FDA-approved) for all lymphomas.. Beumer: Millenium: Other: Research support. Forman: Mustang Therapeutics: Research Funding. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01567709&atom=%2Fbloodjournal%2F126%2F23%2F2731.atom

Background: Amplification or overexpression of AURKA, a key mitotic regulator, is seen in various tumors and is associated with poor outcome in relapsed/refractory (r/r) lymphoma patients. MLN8237 is an oral inhibitor of AURKA, leading to mitotic spindle defects, mitotic delay, and apoptosis in lymphoma cell lines and mouse models. Human studies have shown promising responses in hematologic malignancies. Vorinostat is an oral HDAC inhibitor FDA-approved for cutaneous T cell lymphoma, and is under study in other lymphomas. AURKA inhibitors, MK0457 and MK5108, when combined with vorinostat and applied to Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) cell lines, convincingly showed synergistic pro-apoptotic effects compared with the AURKA inhibitor alone (Kretzner 2011, Cancer Res 71:3912). Our phase I multicenter study assessed the safety and tolerability of MLN8237 combined with vorinostat in patients with lymphoid malignancies [NCT01567709]. Methods: Patients ³18 years old with r/r mature lymphoid malignancies (HL, B-NHL, T-NHL), measureable disease, ECOG performance status 0-2, neutrophils ³1500/µL, platelets ³100,000/µL, and adequate kidney and liver functions were eligible. The primary endpoints were maximum tolerated dose (MTD) of MLN8237 when combined with vorinostat and description of adverse events (AEs). Secondary endpoints were toxicity, clinical response, pharmacokinetic (PK) analysis and correlative studies. A 3+2 modified rolling design was employed. Enrollment was initiated on Schedule I: MLN8237 escalated from 30-50 mg twice daily (BID) on days 1-7 and vorinostat given at 200 mg BID on days 1-14 of a 21-day cycle*. AEs in patients treated on Schedule I dose levels 1 and 2 led to many dose delays, primarily due to gastrointestinal intolerance and myelosuppression. The protocol was amended to the interrupted dosing Schedule II: MLN8237 escalated from 20-50 mg BID on days 1-3 and 8-10, and vorinostat given at 200 mg BID on days 1-5 and 8-12 of a 21-day cycle. Results: As of August 1 2014, 23 patients have been treated (3 HL, 6 DLBCL, 7 FL, 1 MCL, 1 MZL, 2 B-CLL/SLL, 2 ALCL, 1 NK/T cell). Median age was 61 years (range 28-82). Median number of prior therapies was 4 (range 1-8); 9 patients (39%) underwent prior stem cell transplantation. See Table for treatment summary. MTD of the combination is 20mg BID for MLN8237 and 200mg BID for vorinostat in an interrupted schedule. The most common grade 1-2 AEs (>/=30%) were fatigue (78%), nausea (65%), diarrhea (65%), vomiting (56%), hypokalemia (48%), hypertension (47%), anemia (43%) high alkaline phosphatase (39%), hyponatremia (39%), thrombocytopenia (35%), anorexia (31%), alopecia (31%), constipation (30%), high ALT (30%), high AST (30%), high creatinine (30%), leukopenia (30%). Commonest (>5%) >/= grade 3 drug-related AEs were thrombocytopenia (66%), neutropenia (60%), leukopenia (52%), lymphopenia (43%), anemia (34%), febrile neutropenia (13%), and oral mucositis (8%). There were no study-related deaths. 6 patients stopped protocol treatment due to AEs/intolerance and 7 due to progressive disease (PD). Most patients (8) had stable disease (SD) and 1 patient achieved a complete remission (CR). Conclusions: MLN8237 when given in combination with vorinostat is safe and tolerable in an interrupted schedule among heavily pre-treated patients with r/r lymphoid malignancies. The MTD for MLN8237 is 20mg BID on days 1-3 and 8-10, combined with vorinostat at 200mg BID on days 1-5 and 8-12, of 21 day cycles. The commonest AEs are hematologic and gastrointestinal. PK analysis, correlative studies and response assessments are ongoing in a 12-patient expansion cohort. [Trial supported in part by UM1CA186717] Abstract 4483. TableMLN8237 (mg)/ Vorinostat (mg)# treated# completed cyclesMedian (range)# DLTsDLT DescriptionBest responsesSCHEDULE I (continuous)Dose level 1 (30/400*)62 (1-3)1Grade 3 thrombocytopenia requiring a plt transfusion2 SD, 2 PD, 2 N/ADose level 2 (20/200)32 (2-13)2Both pts had grade 4 thrombocytopenia2 SD, 1 PDSCHEDULE II (interrupted)Dose level 1 (30/200)73 (0-18)21 pt had grade 3 febrile neutropenia; 1 pt had grade 3 thrombocytopenia requiring a plt transfusion1 CR, 3 SD, 2 PD, 1 N/ADose level -1 (20/200)71 (1-3)01 SD, 2 PD, 4 too early *Initial vorinostat dosing was 400mg daily but subsequent cohorts were switched to 200mg BID for better tolerance. Disclosures Off Label Use: MLN8237 and vorinostat have not been FDA-approved for all lymphoid malignancies..

Depsipeptide, FR901228, has demonstrated potent in vitro and in vivo cytotoxic activity against murine and human tumor cell lines. In the laboratory, it has been shown to be a histone deacetylase (HDAC) inhibitor. In a phase I trial of depsipeptide conducted at the National Cancer Institute, 3 patients with cutaneous T-cell lymphoma had a partial response, and 1 patient with peripheral T-cell lymphoma, unspecified, had a complete response. Sézary cells isolated from patients after treatment had increased histone acetylation. These results suggest that inhibition of HDAC is a novel and potentially effective therapy for patients with T-cell lymphoma.

Introduction Adult patients (pts) diagnosed with acute lymphocytic leukemia (ALL) are known to have a poor clinical outcome as compared to children. Studies report a 2 year event free survival of 30-40% for Philadelphia chromosome negative (Ph-) patients age >30 yrs and 17% for age >50 yrs. In order to improve outcome for adult ALL, agents that are effective, safe and associated with a low morbidity are needed. Clofarabine, a second generation purine nucleoside analog, has clinical activity as a single agent and in combination with cytosine arabinoside (ara-C) against refractory and relapsed ALL. Clofarabine exerts its cytotoxicity through multiple mechanisms of action, with major effects via inhibition of ribonucleotide reductase (RR) and DNA polymerase-alpha, and incorporation into DNA leading to DNA damage and activation of apoptotic pathways. Histone deacetylases (HDACs) are important regulators of chromatin involved in silencing of tumor suppressor genes. HDAC inhibitors are shown to be apoptogenic in vitro for ALL cell lines and have received FDA approval for the treatment of CTCL and peripheral T cell lymphoma. Pre-treatment with entinostat has been shown to enhance the cytotoxic activity of fludarabine in leukemia cell lines in vitro (Maggio et al, Cancer Research 2004). Given the similarity of clofarabine to fludarabine, and its FDA approval for children with refractory ALL, the combination of entinostat with clofarabine was pursued. Methods A Phase I window of opportunity study using overlapping schedule of entinostat and clofarabine was used in adult pts with ALL (B precursor) or Acute Bilineage Leukemia (ABL). Pts were enrolled onto one of two arms; arm “A” received repeated cycles of entinostat-clofarabine every 21 days as long as there was evidence of response (CR, CRi, or PR following cycle 1) and pts on arm “B” received one cycle of entinostat-clofarabine prior to standard multi-agent chemotherapy. Entinostat was administered orally on day 1 and day 8 (with dose escalation from flat dosing of 4mg to 6mg to 8mg from cohort 1 to 3). Clofarabine was administered intravenously at a fixed dose for all dose cohorts at 10mg/m2 for 5 days (day 3-7). Adults >40 yrs with newly diagnosed, Ph- B-lineage ALL or ABL were eligible. Additionally, adults > 21 yrs with relapsed and refractory, Ph- ALL or ABL were eligible. Eligibility criteria included serum creatinine < 2.0 mg/dl, hepatic enzymes < or = 2.5 ULN and bilirubin Results 23 pts from 3 institutions were enrolled on this study (18 at JHH, 3 at UMD, 2 at UColorado). 17 pts were treated on arm A and 6 pts on arm B. 6 pts were treated in each dose level with responses as follows: in dose level one (1 CR, 5 NR) and dose level two (1 CR, 1 CRi, 4NR) and dose level three (3 PR and 3 NR). The dose level 3 cohort was expanded with a total of 5 additional pts to date (1 PR, 1 SD, 3NR). Thus, the overall response rate on dose level 3 was 4PR, 1SD, 6NR. The 4 pts with CR/CRi/PR were all de novo treated elderly pts from Arm A. Notably, one pt on arm A has been in remission for over 1.5 yrs. Toxicities to date included expected but manageable grade (G) 3 and 4 cytopenias. There were G3 elevations of ALT (N=2) and AST (2) and bilirubin (1) and one bacteremia (1) and G4 cellulitis (1). Planned correlative studies are ongoing and include evaluation of acetylation of target proteins using multiparameter flow cytometry and western blot as well as methylation evaluation. Conclusions Combination therapy with entinostat-clofarabine is feasible and is well tolerated with minimal toxicity. Promising durable responses were observed in older pts that were not otherwise able to receive multi-agent induction chemotherapy upfront. This is notable given the low dose of clofarabine used in every cohort. Correlative studies evaluating protein hyperacetylation and DNA methylation in serial samples from treated pts are in progress. Disclosures: Off Label Use: This clinical study is a Phase 1 investigation and it discusses non-FDA approved doses of both clofarabine and entinostat for adults with Acute Lymphocytic Leukemia. The reason for this is that this study examines these agents in combination in a Phase 1 fashion and we started with low doses of each agent. Ordentlich:Syndax: Employment. Trepel:Syndax: Research Funding.

Background The hypomethylating agent (HMA) azaC reverses epigenetic silencing and is the first agent demonstrated to improve survival in patients with higher-risk MDS (Silverman et al JCO 2002, Fenaux et al Lancet Oncology 2009). Time to initial response with single agent azaC, is 3 to 4 cycles, the CR rate ranges from 7 to 17% and overall response is 45-50%. Vorinostat, a histone deacetylase inhibitor (HDACI) which inhibits class I and II HDAC, has demonstrated single agent activity in patients with MDS with responses of 20% (Garcia-Manero Blood 2006). In vitro the 2 agents are synergistic in reactivating epigenetically silenced genes. The effect is sequence-dependent requiring exposure to the HMA first followed by the HDAC. We conducted a phase I pilot study of escalating and de-escalating doses of the 2 drugs in combination in 8 cohorts and demonstrated broad activity with responses ranging up to 80% across all of the cohorts tested (Blood 2008). Purpose To determine the response rate of patients treated with the combination of vorinostat and azacitidine at the doses established as safe and effective in Phase I in an expanded cohort of patients with MDS. Methods In the phase II component 3 schedules of the combination were chosen based on response and adverse event profile for further evaluation. Eligible patients were entered into one of 3 cohorts with the combination (see table 1): cohort 1: azaC 55 mg/m2/d 1-7 SC, vorinostat 200mg po Bid d3-16; cohort 2: azaC 75 mg/m2/d 1-7 SC, vorinostat 300mg po Bid d3-9; cohort 3: azaC 55 mg/m2/d 1-7 SC, vorinostat 300mg po Bid d3- 9. Patients with IPSS int-1, -2 and high-risk disease were eligible. Patients with secondary MDS were eligible, those with AML were excluded. Using a simon 2 stage design 13 patients were entered in each cohort and assessed for response, scored according to IWG 2006 criteria and toxicity. Results 40 patients have been entered and 39 (21 male, 18 female) are evaluable for toxicity and 33 for response, 1 pt was registered but never treated. IPSS classification among the 39 patients: int-1 8; int-2 12; high-risk 12; unclassified 7, with median age 67 (23-79). Responses among evaluable patients have occurred in 23 of 33 (70%); 10 CR, 4 CRi, (CR+CRi=42%) 9 HI, 5 SD, 5 NR. Median time to response is 2 cycles (8 weeks). Responses by cohort (table 1) are 70%, 73% and 67% for cohort 1, 2 and 3, respectively. Analysis for the MDS clone at the time of best response demonstrated the persistence of the clone in 45% of patients as marked by cytogenetic or FISH abnormalities, suggesting a modulating rather than cytotoxic effect of the combination on the clone. Cycles administered, range from 1 to 26+ with a median of 6 cycles. Median duration of response is 16 months overall and 9.5, 23 and 27 months, respectively for cohorts 1, 2 and 3. Eighteen patients have come off study for: death or due to disease complications (6); co-morbidities (2); consent withdrawal (5); and withdrawal for stem cell transplant (3). Median OS is 21.1 months and is 10.1, 37.4 and 19 months for cohorts 1, 2, and 3 respectively (NS). Grade 3 fatigue occurred during cycle 1, 2 or 3 in cohort 1 (8%), cohort 2 (16%) and cohort 3 (8%). GI toxicity grade 3 (vomiting, diarrhea, dehydration) occurred in 8% of patients in each of the cohorts. There was no suggestion of cumulative toxicity for either fatigue or GI adverse events. Correlative biologic study analysis is underway. Conclusion The combination of azaC and vorinostat can be safely administered, and is well tolerated in repetitive cycles. The dose of azaC in cohort 2 adheres to the FDA approved dose and schedule. OR and CR rates and time to initial response are comparable among the cohorts and these data suggest that the combination is superior to published results of azaC alone. Cohorts 2 and 3, with vorinostat administered for 7 days, appears to be associated with longer response duration and OS. An intergroup study (SWOG-S1117) comparing azaC and vorinostat to either azaC alone or combined with lenalidomide is underway utilizing the doses and schedule in cohort 2 from this study. Correlative studies that may shed light on mechanism of action or guide patient selection are being conducted. Supported in part by NYCC- N01 CM-62204 and the Henry and Mickey Taub Foundation. Disclosures: Silverman: Merck: Research Funding. Off Label Use: vorinostat for treatment of patients with a myelodysplastic syndrome investigational use.

Background PTCL is a heterogeneous group of mature, post-thymic, T- and natural killer–cell disorders associated with a poor prognosis in most subtypes. Anthracycline-based therapies (eg, CHOP) are most often used in the frontline treatment of PTCL, although they do not typically lead to durable remissions. Older patients may not be eligible for additional chemotherapeutic regimens due to comorbidities and/or poor performance status. Thus, it is important to identify appropriate treatment strategies for older patients with PTCL, particularly in the salvage setting. Romidepsin is a potent class I histone deacetylase inhibitor approved by the FDA for the treatment of patients with PTCL who have received ≥ 1 prior therapy and patients with cutaneous T-cell lymphoma who have received ≥ 1 prior systemic therapy. Approval of PTCL was based on results from the pivotal study of romidepsin for the treatment of relapsed/refractory PTCL (N = 131) that demonstrated durable clinical benefit and tolerability and was supported by a similar study from the National Cancer Institute (N = 47). The objective herein is to present efficacy and safety data for romidepsin specific to older patients (≥ 60 years) with relapsed/refractory PTCL in the pivotal and supportive trials. Methods In both trials, patients with PTCL who relapsed from or were refractory to ≥ 1 prior systemic therapy received romidepsin 14 mg/m2 as a 4-hour intravenous infusion on days 1, 8, and 15 of 28-day cycles. For the pivotal trial, the primary endpoint was confirmed/unconfirmed complete response (CR/CRu) determined by an independent review committee. For the supportive trial, the primary endpoints were objective response rate (ORR) and rate of CR by investigator assessment. In this analysis, efficacy and safety data for patients ≥ 60 years old were examined and compared with those for the overall study population. Results In the pivotal study, the overall median age was 61 years (range, 20-83); 71/130 patients (55%) were ≥ 60 years old (median 67 years [range, 60-83]) with a median of 2 prior systemic therapies (range, 1-8), including prior stem cell transplant in 7 patients. Response rates in the older population were similar to those seen overall: 25% ORR for both populations, including 14% and 15% with CR/CRu for older vs overall populations, respectively. Also, in both the older and overall populations, the median DOR was 28 months, with the longest response ongoing at 48 months (median follow-up 22.3 months). Of the 10 older patients who achieved CR/CRu, 6 had a DOR of ≥ 12 months. Survival was also similar, with 5 and 4 months PFS and 12 and 11 months OS for the older vs overall populations, respectively. In the supportive trial, the overall median age was 60 (range, 27-84); 23/47 patients (49%) were ≥ 60 years old (median 68 years [range, 61-84]) with a median of 2 prior regimens (range, 1-8), including prior stem cell transplant in 7 patients. Response rates in the older population were similar to those seen overall: 32% and 38% ORR, including 14% and 18% with CR, respectively. The median DOR was 5 months (range, 3-49) and 9 months (range, 2-74+) for the older vs overall populations, respectively. One 79-year-old patient with 6 prior systemic therapies achieved CR on romidepsin and stopped therapy after 6 cycles in consideration of his age. Off therapy, disease progression was observed; romidepsin was restarted per protocol and patient achieved a second CR, receiving an additional 22 cycles of therapy. In both the pivotal and supportive trials, rates of grade ≥ 3 adverse events were similar for the overall vs older patient populations (Table). Conclusions In phase 2 trials of romidepsin for the treatment of relapsed/refractory PTCL, patients aged ≥ 60 years comprised approximately half of patients. Both efficacy and safety were similar for the older vs overall populations. Thus, data were not skewed due to age, and romidepsin is suitable for use in elderly patients with relapsed/refractory PTCL. Disclosures: Shustov: Celgene Corporation: Honoraria, Research Funding, Speakers Bureau. Coiffier:Celgene Corporation: Consultancy; Spectrum Pharmaceuticals: Consultancy. Horwitz:Celgene Corporation: Consultancy, Research Funding; Spectrum Pharmaceuticals: Consultancy, Research Funding; Seattle Gen: Consultancy, Research Funding; Infinity Pharmaceuticals: Research Funding; Kyowa Hakko Kirin Pharma: Research Funding; Millenium: Consultancy, Research Funding; Genzyme: Consultancy; Janssen: Consultancy. Sokol:Celgene Corporation: Consultancy, Speakers Bureau; Gloucester: Research Funding. Pro:Celgene Corporation: Honoraria. Nielsen:Celgene Corporation: Employment, Equity Ownership. Balser:Celgene Corporation: Consultancy. Prince:Celgene Corporation: Honoraria, Research Funding. Allen:Celgene Corporation: Honoraria. Bates:Celgene Corporation: Research Funding.

Abstract 1657 Poster Board I-683 Background The histone deacetylase (HDAC) inhibitors are a class of epigenetic agents undergoing clinical testing. HDAC inhibitors modulate expression of genes involved in cell cycle regulation, leading to induction of differentiation or apoptosis. Romidepsin, a novel pan-HDAC inhibitor, has previously demonstrated activity as a single agent in patients (pts) with cutaneous T-cell lymphoma (CTCL) in two phase 2 studies. Aims To evaluate the efficacy and tolerability of romidepsin in the treatment of advanced PTCL and as an exploratory endpoint, to examine the molecular effects of romidepsin in both PTCL and CTCL. Methods This Phase 2, open-label, multi-arm, multicenter study enrolled 46 PTCL pts from the NCI and 9 extramural sites. CTCL pts were also enrolled in this study. This study is now closed to accrual. Pts with relapsed or refractory PTCL who had received at least one prior standard chemotherapy regimen were eligible. Pts received romidepsin 14 mg/m2 as a 4-hr infusion on days 1, 8 and 15 every 28 days. Responses were assessed using elements of the IWG criteria and RECIST, as appropriate, for pts with lymph node involvement and extranodal disease. Results 46 pts (24 [52%] men and 22 [48%] women) with a mean age of 59 (range: 28 to 84) years were treated with romidepsin. 32 pts received ≥ 2 cycles of therapy. Mean number of prior therapies was 4.8 (range 1 to 14). Objective disease response rates (ORR) are summarized in the table. The mean number of cycles of treatment was 6.8 (range 1-47) and the overall median duration of response was 9.0 months (range 1.8 months to 5.8 yrs) for all pts. The median duration of response for the 5 pts who achieved CR was 6.0 months (range 2.8 months to 5.8 yrs).The most frequent drug-related AEs (all grades, all cycles) were generally mild and included: nausea (74%; 9% ≥grade 3), fatigue (72%; 20% ≥grade 3), decreased platelets (72%; 35% ≥grade 3), cardiovascular/general-other (72%; 0% ≥grade 3) and decreased AGC (65%; 43% ≥grade 3). One death, in a pt with significant cardiovascular disease who had achieved a CR, was considered possibly related to treatment. Conclusions This study demonstrates tolerability and durable clinical benefit (ORR of 33% and median duration of response of 9.0 months) of romidepsin in pts with recurrent or refractory PTCL. Based on these promising results, a Phase 2B protocol investigating romidepsin in pts with relapsed or refractory PTCL is ongoing at multiple international centers. Disclosures Kirschbaum: Gloucester Pharmaceuticals: Consultancy.

3709 Poster Board III-645 Background Romidepsin is an anti-neoplastic agent that has been identified as a novel pan-HDAC inhibitor with single-agent activity in cutaneous T-cell lymphoma (CTCL) in 2 studies (GPI-04-0001 and NCI 1312). In the GPI study the overall response rate (ORR) was 34% including 6 complete clinical responses (CCRs) in 96 pts with CTCL and the median duration of response (DOR) was 14.9 months (mo). In the NCI study, the ORR was 35% including 4 CCR in 71 pts and the median DOR was 11 mo. NCI 1312 has also reported single-agent activity in patients with peripheral T-cell lymphoma with an ORR of 31% including 4 CRs in the 48 pts analyzed. HDAC inhibitors have variably been reported to have cardiovascular effects, in particular, QTc prolongation. To systemically and rigorously evaluate the potential cardiac effects of romidepsin, a cardiac safety monitoring plan was developed after discussions with the FDA Division of Oncology Drug Products. Subsequently, a romidepsin QTc Clinical/Statisitcal Analysis Plan was developed following the ICH E14 Guidelines, to provide a comprehensive evaluation of the cardiac effects of romidepsin. Methods An analysis of romidepsin cardiac safety, including review and analysis of ECG findings, was performed. The primary objective of these analyses was to evaluate the effect of romidepsin on the change from baseline on the corrected QT interval of the ECG using the Fridericia QT correction method (QTcF) during Cycle 1 Day 1 exposure to study drug. A total of 4909 ECGs from 110 patients in 3 clinical studies, GPI study, NCI study, and Study GPI-06-0005 (a Phase 1 PK study) were evaluated. The 110 patients comprised 103 patients with T-cell lymphoma and 7 patients with advanced cancer treated with romidepsin at 14 mg/m2 as a 4-hour infusion on Days 1, 8, and 15 of a 28-day cycle. Using a nonlinear mixed effects model the pharmacodynamics of romidepsin was characterized for exposure-QTc effects. Results In an analysis of ECG data, a mean increase of 5.0 msec (90% CI upper bound 7.7 msec) in QTcF interval was measured following infusion of romidepsin; this value includes a contribution to QTc prolongation by pre-dose antiemetics. Data from the NCI study showed that the QTcF change persisted to 24 hours, with a return to baseline by 48 hours, whereas in the smaller GPI-06-0005 study, QTcF peaked 2 hours post infusion and there was no QTcF effect seen at 24 hours. Categorical analyses showed no patients with a change from baseline >60 msec or an absolute QTcF >480 msec. No cardiac events of Torsade de Pointes were reported. Per the ICH E14 guidance,56 a threshold for regulatory concern for mean QTc prolongation is 5 msec with an upper bound of the 95% CI of 10 msec. Prolongations of >60 msec over baseline or a value >500 msec are also considered cautionary. Treatment-emergent morphological changes on ECG (minor ST-segment depression, T-wave flattening, biphasic T-waves, and T-wave inversion) have been observed during treatment with romidepsin; however the overall frequency of these minor, asymptomatic changes was similar to the frequency of pre-exposure abnormalities on each dosing day, suggesting a high degree of background noise in these data. Furthermore, there was no association between these ECG findings and any clinical, functional, or laboratory findings of cardiac abnormalities. | Category | Study | | |:------------------------------- | -------------------- | --------------------- | ----------------- | | GPI Study N=87 n (%) | NCI Study N=41 n (%) | GPI-06-0005 N=7 n (%) | Total N=135 n (%) | | QTcF change from baseline[1][1] | | No increase | 31 (36) | 10 (24) | 2 (29) | 43 (32) | | 1-29 msec increase | 46 (53) | 27 (66) | 4 (57) | 77 (57) | | 30-60 msec increase | 2 (2) | 4 (10) | 1 (14) | 7 (5) | | >60 msec increase | | | | | | QTcF not available | 8 (9) | | | 8 (6) | | QTcF absolute value: | | >450 msec | 2 (2) | 2 (5) | | 4 (3) | | >480 msec | | | | | | >500 msec | | | | | * [↵][2]1 For GPI studies, baseline values are those post co-med, or if missing post co-med, then pre-comed baseline is used. Categorical Analysis of QTc Change on Cycle 1, Day 1 Conclusions The findings from the romidepsin studies show that the observed QTc changes were below the threshold levels of concern outlined in ICH E14. Pharmacodynamic modeling showed no evidence of a relationship between the plasma concentration of romidepsin and changes in the QTc interval. Since hypokalemia and hypomagnesemia are associated with ECG abnormalities, supplementation with potassium and magnesium to achieve normal levels prior to romidepsin administration is recommended. Disclosures: Cabell: Gloucester Pharmaceuticals: Consultancy. Whittaker: Gloucester Pharmaceuticals: Research Funding. Nichols: Gloucester Pharmaceuticals: Employment, Equity Ownership. Nix: Gloucester Pharmaceuticals: Employment. Burris: Gloucester Pharmaceutcals: Research Funding. [1]: #fn-1 [2]: #xref-fn-1-1

Abstract 3701 Poster Board III-637 Background Romidepsin is an anti-neoplastic agent that has been identified as a novel pan-HDAC inhibitor with single-agent activity in T-cell lymphoma. In a combined analysis of 167 patients (pts) with cutaneous T-cell lymphoma (CTCL) from 2 clinical studies (GPI and NCI studies), the overall response rate was 35%, including 10 pts with a complete clinical response (CCR). Median duration of response was 13.8 months and 42% of pts with advanced disease (stage ≥IIB) responded [Demierre et al. J Clin Oncol 27:15s, 2009 (suppl; abstr 8546)]. The most common hematologic abnormalities in these pts included anemia (41%), thrombocytopenia (34%), neutropenia (27%), and lymphopenia (26%). Most hematologic toxicities were Grade 1 or 2, although 'Grade 3 events were observed. These events were reversible and a small portion of the patients discontinued the study drug because of these events (2%). This report details an analysis of platelet counts in pts receiving romidepsin and an investigation into the mechanism of thrombocytopenia in nonclinical studies. Methods Pts with CTCL who received ≥1 prior systemic therapy failure and had an ECOG PS of 0-2 were enrolled in 2 single-arm, open-label, multicenter and international clinical studies. Treatment with QTc prolonging therapies or CYP34A inhibitors was prohibited and pts with significant cardiovascular abnormalities were excluded. Romidepsin at 14 mg/m2 was administered as a 4-hr IV infusion on days 1, 8, and 15 of a 28 day cycle. Nonclinical studies were conducted in mice to investigate the mechanism of romidepsin effects on platelets. Romidepsin was administered to female BALB/c mice at doses of 1 or 4 mg/kg by tail-vein injection on days 1, 5 and 9. Blood samples were collected every 2 days from alternating groups of mice to minimize effects of bleeding on platelet counts. Results In clinical studies, there is a mean decrease in platelet counts during the treatment period of each cycle, and a return to baseline levels or above between cycles observed in both clinical studies as described in the table below. No clinically meaningful change has been observed in the central tendency over 4 cycles of treatment in both studies. In the mouse studies, dose-dependent effects were seen on both WBC and platelet counts. Day 2 WBC counts dropped to 45% and 10% of normal at the 1 and 4 mg/kg doses, respectively. WBC counts remained low until after the dosing period in the 1 mg/kg romidepsin group, but recovered more quickly in the 4 mg/kg group. Day 2 platelet counts were 70% of normal at the 1 mg/kg dose and remained near this level until day 10, followed by recovery to normal at day 15. At the 4 mg/kg dose, profound thrombycytopenia was induced, with platelet counts only 20% of normal on days 4-6. Platelet counts slowly recovered to 70% of normal by day 15. Plasma thrombopoietin levels were normal throughout the experiment for the 1 mg/kg group, and showed a large increase to 275% of normal on day 6 in the 4 mg/kg group, which is the expected response to thrombocytopenia as a signal to increase platelet production and indicates that platelet reduction is not attributable to defective TPO production. Bone marrow megakaryocyte populations are being examined to determine the effects of romidepsin on these platelet-producing cells. Conclusions Following romidepsin administration, a saw tooth pattern is observed in the reduction and recovery of platelets. Recovery of platelets appears to occur more rapidly in humans than in mice; however, the effects are reversible after dosing in clinical studies and in murine models. In the clinical data the recovery pattern suggests that the transient effects are direct and are not effects on bone marrow. Disclosures: Whittaker: Gloucester Pharmaceuticals: Research Funding. Prince:Gloucester Pharmaceuticals: Consultancy. Demierre:Gloucester Pharmaceuticals: Consultancy, Honoraria. Lonial:Gloucester Pharmaceuticals: Honoraria. Kim:Gloucester Pharmaceuticals: Consultancy, Honoraria. Nichols:Gloucester Pharmaceuticals: Employment, Equity Ownership. Nix:Gloucester Pharmaceuticals: Employment.

Background: Responses to the novel HDAC inhibitor romidepsin were observed in patients (pts) with T-cell lymphoma in a phase 1 NCI trial. Aims: To evaluate the efficacy and tolerability of romidepsin in the treatment of advanced cutaneous T-cell lymphoma (CTCL). As an exploratory endpoint, to examine the molecular effects of romidepsin in both CTCL and peripheral T-cell lymphoma (PTCL). Methods: This Phase 2, open-label, multi-arm, multicenter study enrolled 71 CTCL pts from the NCI and 9 extramural sites. PTCL pts were also enrolled. Clinical results for pts with CTCL and biomarker analyses for both PTCL and CTCL are presented here; clinical results for the PTCL pts are presented elsewhere. This study is now closed to accrual. Pts with recurrent CTCL (Stages IA-IVB) received romidepsin at 14 mg/m2 as a 4-hr infusion on days 1, 8 and 15 q 28 days. Responses were assessed using a composite endpoint that evaluated overall changes in skin (modified Physicians Global Assessment), lymph nodes, extranodal visceral lesions and abnormal circulating T-cells. Molecular endpoints evaluated in NCI pts were: histone acetylation in peripheral blood mononuclear cells (PBMCs); MDR-1 gene expression in PBMCs and in biopsy samples; and blood fetal hemoglobin levels. Results: 71 pts (48 men and 23 women) with a mean age of 56 yrs (range 28–84) were enrolled and received romidepsin; 63 pts received ≥2 cycles of therapy. Mean number of prior therapies was 3.4 (range 1–10). Objective disease response rates (ORR) are presented in the table. Overall disease control (CR+PR+SD90) was 62% in all pts and 70% in pts who received ≥2 cycles. Target skin lesions were followed and changes were generally similar to overall skin changes. Median duration of response (DOR) was 11 months (mo) and the maximum DOR as of data cut-off was 5.5+ yrs. The most frequent drug-related AEs (all grades, all cycles) were generally mild, including nausea (82%; 4% ≥grade 3), fatigue (73%; 14% ≥ grade 3), electrocardiogram T-wave amplitude decreased (69%, 0% ≥grade 3); hemoglobin decreased (59%, 9% ≥grade 3), and platelet count decreased (59%; 13% ≥grade 3). Six pts died within 30 days of study drug administration: 2 after salvage chemotherapy, 1 with ARDS due to PD, 2 with infection, and 1 of unknown cause; 2 of these deaths were considered possibly related to treatment. An increase in all biomarkers measured was observed, although not in all pts. Correlation between global histone H3 acetylation (AcH3) at the 24-hr time point and Cmax, AUC, and clearance was observed (r = 0.37, 0.36, and −0.44, respectively, p = 0.03). Pts with major responses were more likely to have higher levels of AcH3 at 24 hr. Conclusions: This study demonstrates tolerability and durable clinical benefit (ORR of 35% and median DOR of 11 mo in all pts) of romidepsin in pts with recurrent CTCL. Significant responses were observed at all stages of disease, including an ORR of 32% in all pts with stage ≥IIB and 20% in all pts with stage IV. Time to response was rapid, within 2 mo in most pts, and responses were durable, >6 mo in most pts. Molecular analyses confirmed that romidepsin inhibits its target deacetylases in pts. Increases were not observed in all pts, perhaps reflecting, in part, variable drug exposure or variable response to HDAC inhibition. Correlation of global acetylation with PK parameters suggests that increased drug exposure results in increased global acetylation. All Pts N=71 Pts ≥ 2 cycles N=63 ORR (CR+PR), n (%) 25 (35%) 25 (40%) PR, n (%) 21 (30%) 21 (33%) CR, n (%) 4 (6%) 4 (6%) SD90, n(%) 19 (27%) 19 (30%) Overall disease control (CR+PR+ SD90) 44 (62%) 44 (70%) DOR 11 (1+ mo, 5.5+ yrs) 11 (1+ mo, 5.5+ yrs) OR for pts with stage ≥ IIB, n (%) 20/62 (32%) 20/55 (36%) SD90= stable disease for ≥90 days

Histone deacetylase inhibitors induce changes in gene expression that lead to cellular differentiation and reversal of the transformed phenotype. Interest in clinical development of these agents has been spurred by the responses observed in patients with T-cell lymphoma to romidepsin, previously FK228 and depsipeptide. To date, 61 patients with CTCL and 34 with PTCL have been accrued to our ongoing multiinstitutional trial of romidepsin for patients with recurrent or refractory cutaneous or peripheral T-cell lymphoma. Romidepsin is administered as a 4 hr infusion on days 1, 8, and 15 of a 28 d cycle with a starting dose of 14 mg/m2. Complete and partial responses have been observed in patients with CTCL, with a complete response in a patient with Sézary syndrome ongoing for over 45 months and a partial response ongoing for over 60 months. Both complete and partial responses were observed in patients with various subtypes of PTCL, including PTCL, unspecified, ALK-/CD30+ anaplastic large cell lymphoma, and enteropathy-associated T-cell lymphoma, with a complete response ongoing for over 34 months. Responses will be updated for presentation. Molecular endpoint analysis was performed in normal and malignant circulating peripheral mononuclear cells, PBMCs, and in tumor samples. Immunoblot analysis demonstrated increased histone acetylation in PBMCs of 2-fold or greater from 19 of 33 patients at 4 or 24 hrs. RT-PCR of RNA demonstrated a 2-fold or greater increased expression of MDR-1 in PBMCs from 27 of 45 patients, 11 of which were greater than 4-fold. Microarray performed on CTCL patient samples detected significant changes with treatment. In addition, changes in the 5 gene signature for CTCL previously published (Nebozhyn et al. Blood2006; 107:3189) were also detected when analyzed by QPCR. MDR1 expression was evaluated by RT-PCR in 30 patient’s tumors. MDR1 expression level was not significantly increased at the time of progression, mean 3.2 and 4.9 (s.d. 3.1 and 5.5, respectively) in MDR1 units as defined in Zhan et al. Blood 1997 89:3795. Since differentiating agents have been shown to induce expression of fetal hemoglobin in the laboratory, fetal hemoglobin levels were assayed in patients on this clinical trial. Increased circulating fetal hemoglobin of 2, 5, and 10 fold was observed in 34, 24, and 15 patients, respectively, from 44 patients evaluated. SNP analysis is being performed on the MDR1 gene for comparison with pharmacokinetics and induction of fetal hemoglobin and MDR1 itself. In conclusion, romidepsin has significant single agent activity in patients with CTCL or PTCL. Molecular effects can be assayed in patients treated with romidepsin and related agents. Further clinical development of these agents should include combination trials and the identification of molecular markers of response.

Histone deacetylase inhibitors, such as depsipeptide or FK228 (Gloucester Pharmaceuticals), form a new class of antineoplastic agents found to have activity in T-cell lymphoma. A multi-institutional phase II trial of depsipeptide is ongoing; accrual is complete for the first cohort, comprised of patients with cutaneous T-cell lymphoma who had disease that had progressed or was refractory to prior therapy and who had fewer than three prior regimens of systemic cytotoxic chemotherapy. Depsipeptide is administered as a 4 hr infusion on days 1, 8, and 15 of a 28 d cycle with a starting dose of 14 mg/m2. The first 3 patients enrolled were treated on the earlier NCI schedule, on days 1 and 5 of a 21 day cycle. Twenty-seven patients with a median age of 57 (31–77) were enrolled in this cohort and received a median of 5 (1–56) cycles and 14 (2–111) doses. Patients had received a median of 3 (1–11) prior therapies. These included PUVA in 15 patients, interferon in 8, bexarotene in 13, denileukin diftitox in 6, oral steroids in 6, topical nitrogen mustard in 7, or cytotoxic chemotherapy in 15. Disease extent at time of enrollment included 6 patients with tumor stage, 6 with generalized erythroderma and 3 with Sézary syndrome. Three patients, all with Sézary syndrome, achieved a complete response and five patients had partial responses for an overall response rate of 30%. With patients continuing to respond, the median duration of response is 18 (6–48) months. Partial responses were observed in patients with plaque/patch stage (1), generalized erythroderma (1), and tumor stage disease (3). An additional five patients had stable disease with a median duration to date of 6 months (4–14). Two patients had unconfirmed partial responses, three patients were not evaluable for response, one patient is too early to evaluate for response and seven patients had documented progression of disease. Overall depsipeptide was well tolerated, the main toxicities observed were nausea, taste changes, fatigue, neutropenia, thrombocytopenia. Non-specific ST-T wave changes were noted by ECG, without changes in cardiac function. No evidence of cardiac damage has been detected by serial echocardiograms, MUGA scans or troponin assays. Eight patients with objective responses receiving depsipeptide were followed for 12 to 53 months without evidence of cumulative toxicity. Induction of histone acetylation of more than two-fold was observed in paired samples of PBMNCs obtained from 9 of the 18 individual patients tested. RT-PCR analysis of MDR-1 demonstrated unchanged expression in tumor biopsies taken from 8 patients at the time of tumor progression when compared to prior to treatment initiation. While it is generally recognized that patients with Sézary syndrome are more refractory to available therapies, remarkably all patients enrolled with Sézary syndrome achieved a complete response to single agent depsipeptide. In conclusion, depsipeptide is the first of a new generation of histone deacetylase inhibitors to demonstrate consistent clinical activity with durable responses achieved in patients with CTCL. Patients are being accrued to a replicate arm for the purpose of confirming the observed response rate.