ALBERT

Description: 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.

Description: 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.

Description: Disease-free survival (DFS) is short in patients ≥ age 60 or with secondary AML, adverse cytogenetics, or leukostasis at presentation. In such patients, median CR duration is ≤ 8 mos and only 20% achieve 1 yr DFS. Historically, maintenance therapy with low doses of cytotoxic chemotherapy has not prolonged DFS. We tested the hypothesis that Tipifarnib (T) might be active as maintenance therapy for adults with poor-risk AML in first CR after induction and consolidation therapies. Oral T 400 mg bid for 2/3 wks was begun median 2.4 mos (range 1.0–4.1) after start of final consolidation cycle and given for up to 48 wks (16 cycles) to 36 adults with median age 63 (range 27–82), secondary AML 31%, adverse cytogenetics 47%, leukostasis 17%, ≥ 2 risk factors 40%. T was well-tolerated, with only 4 of 36 unable to complete 2 cycles because of constitutional symptoms (1 rash, 3 non-compliant). To date, 256 cycles have been administered (median 8 per pt, range 1–16), with hospitalization required during only 4 (1.5%) cycles (infection 3, bowel obstruction 1). Dose reductions for myelosuppression (400 mg bid to 300 mg bid) occurred in 17/32 (53%) by cycle 3, and 2 (6%) needed platelet transfusions. A total of 9 patients completed all planned 16 cycles of T with a median CR duration of 24 mos (range 15–36+). Five of the 9 remain in continuous CR (CCR) 19+-36+ mos, median 26+), compared with 4 who relapsed after CR of 15–24 mos (median 22). There are 8 additional pts in CCR and still receiving T (2–12 cycles) with CCR 4+-12+ mos. A total of 15 patients progressed while on T at median 6.5 mos CR (range 3.5–12). Median CR duration for all patients is 10+ mos (range 3.5–36+), with 88% ≥ 6 mos and 48% ≥ 12 mos. In 13 “comparable” poor-risk pts (age ≥ 60 46%, secondary AML 25%, adverse cytogenetics 46%, leukostasis 23%) who were eligible for but declined T, 4 are in unmaintained CCR at 14+-25+ mos, 9 have relapsed at median 7.5 mos (5–13 mos). Treatment with T did not have a negative impact on reinduction chemotherapy at relapse, as 6 of 9 patients achieved second CR. Administration of T in CR after induction and consolidation therapy has low toxicity and is associated with prolonged DFS in some adults with poor-risk AML. Phase 3 studies are warranted in this patient population.

Description: Farnesyltransferase inhibitors (FTI) make up a novel class of anti-cancer agents that competitively and selectively inhibit farnesyl protein transferase. Early trials of the orally bioavailable non-peptidomimetic FTI tipifarnib (ZARNESTRATM, Johnson & Johnson PRD) demonstrated both clinical responses and excellent tolerability in patients with poor-risk or refractory acute myeloid leukemia (AML). (Karp, et al. Blood97:3361, 2001). We herein report updated results of a multicenter phase 2 trial of tipifarnib in an elderly, previously untreated poor-risk AML population who refused or were deemed unfit for conventional induction chemotherapy. Tipifarnib was administered orally in the outpatient setting at a dose of 600 mg BID for 21 days, followed by a 1–3 week recovery period. Up to 4 cycles of tipifarnib were permitted in patients with complete responses (CR). The primary endpoint was overall response rate (CR + PR). Secondary endpoints included toxicity rates, measurement of markers of farnesylation (HDJ-2) in bone marrow cells, measurement of signaling intermediates ERK and AKT, and RNA microarray expression patterns. Accrual to the trial is complete. 170 patients have been enrolled, 148 of whom are evaluable for response (AML=160; high-risk MDS=4; high-risk CMML=6). The median age was 73 years (range 34–85), and 76 patients (45%) were age = 75. M/F ratio was 2:1. An unfavorable karyotype and/or antecedent MDS was present in 47% and 79% of patients, respectively. The median number of cycles received was 1, and the median number of days of drug received was 36 days. Dose reductions were implemented in 38% of patients, more commonly in cycles subsequent to cycle #1. The overall response rate (CR + PR) was 34%. CR occurred in 18% of patients. Responses were evenly distributed across study centers. In patients ≥ 75 years, the overall response rate was 30% (CR 20 %). Median CR duration was 6.4 months (range 1.5–11+ months). Median overall survival was 5.6 months for all patients. CR patients had a median survival of 14.4 months, with 63% alive at 12 months. In non-responders, median survival was 3.1 months. The incidence of grade = 3 tipifarnib-related non-hematologic adverse events was 43%, comprised mainly of infectious and gastrointestinal complications. The hospitalization rate for tipifarnib-related toxicity was 18% (median duration: 12 days). The death rate from tipifarnib-related toxicity at 6 weeks was 5%. Microarray analysis of pre-and post-treatment bone marrow samples is being performed to identify both predictive and pharmacodynamic gene markers of response to tipifarnib. In summary, tipifarnib is a novel outpatient treatment with activity in previously untreated poor-risk AML. The low hospitalization rate may reflect the low incidence of severe non-hematological toxicity.

Description: Tipifarnib (T) exhibits modest activity in elderly adults with newly diagnosed acute myelogenous leukemia (AML). Based on preclinical synergy, a phase 1 trial of T plus etoposide (E) yielded 25% complete remission (CR). We selected 2 comparable dose levels for a randomized phase 2 trial in 84 adults (age range, 70-90 years; median, 76 years) who were not candidates for conventional chemotherapy. Arm A (T 600 mg twice a day × 14 days, E 100 mg days 1-3 and 8-10) and arm B (T 400 mg twice a day × 14 days, E 200 mg days 1-3 and 8-10) yielded similar CR, but arm B had greater toxicity. Total CR was 25%, day 30 death rate 7%. A 2-gene signature of high RASGRP1 and low aprataxin (APTX) expression previously predicted for T response. Assays using blasts from a subset of 40 patients treated with T plus E on this study showed that AMLs with a RASGRP1/APTX ratio of more than 5.2 had a 78% CR rate and negative predictive value 87%. This ratio did not correlate with outcome in 41 patients treated with conventional chemotherapies. The next T-based clinical trials will test the ability of the 2-gene signature to enrich for T responders prospectively. This study is registered at www.clinicaltrials.gov as #NCT00602771.

Description: Preclinical studies suggest that neoplastic cells may be particularly sensitive to simultaneous interruption of cell-cycle and survival signaling pathways. In accord with this concept, we have shown that flavopiridol (F), a CDK inhibitor, interacts with bortezomib (B), a proteasome inhibitor, to induce mitochondrial injury and apoptosis in human leukemia, myeloma, and lymphoma cells (Dai et al, Oncogene22:7108, 2003; Dai et al, Blood104:509, 2004). These actions were associated with inhibition of NF-kappaB DNA binding, increased expression of phospho-JNK, and downregulation of XIAP and Mcl-1. Based on these findings, a phase I trial has been initiated to identify appropriate doses of B+F for further investigation. Eligible patients (pts) include those with multiple myeloma or indolent B-cell neoplasms, and recurrent or refractory disease following at least 1 prior systemic therapy (excluding allogeneic stem cell transplantation). In the initial stage of the trial, pts received B (iv push) immediately followed by F bolus (1- hour infusion) on d1, 4, 8, and 11 out of a 21-day (d) cycle. Dose levels were, in mg/m2 (B/F): 1.0/15, 1.3/15, 1.3/22, 1.3/30, 1.3/40, 1.3/50, and 1.3/60. Subsequently, a “hybrid” F infusion schedule (30 minute load followed by a 4-hour infusion) was adopted based on evidence of activity of this schedule in chronic lymphocytic leukemia. With the hybrid schedule, all pts receive B (iv push) 1.3 mg/m2 on d1, 4, 8 and 11. Targeted F dose levels using the hybrid schedule are (Fload/Finfusion; mg/m2): 20/20 on d1 and 8; 30/30 on d1 and 8; 30/50 on d1 and 8; 30/30 on d1, 4, 8 and 11; 30/50 on d1, 4, 8 and 11. Dose limiting toxicity (DLT) is defined as NCI CTCAE grade 4 ANC/platelets for 〉 1 week or grade ≥ 3 non-heme toxicity. 38 pts have been enrolled. 29 pts were treated at 7 dose levels with the bolus schedule, after which development of the hybrid schedule was begun. With the hybrid schedule, 11 pts have been treated at 3 dose levels. To date, one DLT (grade 3 lower back pain) was observed at level 5 of the bolus schedule and one DLT (grade 3 fatigue) was seen at the 1st hybrid dose level. The MTD of the hybrid schedule has not been reached. Non-DLT toxicities include herpes zoster (2 disseminated), peripheral neuropathy, fatigue, postural hypotension, syncope, diarrhea and ≤ grade 3 cytopenias. Of 35 pts evaluable for response, there have been 2 complete responses (1 lymphoma and 1 mantle cell lymphoma), 7 partial responses (5 myeloma and 2 lymphoma), 3 minor responses (2 myeloma and 1 extramedullary plasmacytoma), 15 patients with stable disease (5 myeloma, 7 lymphoma, 1 Waldenstrom’s and 2 mantle cell lymphomas). Of the 3 pts who had received prior bortezomib, 2 had minor responses and 1 had disease progression. To date, hyperacute tumor lysis has not occurred with the hybrid schedule, but aggressive prophylaxis and monitoring are integral to the treatment plan. Correlative laboratory studies involving bone marrow CD138+ cells from patients with myeloma revealed a reduction in post-treatment NF-kappaB nuclear localization in 4 of 5 evaluable patients. Variable effects on myeloma cell expression of phospho-JNK, Mcl-1, and XIAP have been observed. Collectively, these findings indicate that a regimen combining bortezomib and flavopiridol, including the use of a hybrid flavopiridol schedule, is well tolerated in patients with progressive B-cell malignancies, and has clear activity in some patients refractory to standard therapy. Pending identification of the MTD and RPTD (recommended phase II dose), phase II evaluation of this therapeutic strategy should define its activity more definitively.

Description: Currently there is no method available to predict response to farnesyltransferase inhibitors (FTI). We analyzed gene expression profiles from the bone marrow of patients from a phase 2 study of the FTI tipifarnib (R115777, ZARNESTRA), in older adults with previously untreated acute myeloid leukemia (AML). The RASGRP1:APTX gene expression ratio was found to predict response to tipifarnib with the greatest accuracy. This two-gene ratio was validated by quantitative PCR in the newly diagnosed AML cohort. We further demonstrated that this classifier could predict response to tipifarnib in an independent set of 54 samples from relapsed or refractory AML, with a negative predictive value and positive predictive value of 92% and 28%, respectively (odds ratio of 4.4). The classifier also predicted for improved overall survival (154 vs 56 days, p = 0.0001), which was shown to be independent of other prognostic factors including a previously described gene expression classifier predictive of prognosis. Therefore, these data indicate that a two-gene expression assay may have utility in categorizing a population of AML patients who are more likely to respond to tipifarnib.

Description: Combination therapy utilizing 2 novel agents with independent mechanisms of action and non-overlapping toxicities may be useful in the setting of advanced cancers. Tipifarnib (T) is an orally bioavailable farnesyltranferase inhibitor with documented single-agent activity in acute myeloid leukemia (AML). Bortezomib (B) is a broad inhibitor of proteasomal function, approved for treatment in multiple myeloma and mantle cell lymphoma. Preclinical studies indicated synergistic activity between these 2 agents for eliciting apoptosis within leukemia and myeloma cell lines and ex-vivo cells adhered to fibronectin. In this phase I combination trial, we studied the effect of combined effect of T plus B in patients with advanced acute leukemias. Objectives: The primary endpoint was toxicity assessment. Secondary endpoints included effect of combined therapy on signaling intermediates, including p-AKT, Bim, Bax, and NF-kB, as well as effects on farnesyltransferase (FT) and the proteasome activity. Eligibility: Patients with AML, ALL, or CML-BC who had received 〈 3 cycles of prior therapy were eligible. Methods: Patients received T on days 1–14 and B on days 1, 4, 8, and 11. Cycles were repeated every 21 days. Dose escalation occurred using cohorts of 3–6 patients. The starting dose was T: 300 mg/m2 and B: 1.0 mg/m2 Bone marrow aspirate was obtained at baseline, day 8, and between day 15 and the start of the next cycle. Measurement of signaling intermediates were performed in Ficoll-enriched leukemic marrow blasts using Western Blot (p-AKT, Bax, Bim) and ELISA (NF-kB). FT and proteasomal activity were directly measured within peripheral blood mononuclear cells (PBMC) using previously described methods. Results: To date, 27 patients have been enrolled at 3 centers. Four patients were ineligible after screening, and 23 patients have been treated. Median age was 69 years (range 48–84) Diagnosis: AML=25, ALL=1, MDS=1. Accrual to the 4th and final dosing cohort has occurred, without maximum tolerated dose being reached at the 4th and final planned dosing cohort (T: 600 mg/m2 and B: 1.3 mg/m2). Six patients received ≥ 2 cycles of treatment. Dose-limiting toxicities to date have included: nausea/diarrhea (1 patient), sensory neuropathy (1 patient), and fatigue (1 patient). Common drug-related (〉 10%) non dose-limiting toxicities included: infection/febrile neutropenia, diarrhea, nausea, vomiting, sensory neuropathy, and fatigue, most of which were grade 1 or 2. FTase inhibition within peripheral blood mononuclear cells (PBMC) was measured serially in 8 patients to date, with a median of 70% inhibition by day 8, and with 5 out of 6 evaluable patients having sustained inhibition at day 22. Proteasome function within PBMCs was reduced by a median of 44.3% in 7 patient samples pre-infusion and 1 hour post-infusion on day 8. Proteasome activity within PBMCs at day 22 was decreased from baseline in 5 out of 7 patient samples tested. Compared to baseline, NF-kB binding activity within leukemic blasts at day 8 was decreased by a median of 39% at in 10 out of 14 paired samples. No significant change in expression of p-AKT, Bax, or Bim, as measured by Western Blot, was detected at day 8. Two patients achieved clinical response; 1 patient had a complete response and another patient had complete response with incomplete count recovery. Four others had stable disease following cycle 1. Conclusion: combined therapy with T + B was well tolerated and demonstrated inhibition of several relevant target signals within leukemic blasts and PBMCs. In addition, clinical activity was seen in 2 patients to date. Accrual to the trial is ongoing and updated clinical and pharmacodynamic data will be presented.

Description: The farnesyltransferase inhibitor tipifarnib exhibits modest activity against acute myelogenous leukemia. To build on these results, we examined the effect of combining tipifarnib with other agents. Tipifarnib inhibited signaling downstream of the farnesylated small G protein Rheb and synergistically enhanced etoposide-induced antiproliferative effects in lymphohematopoietic cell lines and acute myelogenous leukemia isolates. We subsequently conducted a phase 1 trial of tipifarnib plus etoposide in adults over 70 years of age who were not candidates for conventional therapy. A total of 84 patients (median age, 77 years) received 224 cycles of oral tipifarnib (300-600 mg twice daily for 14 or 21 days) plus oral etoposide (100-200 mg daily on days 1-3 and 8-10). Dose-limiting toxicities occurred with 21-day tipifarnib. Complete remissions were achieved in 16 of 54 (30%) receiving 14-day tipifarnib versus 5 of 30 (17%) receiving 21-day tipifarnib. Complete remissions occurred in 50% of two 14-day tipifarnib cohorts: 3A (tipifarnib 600, etoposide 100) and 8A (tipifarnib 400, etoposide 200). In vivo, tipifarnib plus etoposide decreased ribosomal S6 protein phosphorylation and increased histone H2AX phosphorylation and apoptosis. Tipifarnib plus etoposide is a promising orally bioavailable regimen that warrants further evaluation in elderly adults who are not candidates for conventional induction chemotherapy. These clinical studies are registered at www.clinicaltrials.gov as #NCT00112853.

Description: Aims: MSCs are cells being investigated for use in various therapies including facilitation of HSC transplantation and as gene therapy delivery vehicles. We have explored the potential to increase the number of bone marrow (BM) MSCs in vivo, induce mobilization using various cytokine regimens and improve gene transfer into these cells with adeno-associated virus (AAV) in a baboon model. Method: Baboons received cytokines as follows: 1. G-CSF 100mcg/kg/day for 5 days; 2. pegylated G-CSF (pegG-CSF), single dose 300mcg/kg day −5; 3. G-CSF 100mcg/kg/day + stem cell factor (SCF) 50mcg/kg/day for 5 days; and 4. pegylated megakaryocyte growth and development factor (pegMGDF) 1mcg/kg second daily for 10 days + G-CSF 100mcg/kg/day for 5 days starting day −5. Animals underwent BM aspiration at baseline and on the final day of cytokines along with leukapheresis to isolate PBMNCs for detection of peripheral blood (PB) CFU-F. The immunophenotype and differentiation potential of CFU-F derived from animals before and after cytokines was compared. The ability of AAV vectors pseudotyped with capsids derived from AAV of serotypes 1, 2, 3, 4, 5, 6, and 8 to mediate transduction of baboon and human MSCs was assessed. Results: Augmentation of bone marrow MSCs was observed with all cytokine regimens with the fold-increase compared to baseline as follows: 4.1, 2.1, 7.6 and 11.2 after G-CSF, pegG-CSF, G-CSF+SCF and G-CSF+pegMGDF respectively (see Figure 1). The immunophenotype of MSCs obtained after cytokines was identical to baseline cells as was their differentation potential. CFU-F were not detected in baseline PB however they were detected in 3/5 animals after G-CSF+SCF at a frequency of 0.8 to 1.5/mL, but no other cytokine regimen. A similar pattern of transduction efficiency using AAV was shared by human and baboon MSCs (see Figure 2) using control Ad293 cells. Specifically AAV vectors expressing capsids of serotypes 2, 3 and 5 were most efficient in transducing human and baboon MSCs. Those expressing capsids from serotypes 1, 4, 6, and 8 were much less efficient in transducing MSCs from either species. Baboon MSCs were able to be transduced by about 100-fold more than their human equivalent cells using AAV serotypes 2, 3, and 5. Conclusion: This is the first report of mobilization of primate MSCs and, together with the demonstration of in vivo augmentation and AAV gene transfer, offers increased therapeutic opportunities for their safe application in a burgeoning number of diseases. Figure 1: In vivo Bone Marrow CFU-F Augmentation following cytokines Figure 1:. In vivo Bone Marrow CFU-F Augmentation following cytokines Figure 2: Transduction profile of AAV vectors expressing capsids of various serotypes Figure 2:. Transduction profile of AAV vectors expressing capsids of various serotypes