Phase II trial of the histone deacetylase inhibitor romidepsin for the treatment of refractory multiple myeloma

Abstract

Epigenetic dysregulation is a hallmark of cancer, including multiple myeloma. Inhibitors of histone deacetylases (HDAC) induce DNA hyperacetylation by inhibiting removal of acetyl groups from amino tails on histone proteins, thereby uncoiling condensed chromatin favoring transcription of silenced genes, including tumor suppressor genes. Romidepsin is an HDAC inhibitor which exhibits antiproliferative and apoptotic effects against multiple myeloma cell lines. We performed a phase 2 trial of romidepsin in patients with multiple myeloma who were refractory to standard therapy. Treatment consisted of romidepsin (13 mg/m2) given as a 4 hour IV infusion on days 1, 8, and 15 every 28 days. Thirteen patients received a median of 2 cycles of therapy (range 1–7 cycles). Although no patients had an objective response, 4 of 12 patients with secretory myeloma exhibited evidence of M-protein stabilization, and several other patients experienced improvement in bone pain and resolution of hypercalcemia. We conclude that romidepsin, as a single agent, is unlikely to be associated with response rate of 30% or higher in patients with refractory myeloma, although there was some clinical evidence suggesting a biological effect associated with therapy.

INTRODUCTION

Multiple Myeloma (MM) is a clonal plasma cell neoplasm in which malignant cells are arrested at various stages of cell differentiation.¹ Identification of novel oncogenes, chromosomal breakpoints, and immunoglobulin gene translocations has led to improved classification and understanding of the pathogenesis of the disease.² Agents which can modify or regulate the expression of both established and newly discovered oncogenes involved in the clinical course and development of MM may be essential in defining new targets for treatment.³

Recent evidence suggests that the epigenome, which regulates gene expression, may be a promising therapeutic target in MM. Indeed, epigenomic dysregulation of DNA methylation and histone acetylation is a hallmark of cancer, including MM.^4,5,6 Acetylation of histones allows the chromatin structure surrounding the protein to relax, thereby enabling gene transcription. In contrast, histone deacetylation results in tightly wound and compact chromatin which impedes transcription. Histone deacetylases inhibitors induce DNA hyperacetylation by inhibiting removal of acetyl groups from amino tails on histone proteins, thereby de-repressing silenced genes, including tumor suppressor genes. Histone deacetylase (HDAC) is a validated therapeutic target, as the HDAC inhibitors vorinostat and romidepsin are approved for the treatment of cutaneous T-cell lymphoma.⁷ In addition, other HDAC inhibitors, including panobinostat, have also reported positive effects in this malignancy.^8,9,10 The HDAC inhibitor vorinostat has been shown to induce differentiation and apoptosis of human MM cells¹¹, possibility via modulation of multiple targets including the insulin-like growth factor /IGF-1 receptor and IL-6 receptor, antiapoptotic molecules, oncogenic kinases, DNA synthesis/repair enzymes, and transcription factors.¹² HDAC inhibitors such as vorinostat also enhance the effectiveness of standard MM therapies, including bortezomib, dexamethasone, cytotoxic chemotherapy, and thalidomide analogs.¹¹

Romidepsin (formerly known as despipeptide, FR901228, or FK228) is a cyclic peptide HDAC inhibitor which has been shown in vitro to induce apoptosis by downregulation of the BCL-2 family of proteins (BL-XL and MCL-1), and induce G1 cell cycle arrest (by enhancing expression of p21 and p53).¹³ Phase I-II trials have shown that romidepsin is effective for the treatment of cutaneous and peripheral T cell lymphomas.^9,10,14,15 The dosage of romidepsin in its phase I trial was 1-24.9 mg/m²; dose-limiting toxicities included fatigue, nausea, vomiting, thrombocytopenia, and cardiac arrhythmia.¹⁵ At the recommended phase II dose of 17.8mg/m², romidepsin increased histone acetylation in patient-derived peripheral blood mononuclear cells, and also altered cell cycle kinetics of PC3 cells in culture.¹⁵ Although the recommended phase II dose was initially 17.8 mg/m² given over 4 hour infusion on days on days 1 and 5 of a 21-day cycle, a subsequent amendment to the phase II protocol recommended dose reduction to 13–14 mg/m² on days 1, 8, and 15 every 28 days due to better patient tolerability.¹⁶ Based on the aforementioned data, we designed a phase 2 to evaluate the efficacy, safety, and biologic effects of romidepsin monotherapy in patients with refractory or relapsed MM in order to provide a framework for integration romidepsin with other standard therapies.

METHODS

Patient Eligibility

Eligibility criteria included patients with relapsed and/or refractory stage IIa-IIIa MM who had received at least two prior lines of therapy. Other criteria included Karnofsky Performance Scale status of at least 70%, age at least 18 years, and adequate bone marrow (leukocyte count at least 3000/uL, neutrophils at least 1500/uL, platelets at least 100,000/uL), hepatic (total bilirubin less than 2.0 mg/dL, SGOT/SGPT less than or equal to 2.5-fold the upper limits of normal), renal (serum creatinine less than or 1.5 mg/dL or creatinine clearance at least 60ml/min/1.73 m2), and cardiac function (left ventricular ejection fraction at least 50% and normal electrocardiogram). Prior chemotherapy or radiotherapy was permitted if administered more than 4 weeks prior to enrollment (6 weeks for nitrosoureas or mitomycin C). Patients with a history of prior treatment with a histone deacetylase inhibitor or an uncontrolled intercurrent illness (including cardiac arrhythmias) were excluded.

Treatment Regimen

Patients received romidepsin at a dose of 13 mg/m² as a 4-hour infusion on days 1, 8, and 15 of a 28 day cycle. All patients were treated for at least 4 weeks, or equivalently, for at least one full cycle of treatment. After the first cycle, patients with progressive disease were discontinued from the study and all others proceeded to the next 4-week cycle. A maintenance regimen of romidepsin every other week (days 1 and 15) was permitted for those reaching a stable plateau (±25% serum M-protein levels or urine protein excretion over 3 consecutive determinations, each at least 4 weeks apart). Treatment continued until the occurrence of disease progression, an adverse event requiring discontinuation, or withdrawal of patient consent. All patients received 8 mg ondansetron for antiemetic prophylaxis at one hour before and every 8 hours thereafter for 24 to 48 hours after romidepsin infusion. Additional antiemetic treatment was administered based on patient symptoms. Thrombocytopenia was treated conservatively: in the absence of bleeding, platelet transfusions were given only if the platelet count was below 10,000. If the patient developed bleeding, platelet transfusions were administered in accordance with standard practice, to maintain a platelet count of ≥ 50,000/mm³. Potassium and magnesium were administered prior to romidepsin administration for patients who were either below normal or in the low-normal range of serum levels of these electrolytes.

Baseline and Follow-Up Evaluation

Evaluation before beginning protocol therapy included multiple gated acquisition scanning/radionuclear cardiac angiography, skeletal survey, chest x-ray, complete serum and urinary protein electrophoresis and immunofixation, quantitation of serum immunoglobulin and free light chains levels, and 24-hour urinary protein excretion. Laboratory values obtained at screening, weekly during study participation, and at follow-up included complete blood count/differential, full serum chemistries, troponin I levels (or CK isoenzymes, if available), and electrocardiography. Response to treatment was determined by standard criteria for complete response, near-complete response, partial response, stable disease, and progressive disease of MM.¹⁷ All patients who completed at least 1 cycle were assessed for response to treatment. Measurable disease was defined as ≥1.0g/dL serum monoclonal protein, ≥0.1g/dL serum free light chains, ≥0.2 g/24 hours urinary M-protein excretion, and/or measurable plasmacytomas. Criteria for primary response were adopted from the EBMT/ IBMTR/ABMTR¹⁸ and the uniform response criteria for MM (IURC).¹⁷

The National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE), version 3.0 was used to grade adverse events. Toxicity was defined as an adverse event possibly, probably, or definitely related to the study treatment. Data from all subjects who received any study drug is included in the safety analysis. The frequency of subjects experiencing a specific adverse event was tabulated.

Correlative studies

Bone-marrow aspirates (5–10 cc) and biopsy specimens were collected from 3 consenting patients at baseline, 24 hours after the first treatment, and 28 days after the first treatment for correlative studies. The samples underwent ficoll separation, with CD138+ plasma cells selected by radiolabeled microbeads. Phenotype analysis was performed by immunohistochemistry for CD 138/Ki-67 proliferation index, FGFR3, CD31 (PECAM), and cleaved caspase 3.

Statistical objectives and statistical plan

The primary objectives of the study were to evaluate the safety and efficacy of romidepsin as a treatment for patients with advanced refractory or relapsed MM. The study was designed to distinguish between a response rate of 10% or less versus 25% or higher. Simon’s optimal 2-stage design was used in which the probability of a type I error and the probability of a type II error were both set at 10%. If 3 or fewer responses occurred among the initial phase, accrual would be terminated. If there were at least 8 or more total responses reported among the patients, the study would be considered promising. The trial was halted after accrual of 13 patients when no objective responses were noted. A post-hoc analysis indicated that it was unlikely that the response rate would exceed 30% (type I and II error rates of 10%).

Informed Consent and Regulatory Approval

The study was reviewed and approved by the Cancer Evaluation Therapy Program of the National Cancer Institute (P65996, and by the institutional review board at each participating institution (Clinical Trials.gov identifier NCT00066638). All patients provided written informed consent.

RESULTS

Patient characteristics

Thirteen patients were enrolled at 3 participating institutions between December 2003 and May 2006. The characteristics of the patient population are shown in Table 1. All patients had evidence of disease progression prior to enrollment. The median age was 57 years (range 54–74), median disease duration was 6 years (range 2.3–11.3 years), and median number of prior treatment regimens was 3 (range 2–4), including 9 patients (69%) who had had a prior stem cell transplant, 9 patients (69%) who had received thalidomide (N=8) or lenalinomide (N=1), and 6 patients (46%) who had prior bortezomib. Pretreatment fluorescence in situ hybridization (FISH) in 10 patients identified two patients with del 13q14 including one with t(11;14), 1 with tetrasomy 11, one with trisomy 11, and six with no abnormalities. Conventional cytogenetics identified one patient with inversion in chromosome 9.

Table 1.

Patient Characteristics

Median age (range)	57 years (45–73 years)
Male/female	7/6
Median number of years since   Initial diagnosis (range)	6 years (2.3–11.3 years)
Median number prior therapies (range)	3 (2–4)
Prior therapy
Stem cell transplant	9 (69%)
Thalidomide/lenalinomide	9 (69%)
Bortezomib	6 (46%)
M-protein
IgG kappa	6 (46%)
IgG lambda	5 (38%)
Light chain lambda	1 (8%)
Non-secretory	1 (8%)

Treatment Administration and Efficacy

Thirteen patients received an aggregate total of 27 treatment cycles; the median number of cycles given was 2 (range 1–7 cycles). Reasons for discontinuation of therapy included disease progression in 6 patients (46%), an intercurrent bone fracture requiring surgery in one patient (8%), and patient withdrawal in 6 patients (46%) due to lack of response. Twelve of 13 patients were assessable for response by serum protein electrophoresis (SPEP), urine protein electrophoresis (UPEP) or free light chain measurement, which were obtained both at baseline as well as after each cycle of treatment; one patient had non-secretory disease. No patients met the criteria for objective response. Four patients (31%, 95% confidence intervals 9%, 61%) exhibited stabilization of M-protein levels during therapy (Figure 1). There was no consistency in therapy preceding romidepsin indicative of a “priming” effect; the preceding treatment included bortezomib (patient 1), thalidomide plus clarithromycin (patient 2), melphalan (patient 3), and LymphoRad-131 (patient 11). Five patients showed clinical benefit with improvement of hypercalcemia, and two patients showed improvement of pain. Nevertheless, the trial was halted after accrual of 13 patients because it was statistically unlikely that the objective response rate would exceed 30%.

Figure 1.

M-protein levels before and during romidepsin therapy in four patients who exhibited pararprotein stabilization during therapy

Correlative Studies

In 3 patients who exhibited M-protein stabilization, we evaluated bone-marrow samples before therapy, 24 hours after the first romidepsin treatment, and 28 days after the first treatment, at which point patients would have been exposed to a total of 3 romidepsin infusions. Cell cycle marker evaluation in CD138+ selected plasma cells revealed no detectable alteration in cell cycle kinetics in vivo (Figure 2). In addition, evaluation of BCL-2, MCL-1, CD31, and cleaved caspase 3 revealed no detectable modulation in vivo (Figure 3).

Figure 2.

Cell cycle markers including Ki-67/CD-138, CyD1+, CyD3+, p18+, p27+, and p21+ cells before, 24 hours after, and 4 weeks after first romidepsin treatment in 3 patients (all of whom had stabilization of M-protein levels during therapy).

Figure 3.

Percent change in proportion of cells expressing BCL-2, MCL-1, CD31, and cleaved caspase 3 before, 24 hours after, and 4 weeks after first romidepsin treatment in 3 patients (all of whom had stabilization of M-protein levels during therapy).

Adverse Events

There were no grade 4 adverse events. Grade 3 thrombocytopenia occurred in 3 patients (23%). The most common adverse events included grade 1–2 nausea in 7 patients (54%), grade 2 fatigue in 4 patients (31%), and grade 2 taste alteration in 1 patient (8%). Of the 27 cycles administered, 22 were given at full dose, and 5 doses were given at a reduced dose in 5 patients. Electrocardiographic changes were common but clinically insignificant, including asymptomatic and reversible QT interval prolongation, ST-segment depression, and T wave inversion. All of these cardiac conductions abnormalities have been previously described with romidepsin therapy.²¹

DISCUSSION

We evaluated the HDAC inhibitor romidepsin in heavily pretreated patients with MM who were refractory to multiple therapies, often including stem cell transplantation, thalidomide, and bortezomib. Although no patients achieved an objective response, approximately 30% of patients exhibited stabilization of M-protein production indicating a biologic effect. Several patients also exhibited resolution of hypercalcemia or improvement in bone pain, also potentially indicative of a biologic effect. Romidepsin did not appear to modulate apoptosis or cell cycle kinetics in vivo, even in those patients who exhibited serum M-protein stabilization. Even with this demonstrated clinical activity, it still appears that romidepsin monotherapy is unlikely to produce an a significant objective response (in terms of M-protein reduction) in patients with refractory myeloma.

Numerous reports indicate that several HDAC inhibitors have significant antineoplastic effects in myeloma cell lines, including vorinostat¹¹, romidepsin¹³, LAQ824²², and R306465.²³ Our experience, however, represents only the third report evaluating the clinical activity of an HDAC inhibitor as single agent in patients with MM. Richardson et al reported a phase I trial of vorinostat at various doses and schedules in 13 patients with refractory myeloma; one patient exhibited a minor response and 9 patients were reported to have stable disease.²⁴ Gimsing reported disease stabilization in one patient with myeloma treated with the HDAC inhibitor belinostat (PXD101).²⁵ The results of our trial are consistent with these reports, indicating that HDAC inhibitors do not induce tumor regression, but do seem to induce some biological effects that may have clinical relevance.

Romidepsin and other HDAC inhibitors have been shown to enhance the effect of bortezomib in myeloma cell lines^19,26–28, and in some cases also inhibit osteoclasts. Recently, we reported the results of a phase I trial in relapsed and refractory MM, in which vorinostat administered at a dose of 400 mg daily for 8 days in conjunction with bortezomib at 1.3 mg/m2, proved to be safe and effective, with responses in patients who were previously proven bortezomib-unresponsive.²⁹ Similar encouraging results have been reported on a phase 1–2 trial of romidepsin in combination with dexamethasone and bortezomib.³⁰

Based on these results, several trials are currently in progress evaluating the combination of romidepsin and bortezomib in MM, and vorinostat in combination with lenalidomide (NCT00642954, NCT00729118) or pegylated liposomal deoxorubicin plus bortezomib (NCT00744353). Moreover, an ongoing phase III trial is currently evaluating bortezomib alone or in combination with vorinostat (NCT0077347). This and other trials serve to define the future role of HDAC inhibitors in MM.