Leukemia & Lymphoma
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Obatoclax in combination with fludarabine and rituximab is well-tolerated and shows promising clinical activity in relapsed chronic lymphocytic leukemia Jennifer R. Brown, Bethany Tesar, Lijian Yu, Lillian Werner, Naoko Takebe, Evgeny Mikler, Hazel M. Reynolds, Christina Thompson, David C. Fisher, Donna Neuberg & A. S. Freedman To cite this article: Jennifer R. Brown, Bethany Tesar, Lijian Yu, Lillian Werner, Naoko Takebe, Evgeny Mikler, Hazel M. Reynolds, Christina Thompson, David C. Fisher, Donna Neuberg & A. S. Freedman (2015): Obatoclax in combination with fludarabine and rituximab is well-tolerated and shows promising clinical activity in relapsed chronic lymphocytic leukemia, Leukemia & Lymphoma To link to this article: http://dx.doi.org/10.3109/10428194.2015.1048441
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Date: 21 October 2015, At: 17:30
Leukemia & Lymphoma, 2015; Early Online: 1–7 © 2015 Informa UK, Ltd. ISSN: 1042-8194 print / 1029-2403 online DOI: 10.3109/10428194.2015.1048441
ORIGINAL ARTICLE: CLINICAL
Obatoclax in combination with fludarabine and rituximab is well-tolerated and shows promising clinical activity in relapsed chronic lymphocytic leukemia Jennifer R. Brown1,3, Bethany Tesar1,3, Lijian Yu1,3, Lillian Werner2, Naoko Takebe4, Evgeny Mikler1, Hazel M. Reynolds1, Christina Thompson1, David C. Fisher1,3, Donna Neuberg2 & A. S. Freedman1,3 1Department of Medical Oncology, 2Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute,
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Boston, MA, USA, 3Department of Medicine, Harvard Medical School, Boston, MA, USA, and 4Investigational Drug Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA
Abstract Obatoclax is a small molecule mimetic of the BH3 domain of BCL2 family proteins. This phase 1 study combining obatoclax with FR was undertaken in chronic lymphocytic leukemia (CLL) patients relapsed after at least one prior therapy. Obatoclax was given as a 3-h infusion on days 1 and 3 and escalated through three dose levels, with standard dose FR days 1–5. Thirteen patients were enrolled, with a median of two prior therapies. One doselimiting toxicity (DLT) of a 2-week treatment delay for persistent grade 2–3 neutropenia was observed at the highest obatoclax dose (20 mg/m2), but no maximum tolerated dose (MTD) was reached. The overall response rate (ORR) was 85%, with 15% complete responses (CRs) by NCI-96 criteria and 54% by IWCLL 2008 criteria. Median time to progression was 20 months. It is concluded that obatoclax can be safely administered to relapsed CLL patients in combination with FR and shows promising clinical activity.
the most interesting pathways involves BCL-2, which is overexpressed nearly universally in CLL and confers resistance to apoptosis [6]. Inhibition of BCL-2 family members, including MCL-1, has been shown to synergistically induce apoptosis in combination with many cytotoxic and non-cytotoxic therapies in CLL [7–10]. Obatoclax is a small molecule mimetic of the BH3 domain of BCL-2 family proteins and has broad specificity, with activity against BCL-2, BCL-XL, BCL-w and MCL-1 [11]. Its activity against MCL-1 is particularly unique among BCL-2 family inhibitors and may be important for CLL, which frequently over-expresses MCL-1 [11]. Multiple studies have found that obatoclax can induce apoptosis in CLL cells in vitro [7–9,12], in some cases by a BH3 mimetic mechanism. A BH3 mimetic mechanism implies binding of obatoclax to anti-apoptotic BCL-2 family members, leading to release of bound BAX, BAK or activator BH3-only proteins, which then induce apoptosis. However, cytotoxicity induced by obatoclax has also been observed in systems without BAX or BAK expression [9,13,14], suggesting that, at least in some cases, obatoclax is acting by a non-BH3 mimetic mechanism. Obatoclax has in fact been found to induce cellular cytotoxicity by alternative mechanisms, including induction of cell cycle arrest [14,15], reduction of colony formation [14,16] and induction of necroptosis and autophagy in acute lymphoblastic leukemia cell lines [17]. Further work will be required to fully define the mechanism of action of obatoclax in different disease settings. However, its cytotoxicity is clear in vitro, and it has been shown to overcome stromal signaling [8] as well as synergize with both traditional chemotherapeutics and targeted agents in a variety of systems including hematologic malignancies [11]. Clinical testing of obatoclax in hematologic malignancies began with a Phase 1 study predominantly in AML and MDS,
Keywords: Chronic lymphocytic leukemia, phase I–III trials, obatoclax, BCL-2, fludarabine, rituximab
Introduction Chronic lymphocytic leukemia (CLL) is the most common leukemia of adults, but still incurable. Despite recent advances in CLL therapy using kinase inhibitors that impact the B-cell receptor pathway [1–3], most patients achieve only partial remissions (PR). Data suggest that complete remissions (CR) are associated with longer progression-free survival (PFS), even in the relapsed setting, and are certainly a pre-requisite for cure [4,5]. Improved strategies are, therefore, needed to achieve complete remissions (CR). These strategies are likely to involve combination therapy with inhibitors of other key pathways in CLL that may be synergistic. One of
Correspondence: Jennifer R Brown, MD, PhD, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA. Tel: 617-632-4564. Fax: 617-582-7890. E-mail: [email protected] Received 31 December 2014; accepted 12 April 2015
1
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2 J. R. Brown et al. but which also enrolled four CLL patients [18]. This study defined a recommended Phase II dose of 28 mg/m2/day over 24 h for 4 days every 2 weeks, based predominantly on neurologic toxicity including ataxia, dizziness and memory problems, which at this dose were limited to grade 1–2 events. A subsequent Phase 1 study of single-agent obatoclax in heavily pre-treated, largely refractory CLL patients demonstrated again that the dose-limiting toxicity was neurologic, including euphoria and ataxia, leading to a maximum tolerated dose of 28 mg/m2 given over 3 h every 3 weeks. One PR was observed along with biologic activity demonstrated by reductions in lymphocyte counts and improvements in cytopenias [19]. Given this rationale for obatoclax testing in CLL based on in vitro data and its ability to inhibit MCL-1, we undertook this Phase 1 study of the combination of obatoclax with the standard FR regimen in relapsed CLL patients. We chose to combine obatoclax with standard chemoimmunotherapy because of the expectation that synergy with BCL-2 family member inhibition might be greatest with cytotoxic drugs. At the time the study was designed, FR was commonly used in the treatment of both upfront and relapsed CLL. Our study shows that obatoclax administration is safe and welltolerated in relapsed CLL patients and that the combination with FR showed promising clinical activity.
Methods Eligibility This prospective study enrolled patients with CLL/SLL who had been treated with at least one prior line of therapy and again required therapy by NCI-WG 1996 criteria [20]. Prior therapy with fludarabine and/or rituximab was permitted as long as patients had responded and at least 1 year had elapsed since that therapy was completed. There were no restrictions for cytopenias. The study was approved by the Dana-Farber Harvard Cancer Center Institutional Review Board and all patients signed informed consent prior to initiation of therapy. This study is registered at Clinicaltrials.gov as NCT00612612.
Treatment program The planned therapy included standard dose fludarabine 25 mg/m2 on days 1–5 and rituximab 375 mg/m2 on day 1, repeated every 4 weeks according to standard protocol. In the first cycle the rituximab dose could be split with 50 mg/m2 on day 1 and 325 mg/m2 on day 3. Obatoclax was administered as a 3-h IV infusion on days 1 and 3, at three dose levels shown in Table I. This dosing regimen was selected because previous studies had suggested that neurologic toxicity was manageable with this infusion dose and schedule. Obatoclax was administered first, followed by fludarabine, followed by rituximab, in order to allow for correlative science sample collection between each drug. All patients received infectious prophylaxis with trimethoprimsulfamethoxazole and acyclovir (or equivalent). During the first cycle, all patients received allopurinol and intravenous hydration. Pegfilgrastim was administered at the discretion of the treating physician. Six cycles of therapy were planned.
Table I. Treatment and dose-limiting toxicities. Obatoclax 3 h Dose Fludarabine Rituximab infusion level D 1–5 (mg/m2) D1 (mg/m2) D1, 3 (mg/m2)
n
DLTS?
1 2 3
3 3 7
No No Treatment delay 2 weeks for grade 2–3 ANC
25 25 25
375 375 375
10 14 20
Definition of dose limiting toxicity The study used a standard 3 3 dose escalation design, with DLT assessed in the first cycle. Dose limiting toxicity (DLT) was defined as any non-hematologic toxicity of grade 3 or greater severity (excluding asymptomatic grade 3 laboratory abnormalities that are not life-threatening and respond to treatment; grade 3 fatigue; grade 3 nausea, vomiting or diarrhea occurring without optimal prophylaxis; or expected grade 3 rituximab infusion reactions). Any grade 4 non-hematological toxicity, as well as any irreversible grade 2 cardiac, renal or neurologic toxicities, was considered dose-limiting. Grading of non-hematologic toxicities was according to NCI CTC v. 3.0. Dose-limiting hematologic toxicity was defined as febrile neutropenia or failure of adequate hematologic recovery within 2 weeks of the scheduled start date of cycle 2. Adequate hematologic recovery to initiate cycle 2 or subsequent cycles was recovery of neutrophils and platelets to 75% of baseline or ANC 1500 and platelets 100,000, whichever was lower. Grading of hematologic toxicity was according to the IWCLL 2008 update of the 1996 NCI-WG criteria for CLL/SLL [21].
Study evaluations Screening evaluations included laboratory testing for hepatitis B and C and HIV, as well as bone marrow biopsy with karyotype if available and FISH and CT scans of the chest/ abdomen/pelvis. A complex karyotype was considered if three or more abnormalities were identified either by FISH or by an informative karyotype. Clinical response evaluation and CT scans were repeated every two cycles and at the conclusion of therapy. Bone marrow biopsy was performed 2–3 months after the conclusion of all study therapy. The primary end-point of this trial was to determine the maximum tolerated dose of obatoclax in combination with fludarabine-rituximab chemoimmunotherapy in relapsed CLL patients. Secondary clinical end-points included evaluating the toxicity of the three drug combination, determining the overall response rate (ORR) based on NCI-WG 1996 criteria and IWCLL 2008 criteria, i.e. without and with the CT scan data, and determining the progression-free survival (PFS) of the three drug combination.
CLL prognostic factors FISH for the common CLL abnormalities, namely deletions 13q, 11q or 17p, trisomy 12 or rearrangements of chromosome 14, was performed according to standard techniques by the Brigham and Women’s Hospital Clinical Cytogenetics Laboratory [22]. IGHV mutational status and ZAP-70 were performed by the CLL Research Consortium Tissue Core
Obatoclax with FR for CLL 3 according to previously described methods [23]. Unmutated IGHV was considered to be greater than or equal to 98% homologous to the closest germline match and positivity for ZAP-70 was considered to be 20% [23].
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Pharmacodynamic measurements Peripheral blood for analysis of apoptosis by Annexin V and propidium iodide (PI) staining was conducted using the FITC Annexin V Apoptosis Detection Kit I (BD catalog # 556547). Samples were collected on day 1 prior to and after obatoclax, after fludarabine and after rituximab; on day 2 prior to fludarabine; on day 3 prior to and after obatoclax; and on day 4 prior to fludarabine. All analyses for apoptosis were run immediately after sample procurement on Facs Canto II machines and analyzed using FlowJo Software (Version 8) and protein pellets were snap frozen for later analysis. Subsequently, these pellets were lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitor cocktail (Roche, Basel, Switzerland, catalog number 11697498001), resolved on SDS-PAGE gels and transferred onto polyvinylidene fluoride (PVDF) membranes. The blots were then probed with anti-human BCL-2 antibody (BD Pharmingen, San Jose, CA, catalog number 51-1513GR, lot # 000557) or anti-human MCL-1 antibody (Santa-Cruz, Dallas, TX, catalog number S-19, lot # J2913), followed by horseradish peroxidase (HRP)-conjugated secondary antibodies. Beta-actin was probed on all blots as a loading control after stripping the membranes (anti-Beta Actin antibody, Cell Signaling, Danvers, MA, catalog # 8457S, lot # 1). Densitometry analysis of the images was performed using the ImageJ software V1.47 (http://rsb.info.nih.gov/ij/ docs/faqs.html) and BCL-2 and MCL-1 levels were normalized to Beta-actin levels as well as to pre-treatment levels of the respective protein.
planned dose levels (Table I). Their clinical characteristics are detailed in Table II. The median age was 59 (range 35–79) and 54% were male; 38% had stage 3–4 disease at study entry and the median number of prior therapies was 2 (range 1–4). The median beta-2-microglobulin level was 3.5 (range 2.3–6.2) and 54% of patients had an elevated baseline LDH. One patient had 17p deletion and five had 11q deletion, one of whom also had a complex karyotype. Six of nine evaluable patients had unmutated IGHV.
Dose escalation and toxicity The therapy was generally well-tolerated, with only one observed DLT, which was a greater than 2-week delay in initiation of cycle 2 in a patient who had previously had prolonged cytopenias to FR alone (Table I). This dose level was expanded without further dose limiting toxicities, so no MTD was defined. Neurologic toxicity was easily manageable, with two rapidly resolving grade 3 or greater events that were not considered DLTs. Approximately 40% of patients experienced grade 1–2 euphoria, ataxia or dizziness acutely during the infusion, with resolution generally within 30 min after the conclusion of the infusion. No long-term sequelae were observed. Other grade 3 or greater toxicities were as expected with FR in a relapsed CLL population and included five events of grade 3–4 neutropenia (38%) and two each (15%) of grade 3–4 thrombocytopenia, fever without neutropenia, laboratory tumor lysis and allergic reaction (Table III). The median number of treatment cycles was five, with early discontinuations for cytopenias (the patient with the DLT after one cycle, two others after three cycles) and one withdrawal of consent after three cycles. No deaths were attributable to study therapy.
Statistical methods All eligible enrolled patients who received a single dose of therapy were evaluable for toxicity. Those patients who completed at least one cycle of study therapy were evaluable for efficacy. OS was defined as the time from initiation of study therapy to death from any cause, or censoring on the date last known alive. PFS was defined as the time from initiation of study therapy to disease relapse, progression or death in remission, whichever occurs first, or censoring on the date last known alive without progression. PFS and OS curves were obtained using the Kaplan-Meier method, with 95% confidence intervals calculated using Greenwood’s formula [24,25]. The response rate and 90% confidence intervals were calculated using the exact binomial test. Fisher’s exact test was used to assess the association of response and prior use of fludarabine. Changes in BCL-2 or MCL-1 levels from before and after obatoclax infusion were assessed using the Wilcoxon signed rank test.
Results Patient characteristics Thirteen patients were enrolled on this study between January 2008 and November 2009, to three successive
Table II. Patient characteristics. Age at diagnosis* Age at study entry* Sex - Female Rai Stage 3–4 Median ALC at baseline Median Hb at baseline Median platelets at baseline Elevated LDH at Baseline b2m* Bulky lymphadenopathy ( 5 cm) Time Dx to study therapy* IGHV unmutated ZAP-70 positive ( 20%) Interphase FISH Del 17p Complex Del 11q Trisomy 12 Normal Del 13q Del 6q Median 2 prior therapies, including: Fludarabine combinations Rituximab Alkylator combinations Alemtuzumab Autologous SCT
* Median (range).
51 (34–75) 59 (35–79) 6 (46%) 5 (38%) 43.7 (1.7, 134) 12.1 (7.7, 15.5) 137 (58, 253) 7 (54%) 3.5 (2.3–6.2) 3 (23%) 80 months (18–162) 6 / 9 (67%) 9 / 10 (90%) 1 (7.7%) 1 (7.7%) 5 (38.5%) 1 (7.7%) 2 (15%) 2 (15%) 1 (7.7%) 9 (69%) 10 (77%) 8 (62%) 3 (23%) 1 (7.7%)
4 J. R. Brown et al. Table III. Toxicities.
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1 Neurologic, Grade 3 or Greater: Depression Dizziness Neurologic, Grade 1–2: Dizziness Euphoria Ataxia Anxiety Speech Impairment Confusion Depression Agitation Depressed Consciousness Extrapyramidal Movement Memory Impairment Any Grade 3 or greater: Neutropenia Thrombocytopenia Fever without Neutropenia Laboratory Tumor Lysis Allergic Reaction Increased ALT/AST Anemia (Hemoglobin) Dyspnea Weakness Anorexia
2
Patients 3
1 1 1 1 2 2 1 2 2 1 1
1 (7.7%) 1 (7.7%) 1 1 1 1
4 4 2 2 3 2 1 1 1 2
2 1 1
1 1
3 2 1 1 1 1 1 1
6 (46%) 6 (46%) 5 (38%) 4 (31%) 4 (31%) 3 (23%) 3 (23%) 2 (15%) 2 (15%) 2 (15%) 2 (15%) 5 (38%) 2 (15%) 2 (15%) 2 (15%) 2 (15%) 1 (7.7%) 1 (7.7%) 1 (7.7%) 1 (7.7%) 1 (7.7%)
Response to therapy By NCI-WG 1996 criteria, the ORR at end of therapy was 85% (11/13; 90% CI 59–97%), with CR rate 15% (2/13; 90% CI 3–41%). In addition to the two complete remissions, five additional patients had nodular PRs (38%; 90% CI 17–65%) and two of those were negative for evidence of CLL by immunohistochemistry and flow cytometry. Seven of 11 responders and one of two patients who achieved complete remission had received prior fludarabine combination therapy (responders: FR (n 3), FC (n 1), FCR (n 2), F-alemtuzumab (n 1); CR (FR)). Only two patients had PD as their best overall response and both had received a fludarabine combination (FC and FCR). There was no significant association between use of prior fludarabine and ORR or CR (p 0.99 for both). Given that CT scanning in clinical trials was not yet recommended as a routine part of CLL staging when this trial was designed, a planned study end-point was to evaluate the impact of CT scans on ORR evaluation and indeed we found that incorporating CT scan evaluation into the final restaging, similar to the IWCLL 2008 criteria, decreased the ORR to 54% (7/13; 90% CI 29–78%), all PRs, largely due to the prevalence of significant abdominal lymphadenopathy in this relapsed patient population.
Survival Of the 13 patients, 10 have developed progressive disease, with a median time to progression of 20 months (95% CI 9–40 months) (Figure 1a). Two patients remain in remission at 42 and 53 months after therapy and a third went on to stem cell transplantation in remission. Ten patients have received subsequent therapy with a median time to next treatment of 29 months (95% CI 9–42 months) (Figure 1b). To date, four
100 80 60 40 20 0 0
2
4
6
8 10 12 14 16 18 Time to progression from baseline
20
22
24
13 patients and 10 events
(b) 100 Percentage of patients who did not receive next treatment
Obatoclax dose levels
Percentage of patients free from progression
(a)
80 60 40 20 0 0
6
12 18 24 Time to next treatment from baseline
30
36
13 patients and 10 events
Figure 1. (a) Time to disease progression. Median 20 months. (b) Time to next therapy. Median 29 months.
patients have died, three of disease progression and one of complications of refractory immune thrombocytopenia (ITP). The latter patient had a history of ITP previously, but a normal platelet count throughout therapy. The median (OS) has not been reached with a median follow-up of 49 months.
Pharmacodynamic measurements Quantitation of apoptotic CLL cells in peripheral blood with Annexin V–propidium iodide staining demonstrated increased apoptosis at some timepoint in nine of 13 patients during the first week of cycle one of therapy. Substantial inter-patient variability was seen, but one representative patient example is shown in Figure 2a. Although the BH3 mimetic activity of obatoclax is thought to result from displacement of apoptotic effectors, obatoclax has also been reported to down-regulate MCL-1 levels and to suppress the up-regulation of anti-apoptotic effectors by other agents [11,12,16]. We, therefore, performed sequential analysis of BCL-2 and MCL-1 protein levels by Western blot during the first week of therapy in seven patients. Protein levels at each treatment step were normalized to pre-treatment levels. All seven patients showed a reduction in BCL-2 levels at the end of the first obatoclax infusion, compared to the start (median decrease 27.9%, range 4–76.4%) (p 0.02), and usually maintained these lower levels over time (Figure 2b). Six of seven patients showed a reduction in MCL-1 levels at the end of the first obatoclax infusion compared to the start (median decrease 33.4%, range 9.6–99.1%) (p 0.05), which was maintained in some cases (Figure 2c).
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Obatoclax with FR for CLL 5
Figure 2. Pharmacodynamic Assays. (a) Increased apoptosis in peripheral blood CLL lymphocytes during cycle 1 of therapy, using Annexin V propidium iodide staining. One representative patient shown. (b) and (c) BCL-2 and MCL-1 protein levels in seven patients, respectively, normalized to Beta-actin as loading control, and expressed as a percentage of day 1 pre-dose levels. Median and interquartile range are shown.
Discussion We report on the safety and efficacy of combining the BCL-2 family inhibitor obatoclax with standard FR chemoimmunotherapy in CLL. The planned twice weekly 3-h infusion was well-tolerated with dose escalation proceeding to the highest planned dose level without unexpected toxicity and with relatively little neurologic toxicity. In fact, the expected neurologic toxicity was limited to primarily grade 1 events and occurred exclusively during the infusion, with rapid resolution thereafter. Furthermore, a median of five cycles of chemoimmunotherapy (CIT) were completed, which is typical for CIT, even in the upfront setting [26,27]. The regimen resulted in no grade 3–4 infections and only a 38% rate of grade 3–4 neutropenia and 15% rate of grade 3–4 thrombocytopenia, all quite favorable for CIT in a relapsed CLL population. The patient population enrolled on this study was reasonably high risk, with a median of two prior regimens including fludarabine-based CIT in ∼ 70% and nearly 50% having deletions of 11q or 17p (39% and 8%, respectively). Despite that, the efficacy of the regimen was high, with 85% ORR and 15% CR. We did observe a significantly lower response rate of 54% with the incorporation of CT scanning as per IWCLL 2008 criteria [21]. Although relatively few studies have made direct comparisons such as this one, response rates with
ofatumumab for example in relapsed CLL have been significantly lower in the post-CT era (RESONATE, 4% [2]) than the pre-CT era (47–58% [28]). Despite this difference in response rates, the median time to progression of 20 months on this study, including two patients with extended remissions 3.5 years, was encouraging relative to other reported outcomes of CIT in the relapsed setting [29–31]. These outcomes were achieved with relatively little chemotherapy (FR) and a regimen which was not particularly toxic. These findings suggest the potential of apoptosis inhibitors to enhance responses when combined with relatively little chemotherapy (FR as opposed to FCR for example), a goal which is currently of great interest in the CLL community. Despite our encouraging results, other studies in hematologic malignancies have primarily focused on single agent obatoclax and unfortunately showed no objective responses in follicular lymphoma [32], Hodgkin lymphoma [33], myelofibrosis [34] and a 8% response rate in myelodysplasia [35]. A combination study with bortezomib in mantle cell lymphoma demonstrated a 31% response rate that was not felt to be significantly better than expected with bortezomib alone [36]. In our studies, as in others, pharmacodynamic measurements of effect on apoptosis were challenging to perform, as apoptotic CLL cells are rapidly cleared from peripheral
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6 J. R. Brown et al. blood, leading to variable results in Annexin V/PI staining. Nonetheless, Annexin V staining did intermittently capture evidence of apoptotic cells in most patients during the first days of therapy. Ultimately, the very rapid reduction in peripheral blood white cell count seen in all patients, including two with evidence of laboratory tumor lysis, was the best marker of tumor cell kill. This rapid reduction also complicated our ability to look at anti-apoptotic proteins in the CLL cells themselves, due to limited sample availability in some patients at some time points. Again, individual results varied, but the majority of patients showed decreases in BCL-2 and MCL-1 during the first obatoclax infusion in particular. The small number of patients in our study and variable quality of samples for pharmacodynamic analysis limited our ability to assess potential predictors of clinical response. The solution to this problem in studies of BH3 mimetics is not obvious because of rapid cell clearance; the best approach may be to study baseline CLL cells in vitro with the hope that a baseline property of a given CLL will correlate with clinical outcome. Obatoclax is one of several potential BCL-2 inhibitors that have been studied in the clinic. Recent results in hematologic malignancies with single agent obatoclax have been disappointing, despite encouraging pre-clinical results, but some combination studies are ongoing [11]. The results reported here in CLL are encouraging, but enthusiasm has been tempered by the concern that the mechanism of obatoclax may not be predominantly as a BH3 mimetic and is not well understood. This problem, as well as the poorly understood neurologic infusional toxicity, has limited its further development and any future role in CLL. Nonetheless, an advantage that obatoclax did have was pan-BCL-2 family inhibition, which is not the case with ABT-263, a BH3 mimetic inhibitor of BCL-2, BCL-XL and BCL-w (but not MCL-1) or the next generation ABT-199, a specific BCL-2 inhibitor. ABT-263 demonstrated activity in fludarabine-refractory CLL but was limited by significant thrombocytopenia due to its inhibition of BCL-XL [37] and a combination study of ABT-263 and FCR or BR demonstrated significant toxicity [38]. The next generation molecule ABT-199 has activity only against BCL-2 and, therefore, limited effect on platelets and has shown remarkable single agent activity in CLL [39]. Even as ABT-199 advances in clinical development, it will be important to be aware of the risk of resistance through upregulation of MCL-1, BCL-XL and BCL-2A1 [10]. Although the future of obatoclax is limited, the promising results reported here should serve as a reminder of the ongoing need and potential benefit of developing pan-BCL-2 family inhibitors, in order to maximize clinical benefit and minimize the development of resistance to this important class of drug.
Acknowledgements We are indebted to the nurses and clinical research staff of the Dana-Farber Cancer Institute for their excellent care of these patients and to the patients for their participation in this study. JRB was supported by NIH grant K23 CA115682-01 and is a Scholar of the American Society of Hematology as well as a Scholar in Clinical Research of the Leukemia and Lymphoma Society.
Potential conflict of interest: Disclosure forms provided by the authors are available with the full text of this article at www.informahealthcare.com/lal
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previously treated chronic lymphocytic leukemia. J Clin Oncol 2010;28: 1756–1765. [32] Goy A, Berger M, Ford P, et al. Sequential single-agent obatoclax mesylate (GX15-070MS) followed by combination with rituximab in patients with previously untreated follicular lymphoma. Leuk Lymphoma 2014;55:2932–2934. [33] Oki Y, Copeland A, Hagemeister F, et al. Experience with obatoclax mesylate (GX15-070), a small molecule pan-Bcl-2 family antagonist in patients with relapsed or refractory classical Hodgkin lymphoma. Blood 2012;119:2171–2172. [34] Parikh SA, Kantarjian H, Schimmer A, et al. Phase II study of obatoclax mesylate (GX15-070), a small-molecule BCL-2 family antagonist, for patients with myelofibrosis. Clin Lymphoma Myeloma Leuk 2010;10:285–289. [35] Arellano ML, Borthakur G, Berger M, et al. A Phase II, Multicenter, Open-Label Study of obatoclax mesylate in patients with previously untreated myelodysplastic syndromes with anemia or thrombocytopenia. Clin Lymphoma Myeloma Leuk 2014;14:534–539. [36] Goy A, Hernandez-Ilzaliturri FJ, Kahl B, et al. A phase I/II study of the pan Bcl-2 inhibitor obatoclax mesylate plus bortezomib for relapsed or refractory mantle cell lymphoma. Leuk Lymphoma 2014;55:2761–2768. [37] Roberts AW, Seymour JF, Brown JR, et al. Substantial susceptibility of chronic lymphocytic leukemia to BCL-2 inhibition: results of a phase I study of navitoclax in patients with relapsed or refractory disease. J Clin Oncol 2012;30:488–496. [38] Kipps TJ, Swinnen LJ, Wierda WG, et al. Navitoclax (ABT-263) Plus Fludarabine/Cyclophosphamide/Rituximab (FCR) or Bendamustine/ Rituximab (BR): A Phase 1 Study in Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia (CLL). ASH Annual Meeting Abstracts 2011;118:3904. [39] Seymour JF, Davids MS, Pagel JM, et al. ABT-199 (GDC-0199) in relapsed/refractory chronic lymphocytic leukemia and small lymphocytic leukemia: high response rates among patients with high risk disease features including unmutated IGHV. Haematologica 2014;99(Suppl 1):702.
ISSN: 1042-8194 (Print) 1029-2403 (Online) Journal homepage: http://www.tandfonline.com/loi/ilal20
Obatoclax in combination with fludarabine and rituximab is well-tolerated and shows promising clinical activity in relapsed chronic lymphocytic leukemia Jennifer R. Brown, Bethany Tesar, Lijian Yu, Lillian Werner, Naoko Takebe, Evgeny Mikler, Hazel M. Reynolds, Christina Thompson, David C. Fisher, Donna Neuberg & A. S. Freedman To cite this article: Jennifer R. Brown, Bethany Tesar, Lijian Yu, Lillian Werner, Naoko Takebe, Evgeny Mikler, Hazel M. Reynolds, Christina Thompson, David C. Fisher, Donna Neuberg & A. S. Freedman (2015): Obatoclax in combination with fludarabine and rituximab is well-tolerated and shows promising clinical activity in relapsed chronic lymphocytic leukemia, Leukemia & Lymphoma To link to this article: http://dx.doi.org/10.3109/10428194.2015.1048441
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Date: 21 October 2015, At: 17:30
Leukemia & Lymphoma, 2015; Early Online: 1–7 © 2015 Informa UK, Ltd. ISSN: 1042-8194 print / 1029-2403 online DOI: 10.3109/10428194.2015.1048441
ORIGINAL ARTICLE: CLINICAL
Obatoclax in combination with fludarabine and rituximab is well-tolerated and shows promising clinical activity in relapsed chronic lymphocytic leukemia Jennifer R. Brown1,3, Bethany Tesar1,3, Lijian Yu1,3, Lillian Werner2, Naoko Takebe4, Evgeny Mikler1, Hazel M. Reynolds1, Christina Thompson1, David C. Fisher1,3, Donna Neuberg2 & A. S. Freedman1,3 1Department of Medical Oncology, 2Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute,
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Boston, MA, USA, 3Department of Medicine, Harvard Medical School, Boston, MA, USA, and 4Investigational Drug Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA
Abstract Obatoclax is a small molecule mimetic of the BH3 domain of BCL2 family proteins. This phase 1 study combining obatoclax with FR was undertaken in chronic lymphocytic leukemia (CLL) patients relapsed after at least one prior therapy. Obatoclax was given as a 3-h infusion on days 1 and 3 and escalated through three dose levels, with standard dose FR days 1–5. Thirteen patients were enrolled, with a median of two prior therapies. One doselimiting toxicity (DLT) of a 2-week treatment delay for persistent grade 2–3 neutropenia was observed at the highest obatoclax dose (20 mg/m2), but no maximum tolerated dose (MTD) was reached. The overall response rate (ORR) was 85%, with 15% complete responses (CRs) by NCI-96 criteria and 54% by IWCLL 2008 criteria. Median time to progression was 20 months. It is concluded that obatoclax can be safely administered to relapsed CLL patients in combination with FR and shows promising clinical activity.
the most interesting pathways involves BCL-2, which is overexpressed nearly universally in CLL and confers resistance to apoptosis [6]. Inhibition of BCL-2 family members, including MCL-1, has been shown to synergistically induce apoptosis in combination with many cytotoxic and non-cytotoxic therapies in CLL [7–10]. Obatoclax is a small molecule mimetic of the BH3 domain of BCL-2 family proteins and has broad specificity, with activity against BCL-2, BCL-XL, BCL-w and MCL-1 [11]. Its activity against MCL-1 is particularly unique among BCL-2 family inhibitors and may be important for CLL, which frequently over-expresses MCL-1 [11]. Multiple studies have found that obatoclax can induce apoptosis in CLL cells in vitro [7–9,12], in some cases by a BH3 mimetic mechanism. A BH3 mimetic mechanism implies binding of obatoclax to anti-apoptotic BCL-2 family members, leading to release of bound BAX, BAK or activator BH3-only proteins, which then induce apoptosis. However, cytotoxicity induced by obatoclax has also been observed in systems without BAX or BAK expression [9,13,14], suggesting that, at least in some cases, obatoclax is acting by a non-BH3 mimetic mechanism. Obatoclax has in fact been found to induce cellular cytotoxicity by alternative mechanisms, including induction of cell cycle arrest [14,15], reduction of colony formation [14,16] and induction of necroptosis and autophagy in acute lymphoblastic leukemia cell lines [17]. Further work will be required to fully define the mechanism of action of obatoclax in different disease settings. However, its cytotoxicity is clear in vitro, and it has been shown to overcome stromal signaling [8] as well as synergize with both traditional chemotherapeutics and targeted agents in a variety of systems including hematologic malignancies [11]. Clinical testing of obatoclax in hematologic malignancies began with a Phase 1 study predominantly in AML and MDS,
Keywords: Chronic lymphocytic leukemia, phase I–III trials, obatoclax, BCL-2, fludarabine, rituximab
Introduction Chronic lymphocytic leukemia (CLL) is the most common leukemia of adults, but still incurable. Despite recent advances in CLL therapy using kinase inhibitors that impact the B-cell receptor pathway [1–3], most patients achieve only partial remissions (PR). Data suggest that complete remissions (CR) are associated with longer progression-free survival (PFS), even in the relapsed setting, and are certainly a pre-requisite for cure [4,5]. Improved strategies are, therefore, needed to achieve complete remissions (CR). These strategies are likely to involve combination therapy with inhibitors of other key pathways in CLL that may be synergistic. One of
Correspondence: Jennifer R Brown, MD, PhD, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA. Tel: 617-632-4564. Fax: 617-582-7890. E-mail: [email protected] Received 31 December 2014; accepted 12 April 2015
1
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2 J. R. Brown et al. but which also enrolled four CLL patients [18]. This study defined a recommended Phase II dose of 28 mg/m2/day over 24 h for 4 days every 2 weeks, based predominantly on neurologic toxicity including ataxia, dizziness and memory problems, which at this dose were limited to grade 1–2 events. A subsequent Phase 1 study of single-agent obatoclax in heavily pre-treated, largely refractory CLL patients demonstrated again that the dose-limiting toxicity was neurologic, including euphoria and ataxia, leading to a maximum tolerated dose of 28 mg/m2 given over 3 h every 3 weeks. One PR was observed along with biologic activity demonstrated by reductions in lymphocyte counts and improvements in cytopenias [19]. Given this rationale for obatoclax testing in CLL based on in vitro data and its ability to inhibit MCL-1, we undertook this Phase 1 study of the combination of obatoclax with the standard FR regimen in relapsed CLL patients. We chose to combine obatoclax with standard chemoimmunotherapy because of the expectation that synergy with BCL-2 family member inhibition might be greatest with cytotoxic drugs. At the time the study was designed, FR was commonly used in the treatment of both upfront and relapsed CLL. Our study shows that obatoclax administration is safe and welltolerated in relapsed CLL patients and that the combination with FR showed promising clinical activity.
Methods Eligibility This prospective study enrolled patients with CLL/SLL who had been treated with at least one prior line of therapy and again required therapy by NCI-WG 1996 criteria [20]. Prior therapy with fludarabine and/or rituximab was permitted as long as patients had responded and at least 1 year had elapsed since that therapy was completed. There were no restrictions for cytopenias. The study was approved by the Dana-Farber Harvard Cancer Center Institutional Review Board and all patients signed informed consent prior to initiation of therapy. This study is registered at Clinicaltrials.gov as NCT00612612.
Treatment program The planned therapy included standard dose fludarabine 25 mg/m2 on days 1–5 and rituximab 375 mg/m2 on day 1, repeated every 4 weeks according to standard protocol. In the first cycle the rituximab dose could be split with 50 mg/m2 on day 1 and 325 mg/m2 on day 3. Obatoclax was administered as a 3-h IV infusion on days 1 and 3, at three dose levels shown in Table I. This dosing regimen was selected because previous studies had suggested that neurologic toxicity was manageable with this infusion dose and schedule. Obatoclax was administered first, followed by fludarabine, followed by rituximab, in order to allow for correlative science sample collection between each drug. All patients received infectious prophylaxis with trimethoprimsulfamethoxazole and acyclovir (or equivalent). During the first cycle, all patients received allopurinol and intravenous hydration. Pegfilgrastim was administered at the discretion of the treating physician. Six cycles of therapy were planned.
Table I. Treatment and dose-limiting toxicities. Obatoclax 3 h Dose Fludarabine Rituximab infusion level D 1–5 (mg/m2) D1 (mg/m2) D1, 3 (mg/m2)
n
DLTS?
1 2 3
3 3 7
No No Treatment delay 2 weeks for grade 2–3 ANC
25 25 25
375 375 375
10 14 20
Definition of dose limiting toxicity The study used a standard 3 3 dose escalation design, with DLT assessed in the first cycle. Dose limiting toxicity (DLT) was defined as any non-hematologic toxicity of grade 3 or greater severity (excluding asymptomatic grade 3 laboratory abnormalities that are not life-threatening and respond to treatment; grade 3 fatigue; grade 3 nausea, vomiting or diarrhea occurring without optimal prophylaxis; or expected grade 3 rituximab infusion reactions). Any grade 4 non-hematological toxicity, as well as any irreversible grade 2 cardiac, renal or neurologic toxicities, was considered dose-limiting. Grading of non-hematologic toxicities was according to NCI CTC v. 3.0. Dose-limiting hematologic toxicity was defined as febrile neutropenia or failure of adequate hematologic recovery within 2 weeks of the scheduled start date of cycle 2. Adequate hematologic recovery to initiate cycle 2 or subsequent cycles was recovery of neutrophils and platelets to 75% of baseline or ANC 1500 and platelets 100,000, whichever was lower. Grading of hematologic toxicity was according to the IWCLL 2008 update of the 1996 NCI-WG criteria for CLL/SLL [21].
Study evaluations Screening evaluations included laboratory testing for hepatitis B and C and HIV, as well as bone marrow biopsy with karyotype if available and FISH and CT scans of the chest/ abdomen/pelvis. A complex karyotype was considered if three or more abnormalities were identified either by FISH or by an informative karyotype. Clinical response evaluation and CT scans were repeated every two cycles and at the conclusion of therapy. Bone marrow biopsy was performed 2–3 months after the conclusion of all study therapy. The primary end-point of this trial was to determine the maximum tolerated dose of obatoclax in combination with fludarabine-rituximab chemoimmunotherapy in relapsed CLL patients. Secondary clinical end-points included evaluating the toxicity of the three drug combination, determining the overall response rate (ORR) based on NCI-WG 1996 criteria and IWCLL 2008 criteria, i.e. without and with the CT scan data, and determining the progression-free survival (PFS) of the three drug combination.
CLL prognostic factors FISH for the common CLL abnormalities, namely deletions 13q, 11q or 17p, trisomy 12 or rearrangements of chromosome 14, was performed according to standard techniques by the Brigham and Women’s Hospital Clinical Cytogenetics Laboratory [22]. IGHV mutational status and ZAP-70 were performed by the CLL Research Consortium Tissue Core
Obatoclax with FR for CLL 3 according to previously described methods [23]. Unmutated IGHV was considered to be greater than or equal to 98% homologous to the closest germline match and positivity for ZAP-70 was considered to be 20% [23].
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Pharmacodynamic measurements Peripheral blood for analysis of apoptosis by Annexin V and propidium iodide (PI) staining was conducted using the FITC Annexin V Apoptosis Detection Kit I (BD catalog # 556547). Samples were collected on day 1 prior to and after obatoclax, after fludarabine and after rituximab; on day 2 prior to fludarabine; on day 3 prior to and after obatoclax; and on day 4 prior to fludarabine. All analyses for apoptosis were run immediately after sample procurement on Facs Canto II machines and analyzed using FlowJo Software (Version 8) and protein pellets were snap frozen for later analysis. Subsequently, these pellets were lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitor cocktail (Roche, Basel, Switzerland, catalog number 11697498001), resolved on SDS-PAGE gels and transferred onto polyvinylidene fluoride (PVDF) membranes. The blots were then probed with anti-human BCL-2 antibody (BD Pharmingen, San Jose, CA, catalog number 51-1513GR, lot # 000557) or anti-human MCL-1 antibody (Santa-Cruz, Dallas, TX, catalog number S-19, lot # J2913), followed by horseradish peroxidase (HRP)-conjugated secondary antibodies. Beta-actin was probed on all blots as a loading control after stripping the membranes (anti-Beta Actin antibody, Cell Signaling, Danvers, MA, catalog # 8457S, lot # 1). Densitometry analysis of the images was performed using the ImageJ software V1.47 (http://rsb.info.nih.gov/ij/ docs/faqs.html) and BCL-2 and MCL-1 levels were normalized to Beta-actin levels as well as to pre-treatment levels of the respective protein.
planned dose levels (Table I). Their clinical characteristics are detailed in Table II. The median age was 59 (range 35–79) and 54% were male; 38% had stage 3–4 disease at study entry and the median number of prior therapies was 2 (range 1–4). The median beta-2-microglobulin level was 3.5 (range 2.3–6.2) and 54% of patients had an elevated baseline LDH. One patient had 17p deletion and five had 11q deletion, one of whom also had a complex karyotype. Six of nine evaluable patients had unmutated IGHV.
Dose escalation and toxicity The therapy was generally well-tolerated, with only one observed DLT, which was a greater than 2-week delay in initiation of cycle 2 in a patient who had previously had prolonged cytopenias to FR alone (Table I). This dose level was expanded without further dose limiting toxicities, so no MTD was defined. Neurologic toxicity was easily manageable, with two rapidly resolving grade 3 or greater events that were not considered DLTs. Approximately 40% of patients experienced grade 1–2 euphoria, ataxia or dizziness acutely during the infusion, with resolution generally within 30 min after the conclusion of the infusion. No long-term sequelae were observed. Other grade 3 or greater toxicities were as expected with FR in a relapsed CLL population and included five events of grade 3–4 neutropenia (38%) and two each (15%) of grade 3–4 thrombocytopenia, fever without neutropenia, laboratory tumor lysis and allergic reaction (Table III). The median number of treatment cycles was five, with early discontinuations for cytopenias (the patient with the DLT after one cycle, two others after three cycles) and one withdrawal of consent after three cycles. No deaths were attributable to study therapy.
Statistical methods All eligible enrolled patients who received a single dose of therapy were evaluable for toxicity. Those patients who completed at least one cycle of study therapy were evaluable for efficacy. OS was defined as the time from initiation of study therapy to death from any cause, or censoring on the date last known alive. PFS was defined as the time from initiation of study therapy to disease relapse, progression or death in remission, whichever occurs first, or censoring on the date last known alive without progression. PFS and OS curves were obtained using the Kaplan-Meier method, with 95% confidence intervals calculated using Greenwood’s formula [24,25]. The response rate and 90% confidence intervals were calculated using the exact binomial test. Fisher’s exact test was used to assess the association of response and prior use of fludarabine. Changes in BCL-2 or MCL-1 levels from before and after obatoclax infusion were assessed using the Wilcoxon signed rank test.
Results Patient characteristics Thirteen patients were enrolled on this study between January 2008 and November 2009, to three successive
Table II. Patient characteristics. Age at diagnosis* Age at study entry* Sex - Female Rai Stage 3–4 Median ALC at baseline Median Hb at baseline Median platelets at baseline Elevated LDH at Baseline b2m* Bulky lymphadenopathy ( 5 cm) Time Dx to study therapy* IGHV unmutated ZAP-70 positive ( 20%) Interphase FISH Del 17p Complex Del 11q Trisomy 12 Normal Del 13q Del 6q Median 2 prior therapies, including: Fludarabine combinations Rituximab Alkylator combinations Alemtuzumab Autologous SCT
* Median (range).
51 (34–75) 59 (35–79) 6 (46%) 5 (38%) 43.7 (1.7, 134) 12.1 (7.7, 15.5) 137 (58, 253) 7 (54%) 3.5 (2.3–6.2) 3 (23%) 80 months (18–162) 6 / 9 (67%) 9 / 10 (90%) 1 (7.7%) 1 (7.7%) 5 (38.5%) 1 (7.7%) 2 (15%) 2 (15%) 1 (7.7%) 9 (69%) 10 (77%) 8 (62%) 3 (23%) 1 (7.7%)
4 J. R. Brown et al. Table III. Toxicities.
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1 Neurologic, Grade 3 or Greater: Depression Dizziness Neurologic, Grade 1–2: Dizziness Euphoria Ataxia Anxiety Speech Impairment Confusion Depression Agitation Depressed Consciousness Extrapyramidal Movement Memory Impairment Any Grade 3 or greater: Neutropenia Thrombocytopenia Fever without Neutropenia Laboratory Tumor Lysis Allergic Reaction Increased ALT/AST Anemia (Hemoglobin) Dyspnea Weakness Anorexia
2
Patients 3
1 1 1 1 2 2 1 2 2 1 1
1 (7.7%) 1 (7.7%) 1 1 1 1
4 4 2 2 3 2 1 1 1 2
2 1 1
1 1
3 2 1 1 1 1 1 1
6 (46%) 6 (46%) 5 (38%) 4 (31%) 4 (31%) 3 (23%) 3 (23%) 2 (15%) 2 (15%) 2 (15%) 2 (15%) 5 (38%) 2 (15%) 2 (15%) 2 (15%) 2 (15%) 1 (7.7%) 1 (7.7%) 1 (7.7%) 1 (7.7%) 1 (7.7%)
Response to therapy By NCI-WG 1996 criteria, the ORR at end of therapy was 85% (11/13; 90% CI 59–97%), with CR rate 15% (2/13; 90% CI 3–41%). In addition to the two complete remissions, five additional patients had nodular PRs (38%; 90% CI 17–65%) and two of those were negative for evidence of CLL by immunohistochemistry and flow cytometry. Seven of 11 responders and one of two patients who achieved complete remission had received prior fludarabine combination therapy (responders: FR (n 3), FC (n 1), FCR (n 2), F-alemtuzumab (n 1); CR (FR)). Only two patients had PD as their best overall response and both had received a fludarabine combination (FC and FCR). There was no significant association between use of prior fludarabine and ORR or CR (p 0.99 for both). Given that CT scanning in clinical trials was not yet recommended as a routine part of CLL staging when this trial was designed, a planned study end-point was to evaluate the impact of CT scans on ORR evaluation and indeed we found that incorporating CT scan evaluation into the final restaging, similar to the IWCLL 2008 criteria, decreased the ORR to 54% (7/13; 90% CI 29–78%), all PRs, largely due to the prevalence of significant abdominal lymphadenopathy in this relapsed patient population.
Survival Of the 13 patients, 10 have developed progressive disease, with a median time to progression of 20 months (95% CI 9–40 months) (Figure 1a). Two patients remain in remission at 42 and 53 months after therapy and a third went on to stem cell transplantation in remission. Ten patients have received subsequent therapy with a median time to next treatment of 29 months (95% CI 9–42 months) (Figure 1b). To date, four
100 80 60 40 20 0 0
2
4
6
8 10 12 14 16 18 Time to progression from baseline
20
22
24
13 patients and 10 events
(b) 100 Percentage of patients who did not receive next treatment
Obatoclax dose levels
Percentage of patients free from progression
(a)
80 60 40 20 0 0
6
12 18 24 Time to next treatment from baseline
30
36
13 patients and 10 events
Figure 1. (a) Time to disease progression. Median 20 months. (b) Time to next therapy. Median 29 months.
patients have died, three of disease progression and one of complications of refractory immune thrombocytopenia (ITP). The latter patient had a history of ITP previously, but a normal platelet count throughout therapy. The median (OS) has not been reached with a median follow-up of 49 months.
Pharmacodynamic measurements Quantitation of apoptotic CLL cells in peripheral blood with Annexin V–propidium iodide staining demonstrated increased apoptosis at some timepoint in nine of 13 patients during the first week of cycle one of therapy. Substantial inter-patient variability was seen, but one representative patient example is shown in Figure 2a. Although the BH3 mimetic activity of obatoclax is thought to result from displacement of apoptotic effectors, obatoclax has also been reported to down-regulate MCL-1 levels and to suppress the up-regulation of anti-apoptotic effectors by other agents [11,12,16]. We, therefore, performed sequential analysis of BCL-2 and MCL-1 protein levels by Western blot during the first week of therapy in seven patients. Protein levels at each treatment step were normalized to pre-treatment levels. All seven patients showed a reduction in BCL-2 levels at the end of the first obatoclax infusion, compared to the start (median decrease 27.9%, range 4–76.4%) (p 0.02), and usually maintained these lower levels over time (Figure 2b). Six of seven patients showed a reduction in MCL-1 levels at the end of the first obatoclax infusion compared to the start (median decrease 33.4%, range 9.6–99.1%) (p 0.05), which was maintained in some cases (Figure 2c).
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Obatoclax with FR for CLL 5
Figure 2. Pharmacodynamic Assays. (a) Increased apoptosis in peripheral blood CLL lymphocytes during cycle 1 of therapy, using Annexin V propidium iodide staining. One representative patient shown. (b) and (c) BCL-2 and MCL-1 protein levels in seven patients, respectively, normalized to Beta-actin as loading control, and expressed as a percentage of day 1 pre-dose levels. Median and interquartile range are shown.
Discussion We report on the safety and efficacy of combining the BCL-2 family inhibitor obatoclax with standard FR chemoimmunotherapy in CLL. The planned twice weekly 3-h infusion was well-tolerated with dose escalation proceeding to the highest planned dose level without unexpected toxicity and with relatively little neurologic toxicity. In fact, the expected neurologic toxicity was limited to primarily grade 1 events and occurred exclusively during the infusion, with rapid resolution thereafter. Furthermore, a median of five cycles of chemoimmunotherapy (CIT) were completed, which is typical for CIT, even in the upfront setting [26,27]. The regimen resulted in no grade 3–4 infections and only a 38% rate of grade 3–4 neutropenia and 15% rate of grade 3–4 thrombocytopenia, all quite favorable for CIT in a relapsed CLL population. The patient population enrolled on this study was reasonably high risk, with a median of two prior regimens including fludarabine-based CIT in ∼ 70% and nearly 50% having deletions of 11q or 17p (39% and 8%, respectively). Despite that, the efficacy of the regimen was high, with 85% ORR and 15% CR. We did observe a significantly lower response rate of 54% with the incorporation of CT scanning as per IWCLL 2008 criteria [21]. Although relatively few studies have made direct comparisons such as this one, response rates with
ofatumumab for example in relapsed CLL have been significantly lower in the post-CT era (RESONATE, 4% [2]) than the pre-CT era (47–58% [28]). Despite this difference in response rates, the median time to progression of 20 months on this study, including two patients with extended remissions 3.5 years, was encouraging relative to other reported outcomes of CIT in the relapsed setting [29–31]. These outcomes were achieved with relatively little chemotherapy (FR) and a regimen which was not particularly toxic. These findings suggest the potential of apoptosis inhibitors to enhance responses when combined with relatively little chemotherapy (FR as opposed to FCR for example), a goal which is currently of great interest in the CLL community. Despite our encouraging results, other studies in hematologic malignancies have primarily focused on single agent obatoclax and unfortunately showed no objective responses in follicular lymphoma [32], Hodgkin lymphoma [33], myelofibrosis [34] and a 8% response rate in myelodysplasia [35]. A combination study with bortezomib in mantle cell lymphoma demonstrated a 31% response rate that was not felt to be significantly better than expected with bortezomib alone [36]. In our studies, as in others, pharmacodynamic measurements of effect on apoptosis were challenging to perform, as apoptotic CLL cells are rapidly cleared from peripheral
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6 J. R. Brown et al. blood, leading to variable results in Annexin V/PI staining. Nonetheless, Annexin V staining did intermittently capture evidence of apoptotic cells in most patients during the first days of therapy. Ultimately, the very rapid reduction in peripheral blood white cell count seen in all patients, including two with evidence of laboratory tumor lysis, was the best marker of tumor cell kill. This rapid reduction also complicated our ability to look at anti-apoptotic proteins in the CLL cells themselves, due to limited sample availability in some patients at some time points. Again, individual results varied, but the majority of patients showed decreases in BCL-2 and MCL-1 during the first obatoclax infusion in particular. The small number of patients in our study and variable quality of samples for pharmacodynamic analysis limited our ability to assess potential predictors of clinical response. The solution to this problem in studies of BH3 mimetics is not obvious because of rapid cell clearance; the best approach may be to study baseline CLL cells in vitro with the hope that a baseline property of a given CLL will correlate with clinical outcome. Obatoclax is one of several potential BCL-2 inhibitors that have been studied in the clinic. Recent results in hematologic malignancies with single agent obatoclax have been disappointing, despite encouraging pre-clinical results, but some combination studies are ongoing [11]. The results reported here in CLL are encouraging, but enthusiasm has been tempered by the concern that the mechanism of obatoclax may not be predominantly as a BH3 mimetic and is not well understood. This problem, as well as the poorly understood neurologic infusional toxicity, has limited its further development and any future role in CLL. Nonetheless, an advantage that obatoclax did have was pan-BCL-2 family inhibition, which is not the case with ABT-263, a BH3 mimetic inhibitor of BCL-2, BCL-XL and BCL-w (but not MCL-1) or the next generation ABT-199, a specific BCL-2 inhibitor. ABT-263 demonstrated activity in fludarabine-refractory CLL but was limited by significant thrombocytopenia due to its inhibition of BCL-XL [37] and a combination study of ABT-263 and FCR or BR demonstrated significant toxicity [38]. The next generation molecule ABT-199 has activity only against BCL-2 and, therefore, limited effect on platelets and has shown remarkable single agent activity in CLL [39]. Even as ABT-199 advances in clinical development, it will be important to be aware of the risk of resistance through upregulation of MCL-1, BCL-XL and BCL-2A1 [10]. Although the future of obatoclax is limited, the promising results reported here should serve as a reminder of the ongoing need and potential benefit of developing pan-BCL-2 family inhibitors, in order to maximize clinical benefit and minimize the development of resistance to this important class of drug.
Acknowledgements We are indebted to the nurses and clinical research staff of the Dana-Farber Cancer Institute for their excellent care of these patients and to the patients for their participation in this study. JRB was supported by NIH grant K23 CA115682-01 and is a Scholar of the American Society of Hematology as well as a Scholar in Clinical Research of the Leukemia and Lymphoma Society.
Potential conflict of interest: Disclosure forms provided by the authors are available with the full text of this article at www.informahealthcare.com/lal
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