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Keywords:

  • 2 stage design;
  • clinical trial;
  • lymphoma;
  • phase I;
  • vorinostat

Summary

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure
  8. References
  9. Supporting Information

Given the poor outcomes of relapsed aggressive lymphomas and preclinical data suggesting that ≥2·5 μmol/l concentrations of vorinostat synergize with both etoposide and platinums, we hypothesized that pulse high-dose vorinostat could safely augment the anti-tumour activity of (R)ICE [(rituximab), ifosphamide, carboplatin, etoposide] chemotherapy. We conducted a phase I dose escalation study using a schedule with oral vorinostat ranging from 400 mg/d to 700 mg bid for 5 d in combination with the standard (R)ICE regimen (days 3, 4 and 5). Twenty-nine patients [median age 56 years, median 2 prior therapies, 14 chemoresistant (of 27 evaluable), 2 prior transplants] were enrolled and treated. The maximally tolerated vorinostat dose was defined as 500 mg twice daily × 5 d. Common dose limiting toxicities included infection (n = 2), hypokalaemia (n = 2), and transaminitis (n = 2). Grade 3 related gastrointestinal toxicity was seen in 9 patients. The median vorinostat concentration on day 3 was 4·5 μmol/l (range 4·2–6·0 μmol/l) and in vitro data confirmed the augmented antitumour and histone acetylation activity at these levels. Responses were observed in 19 of 27 evaluable patients (70%) including 8 complete response/unconfirmed complete response. High-dose vorinostat can be delivered safely with (R)ICE, achieves potentially synergistic drug levels, and warrants further study, although adequate gastrointestinal prophylaxis is warranted.

Significant progress has been made in the front-line treatment of both indolent and aggressive lymphomas leading to prolonged remissions, improved survival, and higher cure rates (Coiffier et al, 2002; Fisher et al, 2005; Swenson et al, 2005; Engert et al, 2009; Herrmann et al, 2009; Armitage, 2010). However, patients suffering relapse after these improved initial therapies have a lower likelihood of attaining long-term disease-free survival when historical salvage strategies are applied (Gisselbrecht et al, 2010; Viviani et al, 2011). In the CORAL (Collaborative Trial in Relapsed Aggressive Lymphoma) study, the three-year overall survival for patients with relapsed or refractory diffuse large B cell lymphoma (DLBCL) after receiving RICE (rituximab, ifosphamide, carboplatin, etoposide) or RDHAP (rituximab, dexamethasonem cytarabine, cisplatin) was adversely affected by prior rituximab treatment (21% vs. 47%)(Gisselbrecht et al, 2010). Unfortunately, dose escalation of the traditional cytotoxic agents in the (R)ICE regimen is not possible due to considerable toxicity (Moskowitz et al, 1999; Kewalramani et al, 2004). Novel non-cross-toxic agents may need to be employed to enhance the efficacy of (R)ICE without adding prohibitive toxicity.

Vorinostat (suberoylanilide hydroxamic acid) is an orally bioavailable histone-deacetylase (HDAC) inhibitor approved by the Food and Drug Administration for treatment of cutaneous T-cell lymphoma (CTCL). It regulates gene expression by inhibiting the activity of class I and II HDACs and modifies nonhistone protein targets that regulate cell proliferation, migration, and apoptosis (Maeda et al, 2000; Kim et al, 2001; Marks et al, 2001; Zhao et al, 2006; Ellis et al, 2009; Frew et al, 2009). Several features of vorinostat make it an attractive agent for the treatment of lymphomas. First, it has a moderate toxicity and tolerability profile when used as single agent, suggesting that the addition of vorinostat to intensified chemotherapy should be feasible (Rubin et al, 2006). Secondly, vorinostat has a distinct mechanism of action, which implies that it may be able to deliver additional anti-tumour activity in combinational therapy with limited overlapping toxicities. Thirdly, vorinostat has been found to potentiate the cytotoxicity of agents, such as etoposide (Marchion et al, 2004) and platinum analogues (Rikiishi et al, 2007), in vitro at concentrations of ≥2·5 μmol/l in a sequence-dependent fashion. Unfortunately, these concentrations are not typically attained in patients treated with the approved dose of vorinostat of 400 mg/day (Rubin et al, 2006), (Dickson et al, 2011), (Kelly et al, 2005). We, thus, hypothesized that pulse high-dose vorinostat preceding (R)ICE chemotherapy might achieve the required high vorinostat concentrations to optimally augment the anti-tumour activity in patients with relapsed or refractory lymphomas.

This multi-centre phase I study sought to determine the maximal tolerated dose (MTD), dose-limiting toxicities (DLT), and safety of vorinostat combined with (R)ICE (V-(R)ICE) in patients with relapsed or refractory lymphomas. Secondary endpoints included efficacy, the ability to collect peripheral blood stem cells (PBSC) following this regimen, pharmacokinetic (PK) analyses, and in vitro correlates based on patient-derived PK data. To minimize the numbers of patients treated at low, potentially ineffective vorinostat doses, we chose a novel trial design to allocate patients using a two-stage dose escalation schedule (Storer, 2001). This design also allowed us to estimate MTD using different parameters, not possible for trials using the 3 + 3 design or the continual reassessment method (CRM). Herein, we report the results of this study.

Methods

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure
  8. References
  9. Supporting Information

Patient eligibility

Patients were eligible if they had histologically confirmed relapsed or primary refractory lymphomas, or untreated T-cell non-Hodgkin lymphoma (NHL) or mantle cell lymphoma (MCL). Patients were required to have measurable disease, age ≥18 years, an Eastern Cooperative Oncology Group (ECOG) performance status score ≤2, absolute neutrophil count (ANC) ≥1·5 × 109/l, platelet count ≥100 × 109/l, serum creatinine ≤132·6 μmol/l or creatinine clearance ≥60 ml/min, total bilirubin ≤1·5 times upper limit of normal (ULN), aspartate transaminase <5 times ULN.

Patients were excluded if they had human immunodeficiency virus or active Hepatitis B virus infection, active central nervous system disease, were pregnant or nursing, had prior malignancies within 5 years, had lymphoma refractory to a regimen containing carboplatin, cisplatin, ifosfamide, or etoposide, had prior treatment with HDAC inhibitors, active infection, active cardiac disease, a left ventricular ejection fraction <50%, autologous or allogeneic transplantation within 12 months, or radioimmunotherapy within 6 months. All patients provided informed consent. The institutional review board at each participating institution approved this trial. It was registered at ClinicalTrials.gov (NCT00601718).

Study design, treatment and dose escalation

This phase I, open-label, multicentre, dose escalation trial was administered by the Puget Sound Oncology Consortium. Vorinostat was supplied by Merck & Co., Inc. (Whitehouse Station, NJ, USA). The treatment schema is depicted in Fig 1. Each 21-day cycle consisted of vorinostat given orally at a dose ranging from 400 mg daily to 700 mg twice daily (BID) on days 1–5, ifosfamide at 5 g/m2 intravenously (iv) by 24-h continuous infusion with MESNA 5 g/m2 on day 4, carboplatin dosed at an area under the curve of 5 iv over 1 h on day 4, and etoposide 100 mg/m2 iv over 1 h on days 3–5 (Kewalramani et al, 2004). Patients with CD20 +  disease also received rituximab 375 mg/m2 on any day during days 3, 4 or 5. No intrapatient dose modification of vorinostat was allowed. Standard dose adjustments for ICE during treatment were allowed based on changes in hepatic and renal function. Filgrastim at ≥5 μg/kg/d until neutrophil recovery, or pegfilgrastim 6 mg × 1 given 24–72 h after chemotherapy completion was required. The second cycle was not initiated until the ANC was ≥ 1 × 109/l and the platelet count was ≥50 × 109/l. Therapy could be delayed for a maximum of 3 weeks. Patients received treatment for up to 2 cycles. Prophylactic management for gastrointestinal toxicities was recommended for the last 23 patients on the study (see supportive care).

image

Figure 1. Treatment Schema. Patients received vorinostat in addtion to the RICE or ICE regimen every 21 d for up to 2 cycles. Vorinostat was given daily from days 1 to 5; ifosfamide on day 4, carboplatin on day 4; etoposide given daily from day 3 to 5; rituximab was given once on either day 3, 4 or 5 to patients with CD20 +  disease. (R)ICE, (rituximab), ifosphamide, carboplatin and etoposide.

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DLTs were defined as: any related grade 4 or 5 (National Cancer Institute Common Terminology Criteria for Adverse Events version 3·0; http://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/ctcaev3.pdf) non-haematological adverse event (AE) within 28 d of the last dose of vorinostat; any related grade 3 gastrointestinal AE lasting >7 consecutive days; or failure to complete one full cycle of therapy due to toxicity. Additionally, any related serious AE that did not meet the criteria above for a DLT, could be considered a DLT at the principal investigators' discretion.

Vorinostat was escalated or de-escalated following the ‘two-stage’ design (Storer, 2001). During stage 1, cohorts of single patients were treated at each dose level starting at 400 mg daily × 5 d (level 1). Once a DLT was observed, stage 1 was complete. Stage 2 began with a cohort of 4 patients entered at one lower dose level than the final dose level of stage 1. Subsequent cohorts of 4 patients were entered with doses escalated, maintained, or de-escalated if 0, 1, or ≥2 DLTs were observed, respectively. These rules were followed until 20 patients were treated in stage 2. A dose-toxicity curve was generated to estimate the MTD/phase II dose. The MTD/phase II dose was pre-specified as the dose of vorinostat that could be combined with (R)ICE chemotherapy yielding a DLT rate of ≤25%.

Supportive care

The following prophylactic management of gastrointestinal (GI) toxicities, including diarrhoea, nausea and vomiting, was recommended:

Diarrhoea prophylaxis: Lomotil® one tablet (atropine 0·025 mg/diphenoxylate 2·5 mg) or loperamide 2 mg by mouth twice daily before each dose of vorinostat and up to 2 d after the completion of vorinostat. Lomotil® or loperamide were held if there was no bowel movement within 48 h, and could be resumed with next bowel movement. If patient developed loose stools or diarrhoea while receiving vorinostat and/or prophylactic Lomotil®/loperamide, either drug could be used every 3 to 4 h as needed. The maximal dose for each drug should not exceed 8 tablets per day.

Nausea/vomiting prophylaxis: (i) Ondansetron 8 mg PO before each dose of vorinostat on days 1, 2 and 3, and every 8 h as needed; (ii) Palonosetron 0·25 mg iv prior to the first dose of vorinostat on day 4; (iii) Aprepitant 125 mg by mouth prior to the first dose of vorinostat on day 4 followed by 80 mg by mouth daily on days 5 and 6; 4) Dexamethasone 4 mg by mouth twice a day on days 5, 6, and 7.

Patient evaluation and response assessments

All patients were evaluated before and after treatment with a history and physical examination, laboratory tests including complete blood count, comprehensive metabolic panel, lactate dehydrogenase, and diagnostic computed tomography. Bone marrow biopsy was performed at baseline and post-treatment if the baseline study showed disease involvement. Responses were determined according to the International Workshop NHL criteria (Cheson et al, 1999).

Pharmacokinetic analyses and correlative studies

Blood samples were collected within 6 h after the fifth dose of vorinostat. The plasma vorinostat concentrations were quantified using a validated liquid-chromatography-tandem-mass-spectrometry assay in the laboratory of Dr Merrill J. Egorin at the University of Pittsburgh (Parise et al, 2006).

Human lymphoma cell lines Granta 519, Pfeiffer, and Karpas 299 (American Type Culture Collection, Rockville, MD, USA) were incubated at a concentration of 0·5 × 106 cells/ml in complete media containing 0, 1·5, or 5 μmol/l vorinostat (ChemieTek, Indianapolis, IN, USA) at 37°C with 5% CO2. Cells were prepared 24 h later for cytotoxicity, apoptosis, and immunoblotting.

Cytotoxicity was determined using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay as described previously (Hansen et al, 1989). Briefly, treated cells were seeded at a concentration of 5 × 105 per ml into a flat-bottomed 96-well plate and Thyazolyl Blue Tetrazolium Bromide (Sigma Aldrich St. Louis, MO, USA) was added to each well at a final concentration of 1 mg/ml. After a 2 h incubation (37°C, 5% CO2) lysis buffer containing 50% dimethylformamide, 20% sodium dodecyl sulfate (SDS), 2·5% 1N HCl and 2·5% 80% acetic acid at pH 4·7 was added and the wells were mixed to dissolve the formazin crystals. The plate was read in a BioTek powerwave XS plate reader at 570 nm using Gen5 software (Winooski, VT, USA).

Apoptosis was measured using the fluorescein isothiocyanate (FITC) Annexin V Apoptosis kit (BD Pharmingen, San Jose, CA, USA) according to the directions provided with the kit. Briefly, 500 μl cells were placed in 1·5 ml centrifuge tubes. The cells were washed twice with phosphate-buffered saline (PBS) and re-suspended in 100 μl 1X binding buffer containing 5% annexin V-FITC and 5% propidium iodide. After a 15-min incubation, 250 μl 1X binding buffer was added and the samples were analysed on the guava minicyte flow cytometer.

Immunoblot analyses were performed as previously described (Menzies & Kenoyer, 2006) with the expression of β-actin (ACTB) used as a loading control. Antibodies raised against HDAC4, HDAC3, Acetyl-Histone H4 (Lys 12), Acetyl-Histone H3 (Lys 18), Acetyl-Histone H3 (Lys 9), and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-acetyl-Histone H4 (Lys 16) was purchased from Millipore (Billerica, MA, USA).Briefly, cells were washed 2 times in cold PBS and lysed for 30 min on ice in a buffer containing 50 nmol/l Tris HCl (pH 7·4), 1% Nonidet P40, 0·25% Sodium deoxycholate, 150 mmol/l NaCl, 1 mmol/l EDTA, 1 mmol/l phenylmethylsufonyl fluoride, 1 mmol/l sodium fluoride, 1% protease inhibitor cocktail (Sigma Aldrich, St Louis, MO, USA), and 1% Halt Phosphatase (Thermo Scientific, Waltham, MA, USA). At the end of the incubation the cells were centrifuged for 10 min at 20,000 g, 4°C. The supernatant was assayed for total protein using the bicinchoninic acid assay and 35–50 μg protein was loaded onto a 12% SDS gel and run for 2 h at 100v. The gels were transferred to nitrocellulose and proteins were detected using antibodies listed above. SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) was used to expose CL-Xposure film (Thermo Scientific) which was processed on a Kodak X-OMAT 2000 Processor. ImageJ was used to analyse the data.

Results

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure
  8. References
  9. Supporting Information

Patient characteristics

A total of 29 patients were enrolled between April 2008 and May 2010. Their baseline characteristics are summarized in Table 1. The median age was 56 years; eleven patients (38%) were aged 60 years or older. Lymphoma histologies included: Hodgkin lymphoma (HL) in 8 patients, DLBCL in 7, MCL in 5, T-NHL in 4, follicular lymphoma (FL) in three, marginal zone lymphoma in one, and chronic lymphocytic leukaemia in one. The median number of prior regimens received was 2 (range 0–7). Thirteen (45%) were relapsed and 14 (48%) were chemoresistant with less than a partial response to their last therapy. All 17 B-NHL patients had received prior rituximab, and 2 patients with HL (7%) had received prior autologous transplantation.

Table 1. Patient characteristics
CharacteristicNo. of patients%
  1. B-NHL, B cell non-Hodgkin lymphoma; ASCT, autologous stem cell transplant.

Median age, years (range)56 (23–69) 
Sex
Male2276
Female724
Race/ethnicity  
White, non-Hispanic2793
Native American14
Asian14
Disease pathology
Hodgkin lymphoma828
Diffuse large B cell lymphoma724
Mantle cell lymphoma518
T cell lymphoma414
Follicular lymphoma311
Marginal Zone lymphoma14
Chronic lymphocytic leukaemia14
Stage at treatment
III414
IV2481
Median number of prior therapies (range)2 (0–7) 
No prior chemotherapy27
Chemoresistant1448
Relapsed1345
Prior rituximab (B-NHL only)17100
Prior ASCT27

Treatment, dose escalation, and MTD determination

Fifteen patients completed both cycles of therapy, while 14 did not proceed to a second cycle due to development of a DLT (n = 8), physician or patient choice (n = 4), insurance denial of coverage for the clinical trial (n = 1), or progressive disease (n = 1). Fig 2 illustrates the progress of the two-stage dose escalation schedule. Nine patients were treated in stage 1 and 20 patients were treated in stage 2. The vorinostat dose was escalated in combination with standard (R)ICE from dose level 1 (400 mg daily) to level 6 (700 mg BID) in stage 1 until a DLT was observed and then modulated in cohorts of 4 in stage 2 yielding the results described in Fig 2 and Table SI. The resultant dose-toxicity curve and the pre-specified DLT rate of ≤.025 estimated the MTD/phase II dose to be 500 mg BID × 5 d (Fig 3A). This model also allowed us to estimate a weight-based MTD of 5·5 mg/kg BID × 5 d and a body surface area (BSA)-based MTD of 248 mg/m2 BID × 5 d with the identical DLT rate (Fig 3B,C).

image

Figure 2. Progress of the two-stage dose escalation schedule. V, vorinostat; pt, patient. DLT, dose-limiting toxicity.

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image

Figure 3. Maximal tolerated dose determined by a flat (A) versus a body surface area-based (B) or a weight-based (C) dosing schedule. DLT, dose-limiting toxicity.

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Safety and toxicity

There were no treatment-related deaths. Three patients experienced DLTs at dose level 4, including one grade 4 infection, one grade 4 pulmonary embolism, and one episode of prolonged cytopenia. Five patients experienced DLTs at dose level 5: two grade 3 elevations of transaminases which led to withholding the last vorinostat dose, one grade 4 infection; one grade 3 anorexia lasting >7 d; and one grade 4 hypokalaemia. One patient experienced grade 4 hypokalaemia at dose level 6. Table SI summarizes the DLTs observed.

Twenty-five patients experienced at least one ≥ grade 3 non-haematological treatment-related adverse event with the most common being hypophosphataemia (40%). These events are summarized in Table 2. Nine patients experienced grade 3 treatment-related GI toxicities including nausea, vomiting, dehydration, diarrhoea, anorexia, and haemorrhoids. A GI prophylaxis regimen was recommended to the treating physicians of the last 23 patients of which 21 partially or fully received the treatment. The incidence of grade 3 GI toxicity was reduced by 50% (P = 0·2), from 50% (4 of 8) of those who did not receive prophylaxis to 24% (5 of 21) of those who received prophylaxis. At the MTD, 8 of 11 patients had grade 3 non-haematological treatment-related adverse events, the most common being hypophosphataemia (n = 3, 27%), febrile neutropenia (n = 3, 27%), and infection (n = 3, 27%); two of 11 patients had treatment-related grade 3 GI toxicity, one of them did not receive GI prophylaxis.

Table 2. Incidence of CTC Grades 3 and above treatment-related toxicities in all patients and patients treated at MTD
Adverse event (grade ≥3)All patients = 29 n (%)Patients treated at MTD (= 11) n (%)
  1. CTC, Common Terminology Criteria for Adverse Events, Version 3; MTD, maximal tolerated dose; GI, gastrointestinal; AST, aspartate transaminase; ALT, alanine transaminase; DVT, deep vein thrombosis; INR, International Normalized Ratio; PTT, partial thromboplastin time.

  2. a

    Including one grade 4 adverse event.

  3. b

    Including two grade 4 adverse event.

Febrile neutropenia8 (28%)a3 (27%)a
Infection8 (28%)b3 (27%)a
GI
Nausea6 (21%)2 (18%)
Dehydration5 (17%)1 (9%)
Anorexia3 (10%)1 (9%)
Vomiting2 (7%)0 (0%)
Haemorrhoids1 (3%)0 (0%)
Hypophasphataemia12 (41%)3 (27%)
Hypokalaemia10 (34%)b1 (9%)
Hyponatraemia4 (14%)2 (18%)
Hypocalaemia3 (10%)1 (9%)
Hypercalaemia1 (3%)0 (0%)
Hyperkalaemia1 (3%)0 (0%)
AST/ALT4 (14%)0 (0%)
Elevated Alkaline Phosphatase1 (3%)0 (0%)
Hypoalbuminaemia1 (3%)0 (0%)
Hyperurecaemia1 (3%)1 (9%)
DVT2 (7%)1 (9%)
Elevated INR1 (3%)1 (9%)
Elevated PTT3 (10%)2 (18%)
Elevated D-dimer1 (3%)1 (9%)
Pain4 (14%)2 (18%)
Fatigue3 (10%)1 (9%)
Syncope1 (3%)0 (0%)
Hallucination1 (3%)0 (0%)
Encephalopathy1 (3%)0 (0%)
Confusion1 (3%)0 (0%)
Delirium1 (3%)0 (0%)
Hypotension1 (3%)0 (0%)
Hypoxia2 (7%)1 (9%)
Dyspnea1 (3%)1 (9%)
Acute renal failure1 (3%)1 (9%)
Ulceration1 (3%)0 (0%)

Response and peripheral blood stem cell mobilization

Twenty-nine patients received study treatment and 27 were evaluable for response assessment. Two patients did not undergo post-therapy staging. Responses were observed in 19 patients (71%) with 8 (30%) attaining a complete response/unconfirmed complete response (CR/Cru). Responses by histology included: HL 7/8 (88%), DLBCL 4/6 (67%), MCL 3/5 (60%), FL 3/3 (100%), and T-cell lymphoma 1/3 (33%). Responses were seen in 11/13 (85%) with chemosensitive disease, and 8/14 (57%) with chemoresistant disease. Overall, 26 of 27 patients experienced tumour reduction following this regimen (Fig 4).

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Figure 4. Waterfall plot of best response after therapy (n = 27) by histology and chemoresistance. Two of the treated patients (n = 29) are not included due to lack of proper restaging study immediately after therapy. HL, Hodgkin lymphoma; DLBCL, diffuse large B cell lymphoma; MCL, mantle cell lymphoma; FL, follicular lymphoma; MZL, marginal zone lymphoma; CLL, chronic lymphocytic leukaemia; T-NHL, T-cell non-Hodgkin lymphoma.

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Since many of these patients were destined to proceed to autologous haematopoietic transplantation, we also evaluated the ability to mobilize and collect PBSC after receiving treatment on this study. Mobilization of PBSC was ultimately successful in 20 of 21 patients who attempted this procedure following V-(R)ICE, including 4 who were successfully collected immediately following V-(R)ICE (median 5·52 × 106 CD34/kg), 4 who failed an initial attempt but underwent a second successful mobilization (median 4·38 × 106 CD34/kg), and 12 who did not attempt collection after V-(R)ICE and underwent successful PBSC mobilization and collection after a subsequent regimen (median 7·54 × 106 CD34/kg). Successful mobilization and collection of PBSC after this regimen did not appear to correlate with patient age, gender, number of prior therapies, vorinostat dose, or pretreatment bone marrow involvement.

Vorinostat inhibits lymphoma cell growth in a dose-dependent manner

We measured plasma levels of vorinostat after the fifth oral dose to determine whether the pulse-dosing schedule used in V-(R)ICE could achieve the target serum vorinostat concentration (≥2·5 μmol/l) determined to be required for synergism with platinum and etoposide in vitro (Marchion et al, 2004; Rikiishi et al, 2007). Serum samples from three patients were available for this purpose. The vorinostat concentrations at this time point were 4·1 μmol/l in Patient 1, who had DLBCL and was treated with vorinostat 600 mg BID, 4·5 μmol/l in Patient 2, who had MZL and was treated with vorinostat at the MTD level, and 6·0 μmol/l in Patient 3, who had MCL and was treated at the MTD level These results support that our pulse-dose schedule was able to achieve target levels at the time of introducing cytotoxic agents.

We treated Granta 519 (an MCL line), Peiffer (a DLBCL line), and Karpas 299, (an anaplastic large cell lymphoma line) with increasing concentrations of vorinostat to determine if the vorinostat levels we detected in patients could induce a dose-dependent cell growth inhibition. Fig 5A shows that vorinostat significantly inhibited the growth of all three cell lines, as determined by MTT assays, with maximal inhibition observed in cells treated at a concentration of 5 μmol/l. We also observed a corresponding dose-dependent increase in apoptosis in all three cell lines as determined by Annexin V labeling (Fig 5B). The concentration-dependent growth inhibition also correlated with the level of acetylated-H3 and -H4 proteins detected in these cultures (Fig 5C), corroborating the predicted dose-dependent anti-tumour activity of vorinostat and the potential importance of high-dose vorinostat administration.

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Figure 5. (A). Vorinostat exhibited dose-dependent cytotoxicity of lymphoma cell lines. All cells were treated with vorinostat for 24 h. MTT assay was used to determine the cytotoxicity in treated cells. Representative results from three independent experiments are shown. Granta519 is a mantle cell lymphoma cell line; Peiffer, a diffuse large B cell lymphoma line; Karpas 299, an anaplastic large cell lymphoma line. (B) Dose-dependent apoptotic response of lymphoma cell lines after 24 h treatment with vorinostat. The response was measured by Annexin V assay. (C) Western blotting revealed dose-dependent accumulation of acetylated histones H3 and H4 in lymphoma cells after 24 h treatment with vorinostat. β-actin level was used as loading control.

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Discussion

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure
  8. References
  9. Supporting Information

This phase I trial tested the hypothesis that high-dose pulse vorinostat can be safely combined with the (R)ICE regimen in patients with relapsed lymphoma and determined the pre-specified primary endpoint of the MTD/phase II dose. We also examined the safety, pharmacokinetics, in vitro anti-tumour effects, stem cell mobilization potential, and potential efficacy of this regimen in this heavily pretreated patient population.

We used a novel two-stage dose escalation schedule to conduct this study. Such a design has the advantage of minimizing the numbers of patients treated at a potentially sub-therapeutic dose when compared to the traditional ‘3 + 3’ design (Storer, 2001). This design also has the flexibility to estimate the MTD/phase II dose in different modalities (Storer, 2001). In our study, the MTD/phase II dose for vorinostat administrated daily for 5 consecutive days of a 21-day cycle in combination with (R)ICE regimens was determined to be 500 mg BID as a flat dose. Interestingly, five of the nine patients experiencing DLTs had a BSA less than the mean of 1·73 m2. Four of these five patients also experienced non-DLT grade 3 GI toxicities, whereas, only four of the 15 patients with a BSA above average experienced grade 3 GI toxicities. Our phase I model allowed us to estimate the MTD based on BSA or weight, which may be more appropriate for selected patients and, potentially, a paediatric population (Fig 3). Toxicity and pharmacokinetic data from future trials using a weight- or BSA-based dosing schema will be required to confirm that toxicities are reduced and that therapeutic concentrations are maintained in smaller patients. Such a strategy is also attractive for the paediatric use of high-dose vorinostat.

The overall toxicity profile of this regimen was comparable to (R)ICE with the exception of more frequent GI toxicity and associated electrolyte abnormalities. Studies of CTCL patients receiving 400 mg of vorinostat daily reported a 9·4% incidence of severe GI AEs among 86 patients. Similarly, 12 of 23 patients (52%) with haematological malignancies receiving vorinostat doses ranging from 400 to 800 mg daily encountered severe GI toxicities (Rubin et al, 2006). In contrast, only two grade 3 or 4 AEs of nausea/vomiting were reported in 36 patients who received RICE (Kewalramani et al, 2004) and none were reported in 163 patients who received ICE (Moskowitz et al, 1999). Therefore, the observed increased rate of severe GI toxicities in our trial was anticipated and probably related to vorinostat use. Two adverse factors associated with the occurrence of these severe GI toxicities are receiving vorinostat dose above the MTD level, and lack of GI prophylaxis. Among the 9 patients who experienced treatment-related severe GI toxicities, 4 patients were treated above the MTD level, and 4 of the remaining 5 patients did not receive preemptive GI prophylaxis. In contrast, only one of the 9 patients who were treated at MTD and received GI prophylaxis experienced severe treatment-related GI toxicities. Hence, it appeared that aggressive GI prophylaxis along with frequent metabolic monitoring and prompt electrolyte repletion could reduce the incidence of these AEs substantially, though confirmation will be required in pending studies at the phase II dose.

We also evaluated the ability to mobilize PBSC following this regimen. Unlike the reported 86% collection rate after three cycles of ICE (Moskowitz et al, 1999), 4 of 9 patients (44%) successfully mobilized and collected PBSC immediately after V-(R)ICE treatment. Even though our patients attempting mobilization had received more prior regimens than the original ICE data (2 vs. 1), these observations may suggest a negative impact of high-dose vorinostat on stem cell mobilization. However, this effect appeared reversible, as all patients collected PBSC successfully after a second attempt. The exact molecular mechanism of our observation is unclear. Other HDAC inhibitors have been shown to promote CD34 + stem cell homing by up-regulating CXCR4 mRNA and its encoded protein expression levels (Gul et al, 2009). It is well known that down regulation of CXCR4 plays a crucial step in stem cell mobilization. It is plausible that high-dose vorinostat might temporarily retard mobilization by strengthening the interaction between stem cells and the bone marrow microenvironment. Therefore, one may consider adding plerixafor, a direct CXCR4 antagonist, or using an alternate chemotherapy-based mobilization regimen for stem cell collection in future trials using high-dose vorinostat.

A plasma vorinostat concentration ≥2·5 μmol/l was required for its synergism with platinum and etoposide in vitro (Marchion et al, 2004; Rikiishi et al, 2007).It has been well documented that the median time to maximal plasma concentration of vorinostat after oral administration ranges from 0·5 to 4·2 h, with a serum half life of 1·4 h after either single or multiple-dosing absorption (Kelly et al, 2005; Rubin et al, 2006; Ramalingam et al, 2007). Our in vitro correlates were based on study-derived pharmacokinetic data that vorinostat concentrations of c. 5 μmol/l prior to the introduction of (R)ICE regimen were readily attained. We observed dose-dependent acetylation of histone proteins and cytotoxicity in all three lymphoma cell lines tested (Fig 5). Although the sample size was small, these data support the notion that the pulse dose schedule used in the V-(R)ICE regimen is able to achieve the target plasma vorinostat level (≥2·5 μmol/l). Furthermore, a recent study (Dickson et al, 2011) found that maximal vorinostat concentration ≥2·5 μmol/l was achieved in 86% of patients taking 800 mg/d of vorinostat for three consecutive days, a slightly lower dose than the three patients who had peak vorinostat plasma level measured on our study. Hence, with these and our data, we expect that the majority of patients receiving 500 mg BID would have a target plasma vorinostat level ≥2·5 μmol/l. Future pharmacokinetic study with a larger sample size will address this postulation.

The long-term goal of this project is to set the stage for developing a more effective salvage regimen for aggressive lymphoma. This is particularly relevant in the post-rituximab era, wherein the response rates and outcomes following relapse are poorer. For example, in the CORAL study, the overall response rate (ORR) of DLBCL after 3 cycles of RICE or R-DHAP treatment was only 51% in patients who had prior rituximab exposure as compared to 83% in those who were rituximab-naïve (Gisselbrecht et al, 2010). The patients in our study were more heavily treated, with 16 (56%) patients with at least three prior therapies and 11 (38%) with at least two prior therapies, as compared to patients in the CORAL study with only 1 prior therapy. All 17 B-NHL patients had prior rituximab treatment and 7 (41%) had chemoresistant disease. Although patients only received up to 2 cycles of V-(R)ICE, we observed encouragingly high responses with 71% ORR, and 30% CR/CRu. Importantly, the response rate was 57% for patients with chemoresistant disease, and 67% for patients with DLBCL. Among the 6 evaluable patients with DLBCL, 2 of 3 with chemoresistant disease responded. We also observed high antitumour activity (88% ORR) in patients with HL. Among the responders, two patients had prior autologous transplant and had chemoresistant disease. This suggests that addition of pulse high-dose vorinostat to the (R)ICE regimen was associated with a preliminary high antitumour activity in vivo, and may be promising in overcoming the resistance to chemotherapy or rituximab. This study provides clinical support for further exploration of the role of HDAC inhibition in combination with intensive chemotherapy for improving outcomes for patients with relapsed or refractory lymphomas.

In conclusion, this prospective, multi-centre trial suggests that addition of high-dose vorinostat to a combination therapy of (R)ICE in patients with relapsed or refractory lymphomas was safe, feasible and associated with encouraging response rates, although adequate prophylaxis and prompt treatment of GI toxicity were required. These findings along with our correlative data support the potential benefit of vorinostat at plasma levels ≥ 2·5 μmol/l in combination with platinum and etoposide in vivo and warrant further study.

Acknowledgements

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure
  8. References
  9. Supporting Information

L.E.B, M.M.Z., T.A.G and A.K.G designed the clinical trial and associated experiments; L.E.B., M.M.Z., A.R.S., J.M.P., T.L.C., T.A.W., T.E.B., O.W.P. and A.K.G enrolled patients on study and took care of patients; L.E.B and A.K.G. wrote the manuscript; all authors interpreted data. Special thanks are given to the late Dr Merrill J. Egroin from University of Pittsburgh for his contribution to the vorinostat PK analysis. Appreciation also goes to Tina Bogne, and the patients who participated in this study.

This work was supported by Merck Sharp & Dohme Corp USA, the Washington State Life Sciences Discovery Fund, an NIH PO1 grant (CA44991), a grant from the Lymphoma Research Foundation Mantle Cell Lymphoma Initiative, a SCOR grant (7040) from the Leukemia and Lymphoma Society, the Mary A. Wright Memorial Research Fund, a donation from Frank and Betty Vandermeer. L.E.B is a Special Fellow in Clinical Research of the Leukemia and Lymphoma Society. A.K.G is a Scholar in Clinical Research of the Leukemia and Lymphoma Society.

Disclosure

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure
  8. References
  9. Supporting Information

L.E.B. and A.K.G received funding support from Merck Sharp & Dohme Corp USA. A.K.G. is a member of the speaker bureau for Seattle Genentics and Millenium Pharmaceuticals. All remaining authors have declared no conflicts of interest.

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  6. Acknowledgements
  7. Disclosure
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Disclosure
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
bjh12230-sup-0001-TableS1.docWord document34KTable SI. Summary of dose-limiting toxicities and responses at each dose level

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