Leukemia (2007) 21, 524–528 & 2007 Nature Publishing Group All rights reserved 0887-6924/07 $30.00 www.nature.com/leu ORIGINAL ARTICLE Sequence-dependent synergy of the proteasome inhibitor bortezomib and cytarabine in mantle cell lymphoma O Weigert1,2,3, A Pastore1,2,3, M Rieken2, N Lang1, W Hiddemann1 and M Dreyling1,2 1Department of Internal Medicine III, University of Munich, University Hospital Grosshadern, Munich, Germany and 2GSF-National Research Center for Environment and Health, Haematologicum, Munich, Germany Single-agent bortezomib, a potent, selective and reversible consisted of CHOP (cyclophosphamide, vincristine, doxorubi- inhibitor of the 26S proteasome, has demonstrated clinical cin and prednisone),12 R-FCM (rituximab, fludarabine, cyclo- efficacy in relapsed and refractory mantle cell lymphoma (MCL). 13 Objective response is achieved in up to 45% of the patients; phosphamide, and mitoxantrone) and gemcitabine/oxaliplatin however, complete remission rates are low and duration of with all relapses occurring within 1 year. No response was seen 90 response proved to be short. These limitations may be after radioimmunotherapy with rituximab/ Y-ibritumomab– overcome by combining proteasome inhibition with conven- tiuxetan 5 months earlier. Salvage therapy was started with tional chemotherapy. Here we present two case reports and in single-agent bortezomib 1.3 mg/m2 given intravenously twice vitro data suggesting synergistic efficacy of bortezomib weekly for 2 consecutive weeks, followed by a 1-week rest combined with cytarabine in MCL. Interestingly, efficacy in vitro correlated with sequence of treatment, indicating that period. Because of progressive lymphadenopathy at week 5, pretreatment with cytarabine, followed by proteasome inhibi- treatment was changed to a combination regimen consisting 2 tion, may be the preferred approach. of bortezomib (1.5 mg/m , days 1 and 4) and cytarabine Leukemia (2007) 21, 524–528. doi:10.1038/sj.leu.2404511; (1000 mg/m2 as a 3-h infusion on days 2 and 3) applied in published online 1 February 2007 3-week intervals. After two cycles, the leg edema and Keywords: non-Hodgkin’s lymphoma; mantle cell lymphoma; lymphadenopathy improved dramatically, and a very good bortezomib; cytarabine; combination treatment partial response was confirmed by computer tomography (CT) scanning (Figure 1a and b). Because of NCI/CTC grade 4 neutropenia after cycle 2, the dose of cytarabine was reduced to 75% and granulocyte-colony-stimulating factor (G-CSF) support was initiated. Importantly, there was no infectious complication Introduction and only cycle 4 had to be delayed for 1 week. Because of NCI/ CTC grade 3 sensory peripheral neuropathy after cycle 2, The ubiquitin–proteasome pathway is essential for maintaining bortezomib was omitted during the subsequent two cycles with intracellular protein homeostasis and represents a valid target for polyneuropathy gradually improving; the hematologic toxicity the treatment of malignant disease.1 Various oncogenes and did not differ significantly after bortezomib was held. Except for regulatory proteins for cell cycle progression and apoptosis are alopecia, there was no additional significant non-hematological processed by this pathway.2 Bortezomib is a potent, selective toxicity. Meanwhile, 11 months (343 days) after initiation of and reversible inhibitor of the proteasome with demonstrated combination therapy, the patient suffered from systemic progres- efficacy in relapsed multiple myeloma.3,4 Single-agent bortezo- sion and was started on a bendamustin-containing regimen. mib demonstrated clinical activity in several other hematologic malignancies5 with especially encouraging results in relapsed or 6–8 refractory mantle cell lymphoma (MCL). Objective response Case report 2 is achieved in 29–45% of MCL patients; however, complete A 63-year-old woman with relapsed stage IV MCL presented remission rates are low and median duration of response short. with progressive abdominal lymphadenopathy and reappear- These limitations may be overcome by combining bortezomib ance of extensive watery diarrhea, attributable to exacerbation 9,10 with conventional chemotherapy. Feasibility has been of colonic infiltration, confirmed by colonoscopy after 10 cycles 11 demonstrated for liposomal doxorubicin. We present two of single-agent bortezomib therapy (1.3 mg/m2 intravenous, case reports and in vitro data suggesting a sequence-dependent days 1, 4, 8, 11, every 3 weeks). Four years earlier, the disease synergy of bortezomib and cytarabine in MCL. was diagnosed with colonic polyposis and infiltration of the stomach, the nasopharynx, the right lacrimal gland and bone marrow involvement. Previous treatment consisted of CHOP Case report 1 followed by interferon maintenance,12 R-FCM13 and radio- A 67-year-old man with relapsed stage IV MCL presented with immunotherapy with rituximab/90Y-ibritumomab–tiuxetan. The progressive bilateral leg edema caused by extensive inguinal, best response consisted of partial remission, and duration of iliac and paraaortic lymphadenopathy. Previous treatment response declined from initially observed 21 months with every consecutive line of treatment. Single-agent bortezomib treat- Correspondence: Professor M Dreyling, Department of Medicine III, ment resulted in a decreased frequency of diarrhea by cycle 4, University Hospital Grosshadern, Marchioninistrasse 15, D-81377 which however, failed by cycle 10. Treatment was changed to Munich, Germany. the combination regimen, consisting of bortezomib (1.5 mg/m2, E-mail: [email protected] 2 3These authors contributed equally to this work days 1 and 4) and cytarabine (1000 mg/m as a 3-h infusion on Received 21 April 2006; revised 3 October 2006; accepted 11 days 2 and 3), applied in 4-week intervals with rituximab added October 2006; published online 1 February 2007 on day 0 (375 mg/m2). Because of NCI/CTC grade 1 sensory Synergy of bortezomib and cytarabine in MCL O Weigert et al 525 Proliferation assay Cells were seeded at a density of 5 Â 105/ml in the absence or presence of bortezomib (25 nM). Cells were counted in a hematocytometer, and viability of cells was assessed by Trypan blue exclusion test (Gibco, Invitrogen, Karlsruhe, Germany) at 0, 24, 48 and 96 h of treatment. Apoptosis assay Cells were washed and stained with Annexin V-PE/7–AAD in accordance with the manufacturer’s protocol (BD Biosciences, Heidelberg, Germany) and subjected to flow cytometry (BD FACS Calibur, BD Biosciences, Palo Alto, CA, USA). Apoptosis rate was calculated as follows: 0fraction viable treated cells0 1 À Â100%: 0fraction viable untreated cells0 Spontaneous apoptosis rate was up to 10% for established MCL cell lines and up to 25% for MCL cells derived from patient samples. Figure 1 Clinical efficacy of bortezomib and cytarabine in MCL. CT Cell viability assay and determination of Chou and scans before (a and c) and after two cycles (b and d) of combination Talalay’s combination index treatment with cytarabine and bortezomib demonstrating significant Cell viability correlates with the activity to metabolize the decrease of lymphadenopathy (arrows). tetrazolium salt WST-1 to a water-soluble formazan dye, which is measured spectrophotometrically. Cells were seeded at a density of 1 Â 106/ml in a 96-well microplate in triplicate (100 000 cells/well) and the assay was performed according to peripheral neuropathy, the dose of bortezomib was reduced to the manufacturer’s protocol (Roche Applied Science, Man- 1.0 mg/m2 during cycles 2–4. The diarrhea improved rapidly nheim, Germany). The half maximal inhibitory concentration and disappeared subsequently. After two and four cycles, a (IC ) was used for calculation of Chou and Talalay’s combina- complete disappearance of pathological lymphadenopathy was 50 tion index (CI ) applying CalcuSyn Software (Biosoft, Cam- confirmed by CT scanning (Figure 1c and d). Endoscopically, the 50 bridge, UK) as described previously.14 This index allows polypoid lesions decreased substantially however, residual MCL identification of antagonistic (CI 41.3), additive (CI ¼ infiltration was seen on biopsy specimen. A partial remission 50 50 170.3) or synergistic (CI 0.7) efficacy of combination was diagnosed and no further treatment was applied to the 50 o treatment by considering cell viability curves determined after asymptomatic patient. NCI/CTC grade 3/4 toxicity consisted 12 and 24 h of treatment with cytarabine alone, bortezomib mainly of myelotoxicity, with neutropenia requiring G-CSF alone or in combination (synchronous and sequential incuba- support and one episode of neutropenic fever requiring empiric tion) of both, respectively. antibiotic treatment. There was no treatment delay and no dose reduction of cytarabine. Meanwhile, the patient has remained in documented ongoing remission for more than 7 months (233 Statistical analysis days) after initiation of combination treatment with no disease- A two-sided Student’s t-test was applied when indicated. related symptoms. Results Materials and methods At clinically achievable concentrations, bortezomib (25 nM) Cell culture and reagents inhibited cell growth in all cell lines in a time-dependent Established MCL cell lines consisted of Granta519, HBL-2 and manner, with MCL demonstrated to be more sensitive than the Jeko-1; the T-ALL cell line Jurkat was used as hematologic hematological control cell line Jurkat (Figure 2a, Po0.01 for control cell line; all established cell lines were obtained from MCL vs non-MCL cell lines at 48 and