Letters to the Editor 973 Medical School) for providing modified K562 cell line cells and Nahid Kordelas for 3 Baron F, Storb R. Mensenchymal stromal cells: a new tool against graft-versus- proofreading. host disease? Biol Blood Marrow Transplant 2011; 18: 822–840. 4 Lai RC, Arslan F, Lee MM, Sze NS, Choo A, Chen TS et al. Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury. Stem Cell Res 2010; 4: AUTHOR CONTRIBUTIONS 214–222. PAH, DB and BG designed the study; LK and DB treated the patients; A-KL, SR 5 Ludwig AK, Giebel B. Exosomes: small vesicles participating in intercellular communication. Int J Biochem Cell Biol 2012; 44:11–15. and TRD raised cells and purified exosomes; VR, A-KL and JR characterized 6 Raposo G, Nijman HW, Stoorvogel W, Liejendekker R, Harding CV, Melief CJ et al. exosomes; VR performerd immune assays; LK, VR and BG wrote the paper; B lymphocytes secrete antigen-presenting vesicles. J Exp Med 1996; 183: All authors discussed the data and the manuscript. 1161–1172. 7 Nguyen DG, Booth A, Gould SJ, Hildreth JE. Evidence that HIV budding in primary macrophages occurs through the exosome release pathway. J Biol Chem 2003; DISCLOSURE 278: 52347–52354. A patent entitled ‘Use of preparations comprising exosomes derived from 8 Kimpton CP, Corbitt G, Morris DJ. Comparison of polyethylene glycol precipitation and ultracentrifugation for recovery of cytomegalovirus from urine prior mesenchymal stem cells (MSCs) in the prevention and therapy of inflammatory to detection of DNA by dot-blot hybridisation. J Virol Methods 1990 May; 28: conditions’ has been submitted. 141–145. 9 Kohno T, Mohan S, Goto T, Morita C, Nakano T, Hong W et al. A new improved 1,2,6 2,3,6 3 2,3 L Kordelas , V Rebmann , A-K Ludwig , S Radtke , method for the concentration of HIV-1 infective particles. J Virol Methods 2002; J Ruesing4, TR Doeppner5, M Epple4, PA Horn2,3, 106: 167–173. DW Beelen1,2 and B Giebel2,3 10 Sokolova V, Ludwig AK, Hornung S, Rotan O, Horn PA, Epple M et al. 1Department of Bone Marrow Transplantation, University Hospital, Characterisation of exosomes derived from human cells by nanoparticle tracking University of Duisburg-Essen, Essen, Germany; analysis and scanning electron microscopy. Colloids Surf B Biointerfaces 2011; 87: 2German Cancer Consortium (DKTK), Germany; 146–150. 3 11 Alegre E, Rebmann V, Lemaoult J, Rodriguez C, Horn PA, Diaz-Lagares A et al. Institute for Transfusion Medicine, University Hospital, University of In vivo identification of an HLA-G complex as ubiquitinated protein circulating in Duisburg-Essen, Essen, Germany; exosomes. Eur J Immunol 2013; 43: 1933–1939. 4 Institute of Inorganic Chemistry, University of Duisburg-Essen, 12 Wendler F, Bota-Rabassedas N, Franch-Marro X. Cancer becomes wasteful: Essen, Germany and emerging roles of exosomes in cell-fate determination. J Extracell Vesicles 2013; 2: 5Department of Neurology, University Hospital, University of 22390. Duisburg-Essen, Essen, Germany 13 Bobrie A, Colombo M, Krumeich S, Raposo G, Thery C. Diverse subpopulaions of E-mail: [email protected] or [email protected] vesicles secreted by different intracellular mechanisms are present in exosome 6Both authors share first authorship. preparations obtained by differential ultracentrifugation. J Extracell Vesicles 2012; 1. 14 Ulbrecht M, Martinozzi S, Grzeschik M, Hengel H, Ellwart JW, Pla M et al. Cutting edge: the human cytomegalovirus UL40 gene product contains a ligand REFERENCES for HLA-E and prevents NK cell-mediated lysis. J Immunol 2000; 164: 1 Deeg HJ. How I treat refractory acute GVHD. Blood 2007; 109: 4119–4126. 5019–5022. 2 Le Blanc K, Rasmusson I, Sundberg B, Gotherstrom C, Hassan M, Uzunel M et al. 15 Ringden O, Le Blanc K. Mesenchymal stem cells for treatment of acute and Treatment of severe acute graft-versus-host disease with third party haploiden- chronic graft-versus-host disease, tissue toxicity and hemorrhages. Best Pract Res tical mesenchymal stem cells. Lancet 2004; 363: 1439–1441. Clin Haematol 2011; 24: 65–72.

Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)

The combination of hyper-CVAD plus nelarabine as frontline therapy in adult T-cell acute lymphoblastic leukemia and T-lymphoblastic lymphoma: MD Anderson Cancer Center experience

Leukemia (2014) 28, 973–975; doi:10.1038/leu.2013.312 T lymphoblasts prevents DNA replication and causes apoptosis and makes it an ideal purine nucleoside analogue with T-cell specificity.5,6 Clinical studies of nelarabine in pediatric and adult patients with relapsed/refractory T-ALL/LL have confirmed a Therapeutic options in adult T-cell acute lymphoblastic leukemia substantial level of activity,7–10 andin2005theFoodandDrug and T-lymphoblastic lymphoma (T-ALL/LL) are limited.1 We have Administration (FDA) granted accelerated approval for earlier demonstrated that the hyper-CVAD regimen achieves high nelarabine in this patient population.11 One trial has shown rates of complete remission (CR) and improves clinical outcomes in that nelarabine could be safely combined with intensive patients with T-ALL/LL.2 However, B50% of patients relapse and chemotherapy (BFM-86) in the frontline therapy of pediatric further improvements of the therapeutic armamentarium are needed. T-ALL.12 We therefore studied the efficacy and safety of Nelarabine is a prodrug of 9-b-arabinofuranosylguanine nelarabine in combination with hyper-CVAD in the frontline (ara-G).3,4 Preferential accumulation of ara-G inside the therapy for adult patients with T-ALL/LL.

Accepted article preview online 25 October 2013; advance online publication, 15 November 2013

& 2014 Macmillan Publishers Limited Leukemia (2014) 935 – 979 Letters to the Editor 974 100 100

80 80

60 60

40 40 Total Fail 3 yr CRD Median Mature 4 0 100 Not reached Total Fail 3 yr CRD Median Thymic 11 2 82 Not reached

Percent remission 20 Percent remission 20 37 13 65 64 mos Early 11 6 46 14.5 mos p=ns 0 0 0 12 24 36 48 60 72 01224 36 48 60 72 Time in Months Time in Months

100 100 80 80 60 60 40 40 Total Fail 3 yr DFS Median 20 20 Mature 4 0 100 Not reached Percent disease free Total Fail 3 yr DFS Median

Percent disease free Thymic 11 2 82 Not reached 37 14 61 64 mos Early 11 7 36 14.5 mos 0 0 0 12 24 36 48 60 72 020406080 Time in Months Time in Months

100 100

80 80

60 60

40 40 Total Fail 3 yr OS Median Mature 4 0 100 Not reached Percent survival 20 Percent survival 20 Thymic 12 2 78 Not reached Total Fail 3 yr OS Median Early 13 7 45 22 mos 40 12 63 Not reached p=ns 0 0 0 12 24 36 48 60 72 0 12 24 36 48 60 72 Time in Months Time in Months Figure 1. Time-to-event analysis of patients receiving nelarabine with hyper-CVAD—(a) CRD, (b) CRD according to immunophenotype, (c) DFS curve, (d) DFS according to immunophenotype, (e) OS curve and (f) OS curve according to immunophenotype.

This study is a single-arm, phase 2 study of 40 adult patients with 17 (43%) had LL. The performance status was p2 in 36/40 (90%) T-ALL/LL who were either untreated or minimally pretreated patients; seven patients (18%) had a WBC of 4100 000 K/mlat (maximum of one induction cycle prior) and were enrolled between diagnosis. For 29 patients with available IPT values, the distribution August 2007 and June 2013. The study was approved by the according to IPT was 13 (45%), 4 (14%) and 12 (41%) for early, mature Institutional Review Board and informed consent from the and cortical types, respectively. Twenty-one patients had diploid and patients was obtained in accordance with the Declaration of 14 patients had other karyotypes at diagnosis. Fifteen patients had Helsinki. This trial is registered at ClinicalTrials.gov (NCT00501826). bulky mediastinal disease, and four had CNS involvement. All patients had a confirmed diagnosis of T-ALL/T-LL. Based on the Among the 40 patients, 5 patients were in CR at the time of immunophenotype (IPT), patients were categorized into early, initial presentation. Overall response rate (ORR) was 97% with 32 mature and cortical types.13,14 Standard criteria were used for (91%) patients in CR, which was MRD-negative by multiparameter response assessment.2 Minimal residual disease (MRD) assessment flow cytometry in 8/17 (47%) patients. CR rates were similar was done after the induction chemotherapy at the time of CR. The between T-ALL and T-LL (89% vs 94%, respectively). There were no doses and schedule of hyper-CVAD were as described earlier.2 early deaths (within 4 weeks of therapy).With a median follow-up Patients received two cycles of nelarabine at a dose of 650 mg/m2 of 30.4 months (2.4–69.2 months), 28 patients remain alive and in intravenous daily for 5 days both early (after cycle 4 and 5 of hyper- remission. Thirteen patients relapsed with a median time to CVAD) and later during the POMP (monthly vincristine, prednisone, relapse of 6.5 months (1.4–63.7 months). Site distribution of the mercaptopurine and methotrexate) maintenance (replacing cycles 13 relapses was as follows: 7 hematologic (marrow ± blood), 6 and 7 of POMP). Radiation therapy to patients with bulky 4 extramedullary (nodes, rib, mediastinum), 1 marrow þ adenopathy mediastinal disease was optional. CNS prophylaxis included eight and 1 marrow þ CNS. Probability of CRD at 3 years was 65% intrathecal treatments of methotrexate alternating with ara-C. (95%CI 46.5–78.5%) (Figure 1a). DFS at 3 years was 61% (95%CI The dose of nelarabine was adjusted based on the severity of 42.7–75.7%) with a median DFS of 64 months and 3-year neurotoxicities. Primary endpoint was achievement of CR. Toxicities probability for OS was 63% (95%CI 44–77.3%), with the median were graded according to Common Terminology Criteria for Adverse OS not yet reached. When analyzed by immunophenotype, no events (CTCAE) 3.0 criteria.15 Log-rank test and Kaplan–Meier significant differences were observed with regard to CRD, but estimate were used to estimate the time-to-event outcomes—CR there was a trend for inferior DFS (P ¼ 0.08) and OS (P ¼ 0.16) for duration (CRD), disease-free survival (DFS) and overall survival (OS). patients with early immunophenotype ALL/LL. (Figures 1b–f). Forty patients with T-ALL/LL were enrolled with a median age of When T-LL were compared with T-ALL, patients with T-LL as 38 years (19–78 years). Twenty-three patients (58%) had T-ALL and opposed to those with T-ALL showed a trend of superior CRD, DFS

Leukemia (2014) 935 – 979 & 2014 Macmillan Publishers Limited Letters to the Editor 975 and OS in (P ¼ 0.11, 0.07 and 0.23, respectively). Finally, median AUTHOR CONTRIBUTIONS CRD and proportions of relapses at last follow-up of MRD-negative versus MRD-positive patients was—not reached vs 14.5 months PJ, HK and SF designed the study. PJ, RG, MC-T, DT, SOB and SF analyzed the and 37% vs 67%, respectively. results. PJ, HK, RG, MC-T, DT, SOB and SF wrote the paper. HK, DT, ND, NP, FR, Overall, 8/40 (20%) patients completed all four intended courses MK, JB, SOB, EJ, JC, GB, TK and SF contributed the patient samples. All authors of nelarabine and 14/40 (28%) patients completed two courses of reviewed and gave the final approval for the paper. nelarabine during consolidation phase. Thirty-five (87%) patients had at least one grade X3 infection. Thrombotic episodes P Jain, H Kantarjian, F Ravandi, D Thomas, S O’Brien, T Kadia, occurred in 6 patients (17%) (4 pulmonary embolism and 2 deep J Burger, G Borthakur, N Daver, E Jabbour, M Konopleva, vein thrombosis), 3 (7%) had tachyarrhythmia (2 atrial fibrillation, J Cortes, N Pemmaraju, MA Kelly, M Cardenas-Turanzas, 1 with supraventricular tachycardia). Twenty-two (55%) patients R Garris and S Faderl developed grade p2 peripheral neuropathy, whereas nine Department of Leukemia, The University of Texas MD Anderson (22%) patients developed grade p2 other neurological toxicities Cancer Center, Houston, TX, USA (4 muscle weakness, 1 tremors, 2 memory impairment and altered E-mail: [email protected] mentation, 1 mood alteration and 1 hand paresthesias). The neurological toxicities reversed in all patients, although six patients required a dose reduction of nelarabine to 650 mg/m2 on REFERENCES alternating days (D1, 3, 5) and only one patient required dose reduction during all four courses due to vincristine-associated 1 Marks DI, Paietta EM, Moorman AV, Richards SM, Buck G, DeWald G et al. T-cell grade-3 neuropathy. Nelarabine was withheld in four patients for acute lymphoblastic leukemia in adults: clinical features, immunophenotype, cytogenetics, and outcome from the large randomized prospective trial (UKALL the following reasons: vincristine-associated neuropathy (2 patients), XII/ECOG 2993). Blood 2009; 114: 5136–5145. generalized weakness, older age and patient reluctance (1 patient), 2 Thomas DA, O’Brien S, Cortes J, Giles FJ, Faderl S, Verstovsek S et al. Outcome prolonged cytopenias and infectious complications (1 patient). with the hyper-CVAD regimens in lymphoblastic lymphoma. Blood 2004; 104: Experience with nelarabine as frontline therapy in adult T-ALL/ 1624–1630. T-LL is limited. A pediatric study (AALL00P2)12 of 70 patients with 3 Rodriguez Jr. CO, Stellrecht CM, Gandhi V. Mechanisms for T-cell selective cyto- high-risk newly diagnosed T-ALL combined nelarabine with the toxicity of arabinosylguanine. Blood 2003; 102: 1842–1848. BFM-86 regimen. This pilot study demonstrated that slow early 4 Roecker AM, Stockert A, Kisor DF. Nelarabine in the treatment of refractory T-cell responders achieved a 5-year event-free survival (EFS) of 73%, malignancies. Clin Med Insights Oncol 2010; 4: 133–141. similar to rapid early responders who did not receive nelarabine 5 Gandhi V, Plunkett W, Rodriguez Jr. CO, Nowak BJ, Du M, Ayres M et al. Compound GW506U78 in refractory hematologic malignancies: relationship between (EFS 69%, P ¼ 0.64). cellular pharmacokinetics and clinical response. J Clin Oncol 1998; 16: 3607–3615. Notable in our study is the poor outcome in patients with an 6 Kisor DF, Plunkett W, Kurtzberg J, Mitchell B, Hodge JP, Ernst T et al. Pharmaco- early IPT and T-ALL as compared with T-LL. This difference was kinetics of nelarabine and 9-beta-D-arabinofuranosyl guanine in pediatric and observed in previous studies of hyper-CVAD; patients with T-ALL adult patients during a phase I study of nelarabine for the treatment of refractory do worse and it remains to be determined in how much addition hematologic malignancies. J Clin Oncol 2000; 18: 995–1003. of nelarabine can make a difference. The IPT analysis may not be 7 Berg SL, Blaney SM, Devidas M, Lampkin TA, Murgo A, Bernstein M et al. Phase II completely reliable due to the small numbers of patients per study of nelarabine (compound 506U78) in children and young adults with group. Assessment of MRD was only done early after induction refractory T-cell malignancies: a report from the Children’s Oncology Group. J Clin and our MRD results therefore do not reflect the impact of Oncol 2005; 23: 3376–3382. 8 DeAngelo DJ, Yu D, Johnson JL, Coutre SE, Stone RM, Stopeck AT et al. Nelarabine nelarabine, which was given later. A more comprehensive induces complete remissions in adults with relapsed or refractory T-lineage acute assessment of MRD should have included serial assessments lymphoblastic leukemia or lymphoblastic lymphoma: Cancer and Leukemia before and after nelarabine. Finally, other ways of combining Group B study 19801. Blood 2007; 109: 5136–5142. nelarabine with hyper-CVAD could be conceived such as even 9 Gokbuget N, Basara N, Baurmann H, Beck J, Bruggemann M, Diedrich H et al. High earlier administration or concurrent administration with the actual single-drug activity of nelarabine in relapsed T-lymphoblastic leukemia/ hyper-CVAD or methotrexate/cytarabine elements per se. The lymphoma offers curative option with subsequent stem cell transplantation. reason we did not design the study this way was to avoid Blood 2011; 118: 3504–3511. overlapping neurotoxicities with vincristine. 10 Forcade E, Leguay T, Vey N, Baruchel A, Delaunay J, Robin M et al. Nelarabine for T To conclude, our results show that nelarabine is safe and active cell acute lymphoblastic leukemia relapsing after allogeneic hematopoietic stem cell transplantation: an opportunity to improve survival. Biol Blood Marrow Transpl when combined with hyper-CVAD in adult patients with T-ALL/LL in 2013; 19: 1124–1126. the frontline setting. The combination of nelarabine with hyper-CVAD 11 Cohen MH, Johnson JR, Massie T, Sridhara R, McGuinn Jr WD, Abraham S et al. can achieve durable CRs in T-ALL/LL. The optimal schedule of Approval summary: nelarabine for the treatment of T-cell lymphoblastic nelarabine within a larger and complex multiagent chemotherapy leukemia/lymphoma. Clin Cancer Res 2006; 12: 5329–5335. program such as HCVAD remains a topic of discussion. Eventually, 12 Dunsmore KP, Devidas M, Linda SB, Borowitz MJ, Winick N, Hunger SP et al. Pilot only larger scale randomized trials would be able to determine the study of nelarabine in combination with intensive chemotherapy in high-risk role of nelarabine in the frontline therapy of adult T-ALL/LL. T-cell acute lymphoblastic leukemia: a report from the Children’s Oncology Group. J Clin Oncol 2012; 30: 2753–2759. 13 Bene MC. Groupe d’Etude Immunologique des L, European Group for the CONFLICT OF INTEREST Immunological Classification of L. Pro-T ALL: immunophenotypical analyses. J Biol Regul Homeost Agents 2004; 18: 327–330. The authors declare no conflict of interest. 14 Meijerink JP. Genetic rearrangements in relation to immunophenotype and outcome in T-cell acute lymphoblastic leukaemia. Best Pract Res Clin Haematol 2010; 23: 307–318. ACKNOWLEDGEMENTS 15 Cancer Therapy Evaluation Program. CTCAE criteria, Version 3.0. (Internet). This study was supported partly by GlaxoSmithKline Pharmaceuticals (to SF). CTCAE 2012.

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