Letters to the Editor 1948 patient number places some limitation on interpretation, there is a REFERENCES suggestion that this treatment could still be considered for patients 1 Auner HW, Szydlo R, Rone A, Chaidos A, Giles C, Kanfer E et al. Salvage autologous who may have not previously been referred for auto-HCT but who stem cell transplantation for multiple myeloma relapsing or progressing after up- now have relapsed disease. front autologous transplantation. Leuk Lymphoma 2013; 54: 2200–2204. Our adaptive study design ultimately determined that the 2 Shah N, Ahmed F, Bashir Q, Qureshi S, Dinh Y, Rondon G et al. Durable remission highest doses of lenalidomide (75 and 100 mg) were equivalent to with salvage second autotransplants in patients with multiple myeloma. Cancer 118 – achieve a balance between tolerability and efficacy. Owing to 2012; : 3549 3555. relatively small patient numbers, a longer follow-up time may 3 Lemieux E, Hulin C, Caillot D, Tardy S, Dorvaux V, Michel J et al. Autologous stem cell transplantation: an effective salvage therapy in multiple myeloma. Biol Blood further differentiate the true clinical effect of the dose escalation Marrow Transplant 2013; 19:445–449. and the impact of post-auto-HCT maintenance therapy, which 4 Cook G, Williams C, Brown JM, Cairns DA, Cavenagh J, Snowden JA et al. High- 63% of patients received. Unfortunately (although not surpris- dose chemotherapy plus autologous stem-cell transplantation as consolidation ingly), poor-risk cytogenetics independently predicted for poorer therapy in patients with relapsed multiple myeloma after previous autologous OS, reminding us of the continued need for innovative treatment stem-cell transplantation (NCRI myeloma × relapse (Intensive trial)): a randomised, options for these patients. open-label, phase 3 trial. Lancet Oncol 2014; 15: 874–885. In conclusion, combination of high-dose melphalan with high- 5 Kumar SK, Lacy MQ, Hayman SR, Stewart K, Buadi FK, Allred J et al. Lenalidomide, cyclophosphamide and dexamethasone (CRd) for newly diagnosed multiple dose lenalidomide appears to be a well tolerated and safe 86 – preparative regimen in the setting of salvage auto-HCT. As novel myeloma: results from a phase 2 trial. Am J Hematol 2011; :640 645. 6 van de Donk NW, Wittebol S, Minnema MC, Lokhorst HM. Lenalidomide (Revlimid) agents, maintenance regimens and immunotherapies emerge, it is combined with continuous oral cyclophosphamide (endoxan) and prednisone conceivable that they too may be combined with this auto-HCT (REP) is effective in lenalidomide/dexamethasone-refractory myeloma. Br J platform to offer the relapsed patient yet another chance at a Haematol 2010; 148: 335–337. durable response. 7 Durie BG, Harousseau JL, Miguel JS, Blade J, Barlogie B, Anderson K et al. International uniform response criteria for multiple myeloma. Leukemia 2006; 20:1467–1473. 8 Thall PF, Cook JD. Dose-finding based on efficacy-toxicity trade-offs. Biometrics CONFLICT OF INTEREST 2004; 60:684–693. χ2 Drs N Shah, Bashir, JJ Shah, Hosing and Orlowski receive research funding from 9 Fisher R. On the interpretation of from contingency tables, and the 85 – Celgene. Drs N Shah, JJ Shah and Orlowski have served on Celgene Advisory Boards. calculation of P. J R Stat Soc 1922; :87 94. 10 Freeman GH, Halton JH. Note on exact treatment of contingency, goodness of fit fi 38 – 1 2 2 1 3 1 4 and other problems of signi cance. Biometrika 1951; :141 149. N Shah , PF Thall , PS Fox , Q Bashir , JJ Shah , S Parmar , P Lin , 11 Gelman ACJ, Stern HS, Rubin DB. Bayesian Data Analysis, 2nd edn. Chapman & 1 1 1 1 1 P Kebriaei , Y Nieto , UR Popat , CM Hosing , A Cornelison , Hall/CRC Press: New York, NY, USA, 2004. 1 3 1 1 EJ Shpall , RZ Orlowski , RE Champlin and MH Qazilbash 12 Ibrahim JG, Chen M-H, Sinha D. Bayesian Survival Analysis.Springer:NewYork,NY,2001. 1Department of Stem Cell Transplantation and Cellular Therapy, The 13 Doo NW, Thompson PA, Prince HM, Seymour JF, Ritchie D, Stokes K et al. University of Texas MD Anderson Cancer Center, Houston, TX, USA; Bortezomib with high dose melphalan conditioning for autologous transplant is 2Department of Biostatistics, The University of Texas MD Anderson safe and effective in patients with heavily pretreated and high risk multiple 54 – Cancer Center, Houston, TX, USA; myeloma. Leuk Lymphoma 2013; : 1465 1472. 3Department of Lymphoma and Myeloma, The University of Texas 14 Orlowski RZ. Novel agents for multiple myeloma to overcome resistance in phase III clinical trials. Semin Oncol 2013; 40:634–651. MD Anderson Cancer Center, Houston, TX, USA and 4 15 Kumar SK, Lee JH, Lahuerta JJ, Morgan G, Richardson PG, Crowley J et al. Risk of Department of Hematopatholoy, The University of Texas MD progression and survival in multiple myeloma relapsing after therapy with IMiDs Anderson Cancer Center, Houston, TX, USA and bortezomib: a multicenter international myeloma working group study. E-mail: [email protected] Leukemia 2012; 26:149–157. Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu) Notch-mediated expansion of cord blood progenitors: maintenance of transcriptional and epigenetic fidelity Leukemia (2015) 29, 1948–1951; doi:10.1038/leu.2015.61 provide more rapid short-term myeloid reconstitution when co- infused with a non-manipulated CB unit(s). However, infusion of ex vivo generated HSPCs for long-term hematopoietic reconstitu- Recent advances have been made towards successful generation tion remains a goal in the context of stem cell transplantation and of human hematopoietic stem and progenitor cells (HSPCs) gene therapy, thus the development of methods to assess the derived from embryonic, induced pluripotent or hematopoietic safety of these cultured products are essential. stem cells. However, whether these stem cell populations, which Our group and others have demonstrated successful expansion are generated after ex vivo manipulation, are suitable for clinical of CB HSPCs for both pre-clinical and clinical applications. application depends on the faithful preservation of their Standardized methods for assessing transcriptional quality control transcriptional, epigenetic and functional properties with respect of pluripotent cell lines have been developed; however, there has to their primary cell counterparts. been no standard approach to assessing long-term safety of To overcome the significant delay in neutrophil recovery cultured progenitor cells infused in humans.1 Here we investi- following cord blood (CB) transplantation (CBT), we developed gated the transcriptional and epigenetic states of CB HSPC methods for the ex vivo expansion of CD34+ CB-derived HSPC by generated after ex vivo expansion on Delta1. We found that the culture with the Notch-ligand Delta1. When infused into patients transcriptomes and methylomes of the expanded HSPC overall undergoing a myeloablative CBT, these cells have been shown to recapitulate those of their primary cell counterparts. Accepted article preview online 6 March 2015; advance online publication, 27 March 2015 Leukemia (2015) 1939 – 1958 © 2015 Macmillan Publishers Limited Letters to the Editor 1949 CD34+ CB HSPCs were cultured under conditions identical to cells. To determine whether BEX2 downregulation was the result those in our ongoing clinical trials.2 We validated in vitro and of altered expression of transcription factors known to regulate in vivo growth characteristics of cells used for transcriptional and BEX2, we looked at the expression levels of SOX2, β-catenin, p65 methylation studies to ensure their similarity with our previously and ERBB2 and found no difference between primary and published results. Mean CD34+ fold expansion was 191 ± 62-fold cultured cells. as compared with 184 ± 35 previously. In vivo repopulation in We compared genes upregulated in the cultured cells with an sublethally irradiated (275 rad) NOD-SCID IL-2Rγ-null mice (NSG) established list of proto-oncogenes (www.uniprot.org/uniprot, approved for use by the Fred Hutchinson Cancer Research Center search terms ‘proto-oncogene’, ‘human’) as upregulation of these Institutional Animal Care and Use Committee was at least as genes might be expected to result in unintended cell proliferation. robust as our previously published results. Among the list of 232 proto-oncogenes, 11 were upregulated in For transcriptional analysis, CD34+ CB HSPCs were isolated from cultured cells (Figure 1c). Although many of these genes have six individual CB units within 24 h of collection. Freshly isolated been implicated in hematologic malignancies, they also have CD34+ CB HSPCs were then frozen for subsequent RNA extraction, important roles in normal hematopoiesis, and thus may be whereas remaining cells were placed in culture for 14 days as upregulated in this setting. Because the CB HSPCs were cultured in previously described.2 Upon cell harvest at day 14 of culture, the presence of Delta1, we also determined whether there was CD34+ cells were selected for RNA extraction and analysis on the persistent altered expression of Notch downstream target genes Illumina HT12v3 platform (Illumina Inc, San Diego, CA, USA). All as some of these have known roles in cell cycle entry and data were analyzed using the Bioconductor framework.3 Lumi proliferation. We found no difference in the expression of known package4 was used for reading and normalizing the raw data Notch targets SKP2, HES1, HEY1, CCND1 or BCL2. As shown in output by GenomeStudio software (Illumina Inc.). Limma package3 Figure 1c, MYC expression was upregulated following culture on was used for identifying differentially expressed genes between Delta1 ligand; however, Notch is one of many regulators of MYC freshly isolated and cultured CD34+ CB HSPCs. Significant genes expression. were defined as those with false discovery rate (FDR) o1% and Whereas transcriptional activity represents cellular function, we 4twofold change between non-manipulated and ex vivo expected that DNA methylation states would offer a more detailed expanded cells. picture of the fidelity of the expanded populations with respect to Of 24 912 genes analyzed, 668 genes were upregulated in the cell identity and thus safety for clinical use.