Overcoming the Response Plateau in Multiple Myeloma: A ... · and remains a standard of care in...

Cancer Therapy: Clinical

Overcoming the Response Plateau in Multiple Myeloma: ANovel Bortezomib-Based Strategy for Secondary Inductionand High-Yield CD34þ Stem Cell Mobilization

RubenNiesvizky1, TomerM.Mark1, MaureenWard1, David S. Jayabalan1,3, Roger N. Pearse1,MeganManco1,Jessica Stern1, Paul J. Christos2, Lena Mathews1, Tsiporah B. Shore1, Faiza Zafar1, Karen Pekle1, ZhaoyingXiang4, Scott Ely3, Donna Skerret3, Selina Chen-Kiang3, Morton Coleman1, and Maureen E. Lane1

AbstractPurpose: This phase II study evaluated bortezomib-based secondary induction and stem cell mobili-

zation in 38 transplant-eligible patients with myeloma who had an incomplete and stalled response to, or

had relapsed after, previous immunomodulatory drug-based induction.

Experimental Design: Patients received up to six 21-day cycles of bortezomib plus dexamethasone, with

added liposomal doxorubicin for patients not achieving partial response or better by cycle 2 or very good

partial response or better (�VGPR) by cycle 4 (DoVeD), followed by bortezomib, high-dose cyclophos-

phamide, and filgrastim mobilization. Gene expression/signaling pathway analyses were conducted in

purified CD34þ cells after bortezomib-based mobilization and compared against patients who received

only filgrastim � cyclophosphamide. Plasma samples were similarly analyzed for quantification of asso-

ciated protein markers.

Results: The response rate to DoVeD relative to the pre-DoVeD baseline was 61%, including

39% �VGPR. Deeper responses were achieved in 10 of 27 patients who received bortezomib-based

mobilization; postmobilization response rate was 96%, including 48%�VGPR, relative to the pre-DoVeD

baseline. Median CD34þ cell yield was 23.2� 106 cells/kg (median of 1 apheresis session). After a median

follow-upof 46.6months,medianprogression-free survivalwas47.1months fromDoVeD initiation; 5-year

overall survival rate was 76.4%. Grade �3 adverse events included thrombocytopenia (13%), hand–foot

syndrome (11%), peripheral neuropathy (8%), and neutropenia (5%). Bortezomib-based mobilization

was associated with modulated expression of genes involved in stem cell migration.

Conclusion: Bortezomib-based secondary induction and mobilization could represent an alternative

strategy for elimination of tumor burden in immunomodulatory drug-resistant patients that does not

impact stem cell yield. Clin Cancer Res; 19(6); 1534–46. �2013 AACR.

IntroductionHigh-dose chemotherapy and stem cell transplant

(HDT-SCT) for the front-line treatment of multiple myelo-ma has contributed to improved overall survival (OS; ref. 1)

and remains a standard of care in eligible patients (2).Attainment of a very good partial response or better(�VGPR) with pretransplant induction therapy is associat-ed with improved long-term posttransplant outcomes inpreviously untreated patients with multiple myeloma,including prolonged progression-free survival (PFS),event-free survival, and OS (3, 4); therefore, inductiontherapy should aim to achieve as rapid and as deep aresponse as possible before transplant (2).

The incorporation of novel agents, such as the pro-teasome inhibitor bortezomib (Velcade) and the immu-nomodulatory drugs (IMiDs) thalidomide (Thalomid)and lenalidomide (Revlimid), into front-line pretrans-plant induction regimens has resulted in improved re-sponse rates and long-term outcomes compared withconventional induction approaches (5–7). A range ofcombination regimens have been evaluated in clinicaltrials (2, 8), with triplet regimens incorporating at leastone novel agent showing superior efficacy to doubletregimens (2, 9). Several newer induction regimens,

Authors' Affiliations: 1Center of Excellence for Lymphoma andMyeloma,Division of Hematology and Medical Oncology, Weill Cornell MedicalCollege and New York Presbyterian Hospital; 2Division of Biostatistics andEpidemiology, Department of Public Health, Weill Cornell Medical Collegeand New York Presbyterian Hospital; 3Department of Pathology, and4Genomics Resources Core Facility, Weill Cornell Medical College, NewYork, New York

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Ruben Niesvizky, Center of Excellence for Lym-phoma andMyeloma, Division of Hematology andMedical Oncology,WeillCornellMedicalCollege andNewYorkPresbyterianHospital, 520East 70thStreet, Starr 341, New York, NY 10021. Phone: 212-746-3964; Fax: 212-746-5536; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-12-1429

�2013 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 19(6) March 15, 20131534

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Published OnlineFirst January 28, 2013; DOI: 10.1158/1078-0432.CCR-12-1429

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including bortezomib-dexamethasone (VD), lenalidomidelow-dose dexamethasone (Rd), bortezomib-thalidomide-dexamethasone (VTD), and bortezomib-doxorubicin-dexa-methasone (PAD), have been designated as U.S. NationalComprehensiveCancerNetwork (NCCN) category 1 recom-mendations for transplant-eligible patients with multiplemyeloma based on high-level evidence and uniform con-sensus (10). Alternative induction regimens, such as clari-thromycin (Biaxin)-lenalidomide-dexamethasone (BiRD;ref. 11), bortezomib-lenalidomide-dexamethasone (RVD;ref. 12), RVD plus pegylated liposomal doxorubicin(DOXIL; RVDD; ref. 13), and bortezomib-cyclophospha-mide-dexamethasone (VCD; refs. 14, 15) have also shownvery promising activity in phase I/II studies (2, 8), but theirefficacy remains to be confirmed in phase III trials.While most patients will respond to these induction

regimens, not allwill achieve�VGPR, and further treatmentoptions are required in this setting to maximize transplantoutcomes. According to the Norton–Simon hypothesis(16), the sequential, dose-dense use of agents or regimensthat are not cross-resistant may increase the proportion ofpatients achieving postinduction complete response (CR)or �VGPR, which may translate into improved PFS andOS. Bortezomib and the IMiDs have shown different butoverlapping mechanisms of action (17, 18); therefore, theuse of an IMiD-based regimen after initial bortezomib-based induction therapy, or vice-versa, may increase theproportion of patients achieving pretransplant �VGPR.Several clinical studies of sequential induction regimens

in patients with previously untreated multiple myeloma,including bortezomib-liposomal doxorubicin-dexametha-sone (VDD) ! thalidomide-dexamethasone (TD; ref. 19),VCD ! VTD (20), and vincristine-doxorubicin-dexameth-asone (VAD) ! VTD (21), have already reported encour-aging efficacy.

Bortezomib does not negatively impact stem cell col-lection (22) and enhances the cytoreductive activity ofalkylating agents (14, 15, 23–25). The addition of borte-zomib to a granulocyte colony-stimulating factor (G-CSF)þ cyclophosphamide stem cell mobilization regimenmight therefore offer additional pretransplant cytoreduc-tion while not interfering with potential stem cell yield. Astem cell yield supporting 2 SCTs (8 � 106 to 10 � 106

CD34þ cells/kg) is the collection goal recommended in2009 by the International Myeloma Working Group(IMWG; ref. 26). Identifying induction therapies thatare both effective and capable of achieving this goal areparamount; induction and mobilization therapy beforeHDT-SCT should therefore aim to maximize both cyto-reduction and stem cell yields.

We assessed both the efficacy and the safety of second-ary bortezomib-based induction with DoVeD (bortezo-mib þ dexamethasone � pegylated liposomal doxorubi-cin) as well as the impact of adding bortezomib tomobilization (bortezomib þ cyclophosphamide þ fil-grastim) in patients with multiple myeloma who had astalled incomplete response or were at first relapse afterprior IMiD-based induction therapy. Gene expressionprofiling and cellular signaling pathway analyses werealso conducted on CD34þ cells from patients with mul-tiple myeloma in an attempt to understand the moleculareffects of bortezomib on stem cell mobilization.

Materials and MethodsPatients

All patients aged 18 years or more with active multiplemyeloma (�1.0 g/dL serum M-protein, �0.1 g/dL serum-free light chains, �0.2 g/24-h urinary M-protein excretion,and/or measurable plasmacytomas, with evidence ofmultiple myeloma–related end-organ dysfunction) wereeligible if they had Durie–Salmon stage II/III disease, hadnot received prior treatment with a proteasome inhibitor,and had first-line IMiD-containing induction therapywith amaximum response of partial response or less (�PR)followed by a stalled response or plateau (defined asno significant change in M-protein level for 3 successivemonthly assessments) to continued initial treatment.Patients were also eligible if they had relapsed (determinedin accordance with IMWG criteria; ref. 27) following oneprevious IMiD-containing induction therapy regardlessof initial treatment response. Other eligibility criteriaincluded Karnofsky performance status (KPS)� 70%; abso-lute neutrophil count (ANC) �1,000 cells/mm3; plateletcount �75,000/mm3; aspartate/alanine aminotrans-ferase < 3.0 � upper limit of normal (ULN); serum creat-inine < 2.5 mg/dL; and serum total bilirubin < 2.0 mg/dL.

Translational RelevanceThis study showed substantial activity with sequential

use of non–cross-resistant agents/regimens as inductionin myeloma, with secondary induction with DoVeD(bortezomib–dexamethasone � liposomal doxorubi-cin) resulting in further tumor burden reduction inpatients following stalled or plateaued responses toprimarily immunomodulatory drug–based initialinduction. Furthermore, the approach of adding borte-zomib to standard stemcellmobilization therapy (cyclo-phosphamide and filgrastim) resulted in deeperresponses, providing further evidence for the cytoreduc-tive effect of bortezomib in combination with cyclo-phosphamide. Notably, bortezomib-based mobiliza-tion was associated with very high CD34þ stem cellyields, suggesting that bortezomib may promote stemcell egress. Exploratory gene expression and IngenuityPathway Analysis revealed modulated expression ofgenes with roles in cell migration and associated withthe canonical ephrin signaling pathway in patientsreceiving bortezomib-containing mobilization. Thesepatients also showed significantly decreased plasmalevels of CXCL12 and angiopoietin-1, which couldpotentially be useful as biomarkers of improved CD34þmobilization in bortezomib-treated patients.

Bortezomib-Based Secondary Induction and Mobilization in Multiple Myeloma

www.aacrjournals.org Clin Cancer Res; 19(6) March 15, 2013 1535




Exclusion criteria included history of grade�2 peripheralneuropathy (PN), as defined by National Cancer InstituteCommon Terminology Criteria for Adverse Events (NCI-CTCAE) version 3.0; a history of othermalignancies (exceptfor basal cell or squamous cell carcinoma of the skin orcarcinoma in situ of the cervix or breast) unless disease freefor�5 years; a history of active unstable angina, congestiveheart disease, serious uncontrolled cardiac arrhythmia, ormyocardial infarction within the previous 6 months, orNew York Heart Association (NYHA) Class III/IV heartdisease; and known HIV or hepatitis A, B, or C positivity,or active viral or bacterial infections.

Study designThis single-center, open-label, phase II study was con-

ducted between July 2005 and April 2011. Patients receivedsix 21-day induction cycles of DoVeD (bortezomib 1.3mg/m2 on days 1, 4, 8, and 11; dexamethasone 40 mg ondays 1–4, 8–11, and 15–18; plus, if <PR after 2 cycles or <CRafter 4 cycles, liposomal doxorubicin 30mg/m2onday 4 forthe remaining cycles), followed by one 21-daymobilizationcycle with bortezomib (as above), high-dose cyclophospha-mide (3 g/m2 on day 8), and filgrastim (10 mg/kg/d for 10consecutive days starting 24 hours after cyclophosphamideadministration on day 9). There was no delay betweeninduction cycle 6 and the subsequent stemcellmobilizationcycle. All patients received prophylaxis during inductionand mobilization with trimethoprim/sulfamethoxasole,acyclovir, and omeprazole.

The study was approved by the Institutional ReviewBoard of Weill Cornell Medical College (New York, NY)and was conducted according to the Declaration of Hel-sinki, the International Conference onHarmonization, andthe Guidelines for Good Clinical Practice. All patientsprovided written informed consent.

AssessmentsSafety was monitored throughout the study and adverse

events (AE) were graded by NCI-CTCAE version 3.0.Responses were assessed relative to pre-DoVeD inductionbaseline M-protein levels after every cycle, according toIMWG uniform response criteria (28). PFS and OS weremeasured from the beginning of DoVeD induction therapyand distributions were estimated using Kaplan–Meiermethodology.

Stem cell collection and hematopoietic recovery. Postin-duction stem cell collection was initiated when patients’ANC reached�1.0� 109/L. Leukapheresis was conductedusing indwelling venous catheters and the COBE Spectracontinuous flow cell separator (Gambro BCT) in theautomatic mode for mononuclear cell collection. Large-volume (20–24 L) leukapheresis was conducted to reach atarget of 10 � 106 CD34þ cells/kg. Unmanipulated cellswere characterized for nucleated cell counts and CD34,then cryopreserved at 7.5% in dimethylsulfoxide, andstored at �190�C. The time to posttransplant ANC recov-ery (>0.5 � 109/L) and platelet recovery (>20 � 109/L)were recorded.

Gene expression/signaling pathway/plasma analyses.Analyses were conducted in patients who underwent bor-tezomib-based mobilization in the present study and in acontrol cohort of patients (n ¼ 13) who underwent non-bortezomib–basedmobilization with filgrastim 10 mg/kg/d� high-dose cyclophosphamide (3 g/m2) as part of theirtreatment at Weill Cornell Medical College. This controlcohort comprised patients with Durie–Salmon stage II/IIIMM who had received prior induction therapy with borte-zomib (n ¼ 3), lenalidomide (n ¼ 3), or bortezomib pluslenalidomide (n ¼ 5). Two patients had not received anyinduction therapy. All patients received filgrastim formobi-lization at least 1 month after completing induction.

The gene expression profiles of CD34þ stem cellsenriched from leukapheresis product were comparedbetween 6 and 4 evaluable patients, respectively, who eitherreceived or did not receive bortezomib during stem cellmobilization. Following quality control analysis, one sam-ple from each group was removed after conducting a prin-cipal component analysis, leaving 5 and 3 samples, respec-tively. Levels of CXCL12 (SDF-1a) were compared in 6 and7 patients, respectively, and angiopoietin-1 levels in plasmawere compared between samples from 7 and 9 patients,respectively, who received or did not receive bortezomibduring mobilization.

Microarray gene expression analysis. CD34þ cells werepurified from cryopreserved leukapheresis product usinga human CD34-positive selection kit (Stem Cell Techno-logies). The purity of the CD34þ isolate was assessed byflow cytometry using an anti-human CD34-PE antibody(BD Biosciences). Total RNA was isolated from theCD34þ cells using the RNeasy Mini kit from Qiagen. RNAyield and quality was assayed using a Nanodrop (ThermoScientific) and the Agilent Bioanalyzer 2100 (Agilent).About 25 to 200 ng of total RNA was reverse-transcribedand amplified using the WT-Ovation Pico RNA Ampli-fication System from NuGEN Technologies. AmplifiedcDNA (5 mg) was fragmented, labeled with biotin, andhybridized to Affymetrix HG-U133 plus two microarraychips (Agilent), which contained more than 47,000 tran-scripts, 38,500 well-characterized human genes, and morethan 54,000 probe sets. Raw intensity (.cel) files wereimported and preprocessed using the Robust Multi-ArrayAverage (RMA) algorithm (29).

Chip data were imported into the GeneSpring GX 11.5program (Agilent Technologies). Signal values <0.01 wereset to 0.01, arrays were normalized to the 50th percentile,and individual genes normalized to the median. Anunpaired, asymptotic t test (variances assumed equal) withP < 0.05 was conducted comparing the bortezomib andnon-bortezomib mobilization groups followed by filtra-tion for greater or less than 2.0-fold differences was appliedto determine potential differential expression. Additionalfilters for pathway and biomarker analyses were applied(P < 0.0025, fold change 2.0) using IPA (Ingenuity Sys-tems). The data discussed in this publication have beendeposited in NCBI’s Gene Expression Omnibus andare accessible through the GEO series accession number

Niesvizky et al.

Clin Cancer Res; 19(6) March 15, 2013 Clinical Cancer Research1536




GSE 43124 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc¼GSE43124).Quantitative real-time PCR. Total RNA (50 mg) from

CD34þ cells was reverse transcribed into cDNA usinga qScript cDNA supermix reagent from Quanta Bio-sciences, Inc. according to the manufacturer’s protocol.The reaction mixture was diluted to 100 mL with distill-ed water and 3 mL of the diluted cDNAwas amplified usinggene-specific TaqMan probes and primers from AppliedBiosystems. The following TaqMan gene expression assays(ABI Life Technologies) were used to carry out quantitativePCR (QPCR) on selected genes associated with signi-ficant differential gene expression and pathway analy-ses: AKT2 (Hs01086102_m1), CXCR4 (Hs00607978_s1),PAWR (Hs01088574_m1), RAC1 (Hs01902432_s1),SOD2 (Hs00167309_m1), RNF11 (Hs00702517_s1),PTP4A1 (Hs00743856_s1), PTK2 (Hs00178587_m1), andANKRD36B (Hs00743856_s1). Real-time QPCR was car-ried out in triplicate using the ABI 7900HT machine(Applied Biosystems). Comparative DCt analysis wasconducted to determine the fold change in gene expres-sion between bortezomib (n ¼ 4) and non-bortezomib(n ¼ 3–4) samples.Quantification of CXCL12, angiopoietin-1, and interleu-

kin 8 in plasma. Plasma samples were collected frompatients who received bortezomib-based mobilization orfrom the non-bortezomib–based mobilization controlcohort on the same day that leukapheresis product washarvested for CD34þ cell isolation. Blood samples weredrawn into vacutainers containing K2-EDTA. Blood wascentrifuged at 1,500 � g at 4�C for 10 minutes and plasmawas removed and cryopreserved at �80�C. CXCL12 andangiopoietin-1 concentrations were subsequently quanti-fied using enzyme immunoassay kits (R&D Systems)according to the manufacturer’s protocol and were basedon the average of duplicate samples. Interleukin 8 (IL8)plasma levels were quantified using the mesoscale assayaccording to the manufacturer’s protocol.

Statistical analysesThe observed differences in gene expression between

CD34þ stem cells collected from patients receiving borte-zomib versus non-bortezomibmobilization were tested forsignificance using the Benjamini–Hochberg false discoveryrate for multiple testing correction. A 2-tailed t test wasconducted to test the significance of the difference inCXCL12 plasma levels, and a Mann–Whitney U test wasconducted to determine the significance of differences inangiopoietin-1 and IL8plasma levels between samples frompatients receiving bortezomib versus non-bortezomibmobilization. Kaplan–Meier survival analysis was con-ducted to evaluate PFS and OS. Univariate Cox regressionanalysis was used to measure the association betweenpatient characteristics and PFS. Multivariate Cox regressionanalysis could not be conducted because of the limitednumber of PFS events in the analysis. AllP values are 2-sidedwith statistical significance evaluated at the 0.05 a-level.Ninety-five percent confidence intervals (CI) were calculat-

ed to assess the precision of the obtained estimates. Allanalyses were conducted in SAS Version 9.3 (SAS InstituteInc.) and STATA Version 12.0 (StataCorp).

ResultsPatients and treatment

A total of 38 patients who had reached a plateau or wereinfirst relapse after primary induction therapywere enrolledand constituted the intent-to-treat (ITT) population. Patientbaseline demographics and disease characteristics are sum-marized in Table 1. Briefly, patients had a median age of 61years (range, 27–76), 55% were male, 97% had Durie–Salmon stage II/III disease, and 53% had InternationalStaging System (ISS) disease stage II/III. Twenty-two(58%) patients had cytogenetic abnormalities; 7 (18%)patients had high-risk cytogenetics [del(17p), t(4;14), ort(14;16) by FISH]. More than half the patients had receivedinitial induction therapy with lenalidomide þ dexametha-sone � clarithromycin.

A diagram summarizing patient flow through the study isshown in Supplementary Fig. S1. Patients received amedianof 6 cycles (range, 2–6) of DoVeD induction therapy. Perprotocol, only patients who did not achieve PR by cycle 2 orVGPR by cycle 4 received liposomal doxorubicin. In total,28 (74%) patients received liposomal doxorubicin, 16patients from cycle 3, and 12 patients from cycle 5.

Response to DoVeD induction therapy and followingmobilization

Responses to initial induction therapy are summarizedin Table 1. All 38 patients were evaluable for response afterDoVeD secondary induction. The overall best response rate(ORR,�PR, as measured by�50% reduction inM-protein)to DoVeD from post-primary induction (pre-DoVeD) base-line was 61% (n¼ 23), including 18% CR (n¼ 7) and 39%�VGPR (n¼ 15); in addition, 10 (26%) patients had stabledisease (SD) and 5 (13%) had PD as best response (Sup-plementary Table S1).

Twenty-seven of the 38 patients elected to proceed directlyto HDT-SCT and underwent bortezomib-based mobiliza-tion. Twenty-six of these 27 (96%) patients achieved apost-mobilization response of �PR relative to post-primaryinductionbaseline, including 4 (15%) sCR, 3 (11%)CR, and6 (22%)VGPR (48%�VGPR; Supplementary Table S1). Thisincreased response rate post-mobilization reflected the factthat some patients continued to respond positively to treat-ment during bortezomib-cyclophosphamide–based mobili-zation. Six patientswith SDafterDoVeDinduction improvedto PR after mobilization; in addition, there was 1 VGPR-to-sCR transition and 3 CR-to-sCR transitions after mobiliza-tion. Ten patients underwent stem cell mobilization withnon–bortezomib-based regimens; 6 patients weremobilizedwith filgrastim alone, and 4 were mobilized with cyclophos-phamide þ filgrastim. Of these 10 patients, 1 patient eachachieved CR and VGPR, and 2 patients achieved PR post-mobilization(SupplementaryTableS1).Onepatientwasnotmobilized during the study due to septic arthritis.






Stem cell collection and SCTCD34þ stem cell yields greatly exceeded the study goal

of 10 � 106/kg in 23 of 27 (85%) patients who receivedbortezomib-based mobilization, with a median yield of23.2 � 106 cells/kg (range, 6.8 � 106 to 294.2 � 106)obtained within a median of 1 collection day (range,

1–5). Individual patients’ data are presented in Table 2.The median CD34þ stem cell yield for patients mobilizedwith non-bortezomib–based regimens was 10.72 � 106

cells/kg (range, 4.79 � 106 to 15.77 � 106). No statisticalcomparisons were planned or conducted between thesegroups.

Twenty-five of 27 (93%) patients who underwent borte-zomib-based mobilization subsequently underwent SCTusing standard melphalan 200 mg/m2 conditioning. Fol-lowing transplant, the median time to ANC recoveryamong these patients was 11 days (range, 10–18) andthe median time to platelet recovery was 16 days (range,11–24; Table 2). The median times to ANC and plateletrecovery for patients who received non-bortezomib–basedmobilization were 14 and 15 days, respectively. Of the 10patients who underwent stem cell mobilization with non-bortezomib–containing regimens, 8 subsequently under-went SCT and 2 did not.

SafetyAll 38 (100%) patients completed at least one cycle of

DoVeD; 10 (26%) patients received only bortezomib plusdexamethasone, without the addition of liposomal doxo-rubicin. Safety data are summarized in SupplementaryTable S2. Thrombocytopenia and neutropenia were themost common grade �3 hematologic AEs, with rates of13% and 5% during DoVeD induction and 85% and 96%during mobilization, respectively. Grade�3 febrile neutro-penia was not observed during induction and was observedin only 1 (4%) patient during mobilization. The mostcommon grade �3 nonhematologic AEs during inductionwere hand–foot syndrome (11%) and PN (8%). Diarrheawas the only grade�3nonhematologic AEs reported duringmobilization in 1 (4%) patient. In total, 28 (74%) patientsexperienced PN during DoVeD induction, including 9(24%) with grade 2 PN and 3 (8%) with grade �3 PN.During bortezomib-based mobilization, 6 (22%) patientsexperienced PN but no grade �3 PN was observed. Noadditional grade �3 PN events were observed followinginduction. No serious AEs (SAE) were reported duringinduction therapy.

OutcomesSeven of the 38 (18%) patients had died by the final data

cutoff (April 15, 2011); the median duration of follow-upfrom the start ofDoVeD induction (based on survivors) was46.6 months (range, 6.4–68.8 months). In the ITT popu-lation, median PFS from the start of DoVeD induction was47.1 months (95% CI, 26.2 months, upper limit not esti-mable); the 5-year PFS rate was 28.3% (95% CI, 8.6%–52.2%; Supplementary Fig. S2A). The median OS was notreached; the 5-year OS rate was 76.4% (95% CI, 56.2%–88.2%; Supplementary Fig. S2B).

The effect of multiple patient characteristics (includingmyeloma type, b2-microglobulin level, creatinine level,disease stage, albumin level) on PFS was evaluated byunivariate Cox regression analysis. None of the factorsanalyzed had any significant association with PFS.

Table 1. Patient baseline demographics anddisease characteristics at initiation of DoVeDinduction

Characteristica N ¼ 38

Median age (range), y 61 (27–76)Age <70 y, n (%) 34 (89)Age �70 y, n (%) 4 (11)

Male, n (%) 21 (55)Median b2-microglobulin, mg/L (range) 2.05 (1.0–10.2)Median albumin, g/dL (range) 3.5 (2.0–4.3)Durie–Salmon stage, n (%)Iaa 1 (3)IIa 19 (50)IIIa 17 (45)IIIb 1 (3)

ISS stage, n (%)I 18 (47)II 17 (45)III 3 (8)

Abnormalities by FISH, n (%)Trisomy 11 10 (26)Hyperdiploidy 7 (18)t(4;14) 4 (11)p53 4 (11)del(17p) 1 (3)t(11;14) 5 (13)t(14;16) 2 (5)None 16 (42)

Prior induction therapyLenalidomide þ dexamethasone �clarithromycin

21 (55)

Thalidomide þ lenalidomide þdexamethasone � clarithromycin

8 (21)

Thalidomide þ dexamethasone 3 (8)Thalidomide only 2 (5)Pulsed dexamethasone only 2 (5)Melphalan þ cyclophosphamide 1 (3)Dexamethasone followed bysingle-agent lenalidomide

1 (3)

Best response to prior induction therapyCRb 1 (3)PR 27 (71)Stable disease 10 (26)

aAs assessed by the treating physician, this patient hadactive myeloma based on the IMWG criteria.bCRwithmelphalanþ cyclophosphamide (but with progres-sion of disease after induction).

Niesvizky et al.





Univariate analysis of the factors potentially affectingOS, and multivariate analyses of PFS and OS, could notbe conducted because of insufficient events.

Gene expression/signaling pathway analysesIn an attempt to elucidate an underlying mechanism for

the high stem cell yields observed with bortezomib-contain-ing mobilization, gene expression profiles of enrichedCD34þ cells from bortezomib-mobilized patients in thisstudy were compared with those from patients who receivednon-bortezomib mobilization. In total, 12,727 genes weredifferentially expressed (P � 0.05) between the 2 patientgroups (data not shown). After filtering using a cutoff of P <0.0025, 997 genes were determined to be analysis-ready andused for IPA. The 10most significantly up- or downregulatedgenes identified in microarray analysis of CD34þ cells

(following pathway analysis filters) from patients receivingbortezomib- versus non-bortezomib–containing mobiliza-tion are summarized in Table 3. Nine genes were subse-quently selected for validation by QPCR (SupplementaryTable S3) due to exhibiting either high levels of differentialexpressionbetween patients receiving bortezomib- and non-bortezomib–containingmobilization or an associationwithhigh ranking networks and pathways identified using IPA.The genes validated by QPCR included RAC1, ANKRD36,PTP4A1, AKT2, SOD2, CXCR4, PAWR, PTK2, and RNF11. Ingeneral, the direction of fold change in QPCR data wasconsistent with the direction of change in the microarraydata; 8of 9 genes testedwere in agreementwith respect toup-or downregulation. The level of gene expression changes(fold change up or down) varied between the QPCR andmicroarray datasets (Supplementary Table S3).

Table 2. CD34þ stem cell collection and hematologic recovery post-SCT, by patient, in patients receivingbortezomib-based mobilization

Patient

Number ofcollectiondays

Days from startof mobilizationto start ofcollection

Total CD34þstem cellscollected(�106/kg)

Total CD34þstem cellsinfused(�106/kg)

Days toANCrecovery(>1.5 � 109/L)

Days toplateletrecovery(>20 � 109/L)

1 1 18 21.2 5.78 14 202 1 18 47.4 13.22 11 133 1 19 22 9.87 13 224 1 18 17.9 9.03 10 155 4 19 40.6a 5.44 11 216 1 18 19.9 9.24 10 167 3 19 294.2b 17.73 10 178 2 17 13.8 6.32 13 249 5 18 9.25 4.25, 2.74 11 1810 2 17 21.4 9.05 16 2111 1 24 50.0 No SCTc 16 1612 2 19 66.1 12.83 11 1113 1 18 30.4 7.38 11 1114 2 16 43.6 10.02 12 1515 1 19 51.0 12.72 13 1116 1 17 15.6 5.31 11 1317 1 17 6.8 6.66 12 1318 1 17 31.7 9.20 11 2019 1 17 7.8 7.40 10 1120 1 16 43.9 11.06 10 2121 1 18 23.2 No SCTc 10 1122 1 17 14.0 3.47 16 1623 2 17 13.4 3.96 12 1124 1 16 32.8 7.92 18 1425 1 20 11.5 5.63 11 1826 1 15 29.4 8.2 17 2227 3 18 27.2 10.2 10 14Median (range) 1 (1–5) 18 (15–24) 23.2 (6.8–294.2) 8.2 (3.4–17.7) 11 (10–18) 16 (11–24)

a20.4 � 106 cells collected after 2 days.b86.3 � 106 cells collected after 1 day.cPatient opted to defer HDT-SCT until later.






IL8 was the highest differentially expressed genebetween CD34þ cells from patients receiving bortezo-mib- and non-bortezomib–containing mobilization(Table 3). In addition, IL8 was identified in a biomarkeranalysis of the gene expression data using IPA with a filterfor molecules found in plasma and/or serum (data notshown); we therefore investigated plasma levels of IL8 in2 groups. However, there were no significant differencesin IL8 plasma levels (P ¼ 0.6057, data not shown)between the 2 groups.

Figure 1 summarizes the 5 highest-ranked canonicalpathways exhibiting significant changes in gene expressionbetween bortezomib- and non-bortezomib–containing

mobilization. The highest-ranked canonical pathways(determined using IPA software) were hypoxia signaling inthe cardiovascular system, protein ubiquitination, ephrinreceptor signaling, mitochondrial dysfunction, and actinnucleation by ARP-WASP.

Ephrin receptor signaling is known to play a role in cellmigration (30) and can be differentially expressed in mobi-lized hematopoietic stem cells (31). To determine whetherdifferential gene expression in these highest-ranked path-ways could account for enhanced bortezomib-inducedmobilization of CD34þ cells, the list of genes was filteredfor those with known functional roles in cell migration.Nineteen genes with known roles in cell migration were

Table 3. The 10most significantly up- or downregulated genes [P� 0.0025, fold change (FC) in expression>2.0) identified in gene expression analysis of CD34þ cells from patients receiving bortezomib-versus non-bortezomib–based mobilization

Top upregulated FC up Function/role_up Top downregulated FC down Function/role_down

IL8/Interleukin 8 23.72 Chemokine,chemotaxis,migration

NUMA1/nuclear mitoticapparatus protein

�12.06 Microtubule binding,mitosis

RAC1/Ras-related C3botulinum toxinsubstrate 1

19.45 GTPase, member,protein binding,migration, invasion

MUC6/mucin 6 �11.3 Extracellular matrixconstituent, survival,protein binding

KLF10/Kruppel-likefactor 10

18.44 DNA binding,transcription factor,proliferation,polyubiquitination

CPT1B/carnitinepalmitoyltransferase1B

�10.39 Cell death, proteinbinding, transferaseactivity

VBP1/von Hippel-Lindaubinding protein

17.82 Unfolded proteinbinding

NCOR2/nuclearreceptor corepressor2

�6.72 DNAandproteinbinding,receptor activity,growth, motility, selfrenewal, proliferation

TNFSF4/tumor necrosisfactor 4, apoptosis,proliferation,recruitment

17.2 Cytokine activity, TNFreceptor binding,apoptosis,proliferation,recruitment

MUC4/Mucin 4 �6.35 ERBb2 receptor binding,Extracellular matrix,apoptosis, motility,colony formation

PPT1/palmitoyl-proteinthioesterase 1

16.98 Hydrolase activity,apoptosis,development

MS12/musashihomologue

�5.91 Nucleotide and RNAbinding, development,proliferation

AP1S2/Adapter Protein 1 16.53 Protein transporteractivity, cell cycle,differentiation

INTS3/integratercomplex, subunit 3

�5.66 Protein binding, DNAdamage, cell death,ser/thrphosphorylation

CERS6/Ceremidesynthase 6

15.73 Sequence specific DNAbinding/transcriptionfactor activity,permeabilization

ABCA2/ATP bindingcassette

�5.52 ATPase activity,transmembranemovement, ATPbinding, nucleotidebinding

UBE2A/ubiquitin-conjugating enzyme 2

15.36 Ubiquitin protein ligaseactivity, proliferation,cell death

ANKRD36B/ankyrinrepeat domain 36B

�5.21 Ion channel Inhibitor

GLUD1/Glutamatedehydrogenase

14.78 Protein binding, cellsurvival, apoptosis

KCNIP2/Kv channelinteracting protein

�5.08 Potassium ion channelactivity, Calciumbinding, ER

Niesvizky et al.





determined to be differentially regulated between patientsreceiving bortezomib- and non-bortezomib–containingmobilization. These genes, which are summarizedin Table 4, are also associated with the 5 highest-rankedcanonical pathways.Because of the known involvement of CXCR4 and its

ligand CXCL12 (SDF-1a) in the homing and migration ofmultiple stem cell types (32) and due to its prominent rolein the ephrin signaling pathway with known functionalactivity in migration and chemoattraction (Fig. 2A), welooked at plasma levels of CXCL12 in patients receivingbortezomib- and non-bortezomib–containing mobiliza-tion. While gene expression of the CXCR4 receptor inCD34þ cells was upregulated (P < 0.01, fold change ¼4.74), plasma levels of CXCL12 were significantly lower inpatients receiving bortezomib- (n ¼ 6) versus non-borte-zomib–based (n ¼ 7) mobilization (mean, 1,319 vs. 2,225pg/mL; P < 0.001; Fig. 2B).We also investigated the levels of angiopoietin-1 protein

in plasma as ANGPT1 expression in CD34þ cells was alsosignificantly higher in patients receiving bortezomib- (n ¼7) versus non-bortezomib–based (n ¼ 9) mobilization.Angiopoietin-1 was also identified in a biomarker analysisof the gene expression data using IPA with a filter formolecules found in plasma and/or serum and in the ephrinsignaling canonical pathwaywith known functional activityin cell proliferation. Similar to the CXCL12 results, angio-poietin-1 level was significantly lower in the plasma ofpatients receiving bortezomib- versus non-bortezomib–containing mobilization (mean, 4,296 vs. 11,969 pg/mL;P < 0.001; Fig. 2C), suggesting that lower levels of plasmaangiopoietin-1 and CXCL12 could potentially be useful as

biomarkers of improved CD34þ mobilization in bortezo-mib-treated patients.

DiscussionThis phase II study evaluated the efficacy and safety of

bortezomib-based secondary induction and mobilizationin a cohort of transplant-eligible patients with multiplemyeloma who had plateaued after achieving <VGPR with,or having relapsed following, one previous primary induc-tion therapy, which was mostly IMiD-based (and predom-inantly lenalidomide-based). DoVeD induction showedsubstantial activity in this patient population, and both theinduction and mobilization regimens were well-tolerated,with the observed toxicities being consistent with thosepreviously reported for the individual drugs involved(14, 33).

DoVeD was shown to be successful for breaking pla-teaued responses to primary induction therapy, with anORR relative to pre-DoVeD assessment of 61%, including18% CR and 39% �VGPR. Twenty-seven patients in thestudy had plateaued at PRwith their first induction; DoVeDproved to be a useful regimen for further enhancingresponse to VGPR or better, thereby potentially providingpatients with the associated prognostic benefit associatedwith this level of response. Notably, as evidenced by theresponse rates to DoVeD, addition of liposomal doxorubi-cin in patients with a suboptimal initial response to borte-zomib–dexamethasone appeared effective in improvingtheir responses.

The ORR observed with DoVeD secondary induction inthis small patient population is notable in the context ofprevious phase II and III studies of this combination inpreviously untreated patients with multiple myeloma,which reported ORR rates of 78% to 95%, including42% to 62% �VGPR (4–6, 34–36). Our results indicatethat substantial activity with DoVeD-like regimens in pre-viously untreated multiple myeloma may be retaineddespite patients having previously received IMiD-basedprimary induction. Although other studies have alreadyreported encouraging activity with sequential inductionapproaches in previously untreated multiple myeloma(19–21), this study is unique in showing activity in patientswho received bortezomib-based secondary induction afterachieving maximum incomplete responses to primaryinduction with IMiD-based regimens.

Previous studies have shown an association between theachievement of �VGPR before transplant and improvedsurvival outcomes (3, 4, 9). In this study, after a medianfollow-up of 46.6 months, median PFS was 47.1 months,the 5-year OS rate was 76.4%, and only 7 patients had died.These outcomes compare favorably with respect to previousreports (3), despite the population being preselected forresistance to IMiDs; however, interpretation is limited bydifferences between patients in the treatment coursereceived.

Subsequent to DoVeD secondary induction, furthercytoreduction was seen after a single cycle of stem cell

Figure 1. Top 5 canonical cell signaling pathways associated withgenes differentially expressed in CD34þ cells between patientsreceiving bortezomib- and non-bortezomib–based mobilization. Theleft y-axis displays the �log (P), which is calculated in IPA using aright-tailed Fisher exact test. The right y-axis displays a ratiocalculated using IPA and determined by the number of significantgenes (P < 0.0025, fold change ¼ 2.00) divided by the total number ofgenes that make up the canonical pathway.






Table 4. Differential gene expression in CD34þ cells in the ephrin signaling pathway between patientsreceiving bortezomib- and non-bortezomib–based mobilization.

Symbol Entrez gene name P Fold change Location Function Movement/migration

ABI1 Abl interactor 1 6.14E-04 7.903 Cytoplasm Nucleotide binding Subcellular movementACTR2 ARP2 actin-related protein

2 homolog (yeast)2.33E-03 8.778 Plasma

membraneActin/nucleotidebinding

Subcellular movement

ARPC3 Actin-related protein 2/3complex, subunit 3,21kDa

2.35E-03 4.622 Cytoplasm Protein binding Increased migration ofNIH3T3 and HEK293

CFL2 Cofilin 2 (muscle) 2.33E-03 6.201 Nucleus Protein binding Increased migration ofvascular smoothmuscle cells

GNAQ Guanine nucleotidebinding protein (Gprotein), q polypeptide

2.29E-03 6.116 Plasmamembrane

Enzyme Migration of HUVEC

GNAS GNAS complex locus 1.86E-03 7.411 Plasmamembrane

Enzyme Migration of prostatecells

GNB1 Guanine nucleotidebinding protein (Gprotein), beta 1


Enzyme Migration of smoothmuscle cells, jurkatcells and huvecs, SDF1

GRIN1 Glutamate receptor,ionotropic, N-methyl D-aspartate 1

1.65E-03 �2.799 Plasmamembrane

Ion channel Migration of neurons

GRIN2C Glutamate receptor,ionotropic, N-methyl D-aspartate 2C

1.46E-03 �3.081 Plasmamembrane

Ion channel Projection neurons

ITGB1 Integrin, beta 1 (fibronectinreceptor, betapolypeptide, antigenCD29 includes MDF2,MSK12)


Transmembranereceptor

Migration in cell linesincludes Blymphocytes

MAP2K1 Mitogen-activated proteinkinase kinase 1

1.08E-03 5.886 Cytoplasm Kinase Migration in cell linesincluding Blymphocytes

MAPK1 Mitogen-activated proteinkinase 1

1.59E-03 6.554 Cytoplasm Kinase Migration in cell linesincluding bone marrowcells

NCK2 NCK adaptor protein 2 2.24E-03 4.436 Cytoplasm Kinase NonePGF Placental growth factor 1.62E-03 �2.301 Extracellular

spaceGrowth factor Decreased expression

causes increasedmigration in cellsincluding progenitorcells

RAC1 Ras-related C3 botulinumtoxin substrate 1


Enzyme Migration in cell lines

RAP1A RAP1A, member of RASoncogene family

1.86E-03 5.896 Cytoplasm Enzyme Migration in cell lines

RAP1B RAP1B, member of RASoncogene family

1.25E-03 3.227 Cytoplasm Enzyme Migration in cell lines

SOS2 Son of sevenless homolog2 (Drosophila)

1.61E-03 4.056 Cytoplasm Protein binding/catalytic domain

None

WIPF1 WAS/WASL interactingprotein family, member 1

1.81E-03 6.030 Cytoplasm Actin/proteinbinding

None

Niesvizky et al.





mobilization with bortezomib þ cyclophosphamide þfilgrastim, with 10 of 27 (37%) patients improving theirquality of response compared with post-DoVeD induction,including some converting to sCR. These results add to agrowing body of evidence showing the cytoreductive effect

of bortezomib in combination with cyclophosphamide(14, 15, 23) and highlight the use of continued bortezomibtreatment through mobilization.

The unexpected finding of enhanced CD34þ stemcell yields with the combination of bortezomib,

Figure 2. A, overlay of significant differential gene expression data (P < 0.05, fold change¼ 2.0) onto the canonical ephrin signaling pathway (IPA). CXCR4 andangiopoietin-1 play prominent roles in migration and proliferation, respectively, as highlighted with blue arrows. Green and red highlighting indicate genessignificantly up- and downregulated, respectively. Plasma levels of (B) CXCL12 and (C) angiopoietin-1 were significantly lower (P < 0.001) in patients whoreceived bortezomib- versus non-bortezomib–containing mobilization.






cyclophosphamide, and filgrastim for mobilization sug-gests that bortezomib may promote stem cell egress. Thisled us to undertake exploratory, retrospective gene expres-sion and IPA in an attempt to try to understand themechanism(s) underlying this phenomenon. We validat-ed our microarray gene expression data using QPCR, awidely accepted tool for such validation. The results ofour QPCR validation experiments indicated an overallagreement in the direction of gene expression changeswhen compared with microarray data but a variation inthe level of fold change between the 2 methods, as alsoreported in other studies (37, 38). Such inconsistenciesare likely due to inherent differences between the 2methods, differences in normalization methods, and thelimited amount of RNA available for QPCR (n ¼ 3–4).

The upregulation of IL8 expression levels in CD34þ cellssupports a role for IL8 signaling in stem cell mobilization,and this has been reported in the serum of normal humandonors after G-CSF mobilization (39). IL8-induced stemcell mobilization has also been shown in mice (40). Here,we report that there are no significant changes in IL8 plasmaprotein levels between bortezomib- and non-bortezomib–mobilized patients suggesting that IL8 may play a role inboth mobilization strategies. The significant increase in IL8at the gene expression may be due to differences in expres-sion levels found inCD34þ cells versus secreted IL8 proteinlevels detected in plasma. The mechanisms associated withIL8-induced stem cellmobilization remain to be elucidated.

Among the highest ranked canonical pathways exhibitingsignificant changes in gene expression identified in ouranalyses, the significant dysregulation of the ubiquitinationpathway was an expected result given the use of bortezomibin one of mobilization regimens. Bortezomib prevents theubiquitination of regulatory proteins involved in numerouspathways, including cell-cycle signaling, apoptosis, tran-scriptional regulation, and cell surface receptors and hyp-oxia-inducible factor (HIF)1-a signaling.

Our analyses also revealed modulated expression ofvarious genes known to have a functional role in cellmigration and associated with the canonical ephrin signal-ing pathway in patients receiving bortezomib during mobi-lization versus a control cohort of patients not exposed tobortezomib duringmobilization, thus implicating a role forephrin signaling via CXCL12 and angiopoietin-1 modula-tion in the differential CD34þ cell yields observed in the 2groups. The reason is unclear, but onepotential explanationis that CXCL12 may be more reduced in bone marrowstromal cells than in circulating plasma, which would allowfor CD34þ cell migration. A possible mechanism of actioncould be via the ephrin signaling pathway. Potentially,ephrin-positive hematopoietic progenitor cells (CD34þ)interact with ephrin ligand–producing cells in the bonemarrow stroma, resulting in detachment from the stromalcells and allowing the cells to migrate from the bonemarrow. Similar mechanisms have been reported in cocul-tures of CD34þ/ephrinB4þ cells with stromal cells expres-sing EphB2 ligand; in the presence of EphB2 ligand, CD34þhematopoietic progenitor cells detached from the stroma

(41). Ephrin receptors also exhibit bidirectional signalingmechanisms whereby ephrin proteins can both serve asligands for ephrin receptors but also transduce afferentsignals (upstream or downstream of signaling pathways)upon receptor binding (42). The ephrinB2 receptor and theexpression of ephrinB2/Eph4 complexes have also beenassociated with the migration of endothelial cells via recep-tor-dependent and -independentmechanisms (30, 43). Thedownregulation or inhibition of CXCR4/CXCL12 signalinghas been proposed as the mechanism by which stem cellsexit the bone marrow microenvironment (44). As shownin Fig. 2B, the downregulation of this signaling axis inplasma is evident with bortezomib-containing mobiliza-tion. These findings are similar to those reported in aprevious study in non-Hodgkin lymphoma (NHL) showingthat CXCL12 is significantly decreased in patients withlarger stem cell yields (45).

Another potential mechanism for bortezomib modula-tion of stem cell mobilization is via the downstream effectsof proteasome inhibition on angiopoietin-1 expression(46). Angiopoietin-1 and its receptor, Tie2, are importantformaintaining hematopoeitic stem cell quiescence, as wellas for adhesion of stem cells to the bone marrow microen-vironment in mice (32). Previous studies have shown theability of bortezomib todecrease angiopoietin expression inin vitro models (47). In this study, we confirm that genesdownstream of the angiopoietin signaling pathway werefound to be differentially expressed in CD34þ stem cellsfrom patients receiving bortezomib- versus non-bortezo-mib–containing mobilization. The binding of angiopoie-tin-1 to Tie2 may mediate migration of CD34þ cells andlikely involves several kinases including phosphoinositide3-kinase (PI3K), focal adhesion kinase (FAK), andPAK (48–50). This mechanism likely involves integrins. In our geneexpression data, integrins, PI3K, and PAK were upregulatedin bortezomib-treated versus non-bortezomib–treatedpatients. As with CXCL12, angiopoietin-1 produced frombonemarrow stromal cells could be contributing toCD34þcell migration from bone marrow and is likely to be differ-entially expressed in stromal cells.

In summary, this study has shown very promisingresponse rates, high CD34þ stem cell yields, and manage-able toxicitywith bortezomib-based secondary induction ina relatively small multiple myeloma patient populationwho achieved a suboptimal response to predominantlyIMiD-based primary induction therapy. Early identificationof patients who achieve a suboptimal response to primaryinduction therapy may allow for a timely switch to asecondary induction regimen offering a greater chance ofachieving better responses before transplant (4). Notably,the additionof bortezomib tomobilization or conditioningregimens to further enhance cytoreduction before SCTcould be more broadly applicable; this agent is beinginvestigated as a component of both, supported by preclin-ical data showing synergy with alkylating agents such ascyclophosphamide (in mobilization) and melphalan (inconditioning). Finally, our exploratory gene expression andpathway analyses have suggested the ephrin receptor and its

Niesvizky et al.





ligands as potentially of importance in CD34þ stem cellmigration from the bone marrow, a finding that warrantsfurther investigation.

Disclosure of Potential Conflicts of InterestR. Niesvizky, T.M. Mark, and M. Coleman have served as consultants

and participate as lecturers in the speakers’ bureaus for Celgene and Millen-nium Pharmaceuticals, Inc. R. Niesvizky received research support and drugfor the implementation of this trial. D. Skerrett is a consultant/advisoryboardmember ofMesoblast. K. Pekle has honoraria from Speakers Bureau ofMillenium. F. Zafar is a consultant/advisory board member of MillenniumCorp. No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: R. Niesvizky, R.N. Pearse, T.B. Shore, M.E. LaneDevelopment of methodology: M. Ward, S. Ely, D. Skerrett, M.E. LaneAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): R. Niesvizky, T.M. Mark, M. Ward, R.N. Pearse, T.B. Shore, K. Pekle, Z. Xiang, S. Ely, D. Skerrett, M. Coleman, M.E. LaneAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): R. Niesvizky, T.M. Mark, R.N. Pearse, M.Manco, P.J. Christos, S. Ely, D. Skerrett, M.E. Lane

Writing, review, and/or revision of the manuscript: R. Niesvizky, T.M.Mark, D.S. Jayabalan, R.N. Pearse, M. Manco, J. Stern, P.J. Christos, L.Mathews, T.B. Shore, D. Skerrett, S. Chen-Kiang, M. Coleman, M.E. LaneAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): R. Niesvizky, D.S. Jayabalan, M.Manco, J. Stern, L. Mathews, F. ZafarStudy supervision: R. NiesvizkyExpert patient care: R. Niesvizky

AcknowledgmentsThe authors thank Emma Landers and Steve Hill, medical writers with

FireKite, for writing support during the development of the manuscript,which was funded by Millennium Pharmaceuticals, Inc.

Grant SupportP.J. Christos was partially supported by the following grant: Clinical

Translational Science Center (CTSC; UL1-RR024996).The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received May 6, 2012; revised December 26, 2012; accepted January 18,2013; published OnlineFirst January 28, 2013.

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Niesvizky et al.





2013;19:1534-1546. Published OnlineFirst January 28, 2013.Clin Cancer Res Ruben Niesvizky, Tomer M. Mark, Maureen Ward, et al. CD34+ Stem Cell Mobilization

High-YieldBortezomib-Based Strategy for Secondary Induction and Overcoming the Response Plateau in Multiple Myeloma: A Novel

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