Phase I trial of bortezomib (PS-341; NSC 681239) and \"nonhybrid\" (bolus) infusion schedule of...

$: Phase I trial of bortezomib (PS-341; NSC 681239) and \"nonhybrid\" (bolus) infusion schedule of alvocidib (flavopiridol; NSC 649890) in patients with recurrent or refractory indolent$
Cancer Therapy: Clinical

Phase I Trial of Bortezomib (PS-341; NSC 681239) and"Nonhybrid" (Bolus) Infusion Schedule of Alvocidib(Flavopiridol; NSC 649890) in Patients with Recurrent orRefractory Indolent B-cell Neoplasms

BeataHolkova1,2,Maciej Kmieciak1, E.BrentPerkins1,2, Prithviraj Bose1,2, RachidC.Baz3,G.DavidRoodman4,Robert K. Stuart5, Viswanathan Ramakrishnan6, Wen Wan6, Cody J. Peer7, Jana Dawson3, Loveleen Kang8,Connie Honeycutt1, Mary Beth Tombes1, Ellen Shrader1, Caryn Weir-Wiggins1, Martha Wellons1,Heidi Sankala1, Kevin T. Hogan1, A. Dimitrios Colevas9, L. Austin Doyle9, William D. Figg7,Domenico Coppola3, John D. Roberts1,2, Daniel Sullivan3, and Steven Grant1,2,10,11,12

AbstractPurpose: This phase I study was conducted to determine the dose-limiting toxicities (DLT) and

maximum tolerated dose (MTD) for the combination of bortezomib and alvocidib in patients with B-

cell malignancies (multiple myeloma, indolent lymphoma,Waldenstrommacroglobulinemia, andmantle

cell lymphoma).

Experimental Design: Patients received bortezomib (intravenous push), followed by alvocidib (1-hour

infusion), on days 1, 4, 8, and 11 of a 21-day treatment cycle. Patients experiencing responses or stable disease

continued on treatment at the investigator’s discretion. A standard 3þ3 dose-escalation design was used to

identify the MTD based on DLTs, and pharmacokinetic and pharmacodynamic studies were conducted.

Results: A total of 44 patients were enrolled, with 39 patients assessed for response. The MTD was

established as 1.3 mg/m2 for bortezomib and 40 mg/m2 for alvocidib. The most common hematologic

toxicities included leukopenia, lymphopenia, neutropenia, and thrombocytopenia. The most common

nonhematologic toxicities included diarrhea, fatigue, and sensory neuropathy. Three complete remissions

(8%) and 10 partial remissions (26%) were observed for a total response rate of 33%. Pharmacokinetic

findings with the current dosing regimen were consistent with the comparable literature and the hybrid

dosing regimen. Pharmacodynamic study results did not correlate with clinical responses.

Conclusions: The combination of bortezomib and alvocidib is tolerable, and an MTD has been

established for this schedule. The regimen appears to be efficacious in patients with relapsed/refractory

multiple myeloma or indolent non-Hodgkin lymphoma. As the nonhybrid regimen is less cumbersome

than the previous hybrid dosing schedule regimen, the current schedule is recommended for successor

studies. Clin Cancer Res; 20(22); 5652–62. �2014 AACR.

IntroductionDespite numerous therapeutic options (e.g., cytotoxic

chemotherapy,monoclonal antibodies, radioimmunother-apy, and combinations thereof) for patients with indolent

B-cell non-Hodgkin lymphoma (NHL), cures largelyremain elusive. Allogeneic stem cell transplantation (SCT)using reduced-intensity conditioning (RIC) regimens,which carry a lower treatment-related mortality (TRM) risk

1Massey Cancer Center and 2Department of Internal Medicine, VirginiaCommonwealth University, Richmond, Virginia. 3Chemical Biology andMolecular Medicine Program, H. Lee Moffitt Cancer Center & ResearchInstitute, Tampa, Florida. 4Department of Medicine, University of Pitts-burgh, Pittsburgh, Pennsylvania. 5Department of Medicine, Medical Uni-versity of South Carolina, Charleston, South Carolina. 6Department ofBiostatistics, Virginia Commonwealth University, Richmond, Virginia.7Center for Cancer Research, NCI, NIH, Bethesda, Maryland. 8James A.Haley Veterans' Hospital, Tampa, Florida. 9Cancer Therapy EvaluationProgram, NCI, NIH, Bethesda, Maryland. Departments of 10Microbiologyand Immunology and 11Biochemistry and Molecular Biology, and 12TheInstitute for Molecular Medicine, Virginia Commonwealth University, Rich-mond, Virginia.

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

Current address for J.D. Roberts: Yale University, New Haven, CT; currentaddress for V. Ramakrishnan, Medical University of South Carolina,Charleston, SC; current address for D. Roodman, Department ofMedicine,Indiana University School of Medicine, Indianapolis, IN; and currentaddress for A.D. Colevas, Department of Medicine, Stanford University,Stanford, CA.

Corresponding Author: Beata Holkova, VCU-Massey Cancer Cen-ter, 401 College Street, PO Box 980035, Richmond, VA 23298-0035.Phone: 804-628-2581; Fax: 804-827-0810; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-14-0805

�2014 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 20(22) November 15, 20145652

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Published OnlineFirst September 23, 2014; DOI: 10.1158/1078-0432.CCR-14-0805

than myeloablative regimens, can theoretically producecures via a graft-versus-lymphoma effect, but definitiveevidence of efficacy of this strategy is lacking (1). Similarly,while proteasome inhibitors and immunomodulatorydrugs have dramatically altered the therapeutic landscapein multiple myeloma, the disease remains incurable asidefrom allogeneic SCT. Although RIC regimens have loweredthe high TRM-associated with myeloablative conditioning,convincing evidence is lacking that allogeneic SCT improvessurvival compared with autologous SCT, and the former isonly recommended in the context of clinical trials (2).Novel therapeutic approaches are, therefore, clearly neededfor patients with these diseases.Bortezomib, the first-in-class proteasome inhibitor,

received regulatory approval in the United States in 2003after large phase II and III trials demonstrated promisingsingle-agent activity in patients with relapsed or refractorymultiplemyeloma (3, 4). Itsmechanismof action (MOA) ispartly mediated through NF-kB inhibition that results inapoptosis, decreased angiogenic cytokine expression, andinterference with the tumor microenvironment. AdditionalMOAs include c-Jun N-terminal kinase activation andgrowth factor expressionmodulation (5). Subsequent com-bination phase III studies established the drug for frontlinemultiple myeloma therapy (6). On the basis of a 33%response rate in a large multicenter phase II trial (7),bortezomib was also approved for patients with relapsedor refractory mantle cell lymphoma (MCL). More recently,regimens combining bortezomib with chemoimmunother-apy have yielded high response rates (83%–88%) inpatients with relapsed/refractory indolent B-cell NHL andMCL (8, 9).As perturbations of the cell cycle are almost universal in

human malignancies, the cyclin-dependent kinases (CDK)

have become attractive targets for cancer therapy (10, 11).Alvocidib (flavopiridol), the first CDK inhibitor to enter theclinic, globally represses transcription by non-ATP–com-petitive inhibition of positive transcription elongation fac-tor b (P-TEFb, CDK9/cyclin T1; ref. 12), inducing down-regulation of the short-lived antiapoptotic protein myeloidcell leukemia-1 (Mcl-1), which may represent the principalMOA of the drug in malignant hematopoietic cells (13).Indeed, in multiple myeloma cells, for which Mcl-1 is anessential survival protein (14), alvocidib induces apoptosisthrough Mcl-1 transcriptional repression and downregula-tion (15).

Alvocidib has been administered via a variety of sche-dules (10, 11). While the single-agent activity in patientswith MCL or multiple myeloma using 1-hour intravenousbolus administration was modest to nonexistent (16, 17),marked clinical efficacywasnoted in studies inpatientswithgenetically high-risk chronic lymphocytic leukemia (CLL)using a pharmacologically derived "hybrid" administrationschedule (18, 19). However, hybrid schedule results weredisappointing in a phase I trial in patients with relapsed/refractory acute leukemias (20). Similarly, hybrid dosingdid not improve results compared with a 1-hour bolusinfusion in a phase I trial of alvocidib, fludarabine, andrituximab that reported an overall response rate of 82% inpatients with MCL, B-NHL, or CLL (21). Finally, a recentrandomized phase II comparison of bolus and hybriddosing of alvocidib (followed sequentially by cytarabineand mitoxantrone) in patients with newly diagnosed poor-risk acute myeloid leukemia (AML) showed comparablyencouraging results for both schedules, prompting selectionof a bolus administration for successor studies (22).

Malignant cells are highly susceptible to a strategy inwhich survival signaling and cell-cycle regulatory pathwaysare simultaneously disrupted (23). Furthermore, Mcl-1accumulation is an undesirable molecular consequence ofproteasome inhibitor exposure, attenuating proapoptoticeffects, and arguing that targeting Mcl-1 may increase theeffectiveness of these agents (24). In support of these con-cepts, bortezomib and alvocidib interact synergistically toinduce apoptosis in various leukemic cells (25, 26). In thiscontext, alvocidib also inhibits IkB kinase, and by exten-sion, NF-kB (27), and in this way may cooperate withbortezomib in triggering malignant hematopoietic celldeath (26).

Given these observations, a multicenter phase I trial ofthis novel drug combination was initiated in patients withrecurrent or refractory B-cell neoplasms. Patients receivedbortezomib by intravenous push followed by a 1-hourinfusion of alvocidib on days 1, 4, 8, and 11. On the basisof encouraging results in the CLL trial (18, 19), a hybridschedule of alvocidib as a 30-minute bolus infusion fol-lowed by a 4-hour infusion on days 1 and 8 was exploredand the MTD determined (28). During this time, the orig-inal nonhybrid schedule of this trial was put on holdwithout reaching the MTD and accrual was resumed aftercompletion of the hybrid schedule (28). The results of theoriginal nonhybrid schedule are described in this article.

Translational RelevancePreclinical studies with alvocidib (a cyclin-dependent

kinase inhibitor) and bortezomib (a proteasome inhib-itor) have shown that these agents interact synergisticallyto induce apoptosis in malignant hematopoietic cells.The clinical relevance of this finding for patients with B-cell neoplasms was previously tested in a phase I trial inwhich the dose-limiting toxicities (DLT) and maximumtolerated dose of the combination of these agents wasdetermined, with alvocidib administered by a "hybrid,"pharmacologically derived infusional schedule. Thephase I trial described here extends this study by admin-istering alvocidib according to a standard and consider-ably more easily managed bolus schedule. Here, wereport that the two regimenswere associatedwith similartoxicity and response profiles, including responses inpatients who had previously progressed following bor-tezomib treatment. Consequently, based on the resultsof the present trial, the nonhybrid dosing schedule isrecommended for subsequent phase II evaluation.

Phase I Trial of Bortezomib and Bolus-Infusion Alvocidib

www.aacrjournals.org Clin Cancer Res; 20(22) November 15, 2014 5653



Patients and MethodsDrug supply

Bortezomib (PS-341; NSC 681239; Millennium Pharma-ceuticals, Inc.) and alvocidib (flavopiridol; NSC 649890;Sanofi-Aventis Pharmaceuticals, Inc.) were obtainedthrough the Cancer Therapy Evaluation Program (CTEP)Pharmaceutical Management Branch, NCI (Bethesda,MD).

Patient eligibilityPatients must have had a diagnosis of relapsed or refrac-

tory follicle center lymphoma (follicular or diffuse), MCL,marginal zone B-cell lymphoma (splenic, nodal, or extra-nodal), lymphoplasmacytoid lymphoma/immunocytoma,plasma cellmyeloma, plasmacytoma, plasma cell leukemia,or Waldenstrom macroglobulinemia with no potentialcurative therapeutic options. Patients with a history of acentral nervous system involvement were ineligible. Priorautologous SCT, but not prior allogeneic SCT, was allowed.Prior bortezomib was also allowed.

Additional eligibility criteria included being at least 18years of age, an Eastern Cooperative Oncology Groupperformance status (ECOG) score of 1 or less, no grade 2or greater neuropathy, preserved kidney and liver function,hemoglobin levels of 8 g/dL or higher, an absolute neutro-phil count of 1.5 � 109/L or greater, and platelet counts of100 � 109/L or greater.

Treatment planThis study was designed as a phase I, nonrandomized,

dose-escalation study to determine the MTD for the borte-zomib and alvocidib combination, where alvocidib isadministered as a 1-hour infusion. The dose of alvocidibranged from 15 mg/m2 at dose level 1 to 90 mg/m2 atdose level 9. The dose of bortezomib for dose level 1 was1.0 mg/m2 and for dose levels 2–9 was 1.3 mg/m2. On days1, 4, 8, and 11 of a 3-week cycle, bortezomib was admin-istered via intravenous push over 3 to 5 seconds, after whichalvocidib was administered as an intravenous infusion over1 hour. Treatment was repeated every 3 weeks.

Disease status was assessed after the first 6 weeks oftreatment and every 6 to 8 weeks thereafter. Patientsexperiencing a response or stable disease were allowedto continue treatment indefinitely at the investigator’sdiscretion. Patients received full supportive care includingherpes zoster prophylaxis. No specific precautions fortumor lysis syndrome (TLS) or cytokine release syndrome(CRS) were used before August 2007, but upon resump-tion of accrual to the nonhybrid regimen in May 2010,specific measures were applied. Pretreatment with allo-purinol and overnight in-hospital observation for CRSand TLS monitoring were strongly encouraged for allpatients. Clinical discretion was used to determine wheth-er more stringent tumor lysis precautions were indicatedon the basis of tumor burden and risk. For any signs ofCRS, patients were treated with dexamethasone and pro-phylaxis was encouraged for those with circulating tumorcells documented by routine blood count.

Study design, definition of dose-limiting toxicity, andidentification of the MTD

The standard 3þ3dose-escalation designwas used,with adose level expansion of up to 6 evaluable patients if a dose-limiting toxicity (DLT) was noted. The MTD was defined asthe highest dose level at which fewer than 2 of 6 patientsexperienced a DLT.

A DLT was initially defined as any of the followingoccurring during the first cycle of treatment and determinedto be possibly, probably, or definitely related to studytreatment: (i) grade 4 anemia; (ii) grade 4 absolute neutro-phil count or platelet toxicity of greater than one weekduration; (iii) grade 3 or greater febrile neutropenia; and(iv) any grade 3 or greater nonhematologic adverse eventsexcept grade 3 or greater nausea and vomiting withoutprophylactic and/or symptomatic treatment, grade 3 orgreater diarrhea without maximum opioid and/or octreo-tide treatment, and grade 3 or greater infections, opportu-nistic infection with grade 2 or greater lymphopenia (spe-cifically herpes zoster or herpes simplex infection). A pro-tocol amendment (October 2008) modified the DLT def-inition to include instances inwhich both drugs are omitteddue to dose modifications on at least two days of thescheduled drug administration during the first cycle.

Toxicity evaluationNCI Common Terminology Criteria for Adverse Events

(CTCAE) v3.0 was used for reporting adverse events untilApril 1, 2011, when mandatory conversion to CTCAE v4.0supervened.

Response evaluationResponse criteria used were based on the nature of the

disease as follows: (i) patients with lymphomas were eval-uated using theNCI-sponsoredWorkingGroupLymphomaResponse Criteria (29); (ii) patients with plasma cell mye-loma or plasmacytoma were evaluated according to Euro-pean Group for Blood and Bone Marrow Transplant(EBMT) criteria (30); (iii) patients with plasma cell leuke-mia were evaluated according to the criteria of Vela-Ojedaand colleagues (31); and (iv) patients with Waldenstrommacroglobulinemiawere evaluated according to the SecondInternational Workshop onWaldenstrommacroglobuline-mia criteria (32).

Alvocidib pharmacokinetic studiesSamples from the first 29 enrolled patients were obtained

for alvocidib pharmacokinetic analysis. The samples wereobtained before and after treatment on cycle 1 day 1 andcycle 3 day 8 according to the following schedule: preinfu-sion, immediately postinfusion, and 1, 2, 4, 8, 12, and 24hours postinfusion. Alvocidib concentrations were ana-lyzed as previously described (28).

Enrichment of CD138þ multiple myeloma cells frombone marrow

Paired bone marrow aspirates were obtained from mul-tiple myeloma patients only: (i) before initial study

Holkova et al.

Clin Cancer Res; 20(22) November 15, 2014 Clinical Cancer Research5654



treatment, and (ii) on day 2 of cycle 1, approximately 24hours after initial treatment. CD138þ multiple myelomacells were enriched from bone marrow as previouslydescribed (28).

Protein extraction and Western blot analysisProtein extraction and Western blot analyses with pri-

mary antibodies to pJNK, Mcl-1, XIAP, and GAPDH weredone as previously described (28). A PARP-specific anti-body (BD Pharmingen) was also included. An OdysseyImager (LI-COR Biosciences) was used to quantify bindingof IRDye 680LT-conjugated secondary antibodies (LI-CORBiosciences).

Localization and quantitative immunofluorescence ofp65/RelARelA belongs to a family of transcription factors of NF-kB

complex. Cytospin preparations of enriched CD138þ plas-ma cells were prepared from study patients before andduring treatment. The cells were fixed, stained, and visual-ized by fluorescence microscopy as previously described(28). Analysis of NF-kB within the nucleus was performedusing the Definiens Developer v1.5 software suite (Defin-iens). The nuclear region was determined with automaticthreshold segmentation on the DAPI and Alexa 488 stains;further refined for plasma cells by the size and shape of thenuclei. Mean pixel intensity of the Alexa 488 signal wasdetermined from the nuclei of the plasma cells.

Statistical analysisBasic demographics, adverse events, DLTs, dose levels,

and clinical responseswere summarizedbybasic descriptivestatistics such as frequency, proportion, mean,median, andrange. Pharmacokinetic and pharmacodynamic analysesincluding all biomarkers and p65/ReIA were limited todescriptive statistics.

Human investigation studiesStudies were performed after Institutional Review Board

approval and in accordancewith an assurancefiledwith andapproved by the Department of Health and Human Ser-vices. Informed consent was obtained from each patient.TheClinicalTrials.gov trial registration ID isNCT00082784.

ResultsPatient characteristicsA total of 44 patients, 12 female and 32 male, were

enrolled on study between March 2004 and July 2012(Table 1). The study participants included 35 Caucasians(including 4 Hispanics) and 9 African Americans (includ-ing 1 Hispanic). The median age of patients was 64.5years (range: 40–79). Twenty-four patients were diag-nosed with multiple myeloma, 2 were diagnosed withWaldenstrom macroglobulinemia, and 18 were diag-nosed with NHL (5 of whom had MCL, an NHL subset).The median number of prior treatments was 3, with arange of 1 to 10. Ten patients had received prior auto-

logous SCT and 10 patients had prior bortezomib ther-apy. Patients received a median of 5 cycles of studytreatment, with a range of 1 to 16 cycles.

Safety and tolerabilityMyelosuppression was a common hematologic toxicity

and typicallymanifested itself as grade 3 leukopenia (30%),lymphopenia (25%), neutropenia (34%), or thrombocy-topenia (27%; Table 2). Grade 4 neutropenia (23%) andthrombocytopenia (14%) were also commonly observed.The most common nonhematologic toxicities includedgrade 3 diarrhea (20%) and fatigue (16%). Grade 3 painand grade 3 sensory neuropathy were slightly less common-ly experienced. One patient experienced grade 4 sensoryneuropathy. A patient with follicular lymphoma developedgrade 3 oral mucositis/esophagitis (which lastedmore than7 days) on day 2 of cycle 1, associated with both paraneo-plastic pemphigus and a grade 4 CD4 lymphocyte countwithout HIV infection. The prior treatment regimen of thepatient was rituximab and bendamustine. The most com-mon grade 2 hematologic and nonhematologic toxicities,occurring in at least 20% of patients, are listed in Table 2.Before initiation of prophylactic antiviral treatment with

Table 1. Patient enrollment and characteristics

Gender, nFemale 12Male 32Total 44

Race, nBlack or African American 9White 35

Ethnicity, nHispanic or Latino 5Non-Hispanic 39

Age range, yMedian 64.5Range 40–79

Performance status, n0 211 23

Diagnosis, nMultiple myeloma 24Waldenstrom macroglobulinemia 2NHL (subset of NHL–MCL) 18 (5)

Prior treatment, number of regimensMean 3.4Median 3Range 1–10

Prior treatment, nAutologous stem cell transplantation 10Bortezomib 10

Study treatment, number of cycles initiatedMean 4.5Median 5Range 1–16





acyclovir or comparable agents, 2 patients experiencedgrade 3 herpes zoster.

DLT and MTDIn the initial enrollment phase to the nonhybrid schema,

1 DLT was seen at dose level 5, necessitating expansion to 6DLT-evaluable patients (Table 3). No other DLTs werenoted in that expansion cohort. Patient enrollment contin-ued through 3 DLT-evaluable patients at dose level 7 before

the trial was interrupted and switched to the evaluation ofthe hybrid schema. Upon return to the nonhybrid schema,the 3þ3 dose-escalation design of the study allowed pro-gression through dose level 9. DLT accumulation at subse-quent higher dose levels required a deescalation to lowerdose levels until theMTDwas reached at dose level 5. Grade3 DLTs, experienced by 1 patient each, included back pain,fatigue, peripheral neuropathy, febrile neutropenia, TLS,diarrhea, and esophagitis/oral mucositis. TLS was

Table 2. Hematologic and nonhematologic toxicities occurring during any treatment cyclea

Events/patients

Grade 2b Grade 3 Grade 4

Hematologic toxicitiesAnemia 24/13 1/1 0/0Leukopenia 46/20 29/13 6/3Lymphopenia 43/12 18/11 1/1Neutropenia 33/20 33/15 15/10Thrombocytopenia 50/24 19/12 9/6

Nonhematologic toxicitiesAnorexia 16/12CD4 lymphocytes decreased 1/1 0/0Dehydration 3/3 0/0Diarrhea 33/20 9/9 0/0Esophagitis 1/1 0/0Fatigue 53/27 8/7 0/0Febrile neutropenia 2/2 0/0Hypoglycemia 2/1 0/0Hypotension 1/1 0/0Infection: blood (normal ANC) 0/0 1/1Infection: peripheral nerve (zoster)c 2/2 0/0Mucosal infection 1/1 0/0Mucositis oral/pharyngeal 1/1 0/0Muscle weakness, lower extremity 1/1 0/0Myalgia 1/1 0/0Nausea 10/9 1/1 0/0Neuropathy: sensory 11/9 5/5 1/1Neuropathy: motor 1/1 0/0Oral dysesthesia 1/1 0/0Pain: abdomen 1/1 0/0Pain: back 1/1 0/0Pain: bone 1/1 0/0Pain: extremity 3/3 0/0Pain: kidney 1/1 0/0Pain: neuralgia/peripheral nerve 3/2 0/0Pneumonia 1/1 0/0Syncope 1/1 0/0Tumor lysis syndrome 1/1 0/0Weight loss 1/1 0/0

aOnly those toxicities deemed possibly, probably, or definitely related to the treatment are included in the table.bOnly grade 2 toxicities that occurred in at least 20% of the patients are reported.cCases appeared before a protocol amendment dated January 2005, which included use of prophylactic antiviral treatment for herpeszoster.

Holkova et al.




manifested by short-lived laboratory perturbations (elevat-ed phosphate and uric acid) resolving within 12 hourswithout sequelae or recurrence. One patient (dose level9) experienced neutropenia that prompted dose omissionof 2 scheduled doses of both agents (due to grade 4 ANC onday 4 and grade 3 ANC on day 8). Although grade 4neutropenia lasting less than 7 days did not meet DLTcriteria, omission of 2 scheduled doses of both investiga-tional agents for this adverse event did meet DLT criteria.TheMTD for a schedule of drug administration ondays 1, 4,8, and 11 of a 21-day cycle was determined to be 1.3mg/m2

bortezomib (intravenous push) and 40 mg/m2 alvocidib(1-hour infusion).

Disease responseAlthough this studywas not powered to assess response, 3

complete responses (CR) and 10 partial responses (PR)were observed among the 39patients evaluable for response(overall response rate¼ 33%; Table 4). The overall responserate was similar for both NHL patients (33%) and multiplemyeloma patients (32%), with a 50% response rate in alimited sample of patients with Waldenstrom macroglob-ulinemia. CRs were experienced by 2 (one of whom hadMCL) of the 15 NHL patients (13%) and 1 of the 24multiple myeloma patients (4%). The multiple myelomapatient with the CR had previously been treated withbortezomib. PRs were achieved in 26% of the patients—

3 with NHL (20%), 6 withmultiple myeloma (27%), and 1withWaldenstrommacroglobulinemia (50%). A total of 20patients had stable disease (SD) as best response and 6patients had progressive disease (PD). Among the patientsnot evaluable for response, 3 patients cameoff treatment foradverse events before the disease assessment interval,1 patient came off treatment in cycle 1 due to a secondcancer (renal) and did not have a response assessment, and1 patient came off treatment in cycle 1 for convenience(moved far from the treatment center) and did not have aresponse assessment.

Pharmacokinetic studiesSamples were collected from the first 29 patients enrolled

to the nonhybrid schedule. Alvocidib pharmacokineticswas evaluated using a noncompartmental (model-indepen-dent) approach. A sample immediately at the end of the 1-hour alvocidib infusion was only collected in 11 of the29 patients. Consequently, peak alvocidib concentration(CMAX) could be reported for these 11 patients. Of these 11patients, only 9 had sufficient data to calculate a terminalelimination rate for the determination of half-life, clear-ance, and AUCINF. No lag in CMAX was observed. Maximumalvocidib concentration was observed immediately at theend of the infusion (Supplementary Fig. SS1). Cycle 1 day 1alvocidib pharmacokinetic parameters per dose level areshown in Supplementary Table S1.

Table 3. Dose levels and DLTs

Doselevel

Bortezomib(mg/m2)

Alvocidib(mg/m2)

Patientstreated/DLTs, n DLT

1 1.0 15 3/02 1.3 15 5/03 1.3 22 3/04 1.3 30 3/05a 1.3 40 7/1 Grade 3 back pain6b 1.3 50 6/2 Grade 3 fatigue

Grade 3 peripheral neuropathy7b 1.3 60 8/2 Grade 3 febrile neutropenia

Grade 3 tumor lysis syndrome

The protocol underwent a mandatory amendment at dose level 7 (prior to reaching the MTD of the pre-hybrid schedule) to pursue ahybrid dosing schedule (28). After determining the MTD of the hybrid-dosing schedule, accrual resumed to the pre-hybrid dosingscheduleat dose level 8. Patientswereenrolled throughdose level 9,whenDLTs required retrogradeexpansionofdose levels 8 through6 in order to find the MTD of the pre-hybrid schedule.

8b 1.3 75 6/2 Grade 3 diarrheaGrade 3 esophagitis/oral mucositis

9b 1.3 90 3/2 Grade 3 febrile neutropeniaOmission of 2 scheduled treatments due to neutropeniac

aMTD.bExceeded MTD.cCycle 1 day 4 treatment omitted for grade 4 ANC; cycle 1 day 8 treatment omitted for grade 3 ANC. Because the grade 4 ANC lastedless than 7 days, it was not by itself a DLT by the criteria in the protocol; however, it met DLT criteria by prompting 2 dose omissions incycle 1.





PharmacodynamicsTo determine the feasibility of measuring pharmacody-

namic markers as a predictor of response, pre- and post-treatment samples (24 hours after the first doses of drugs)were obtained from the multiple myeloma patients forquantitative analysis of p65/RelA nuclear localization byimmunofluorescence staining (Fig. 1) and for quantitativeanalysis of pJNK, Mcl-1, PARP, and XIAP by Western blot-ting (Fig. 2). Changes in expression of these proteins werepreviously observed in cells coexposed to alvocidib andbortezomib in vitro (25).

Cytospin preparations of enriched CD138þ plasma cellswere prepared from the pre- and posttreatment bone sam-ples from 8 patients with multiple myeloma including 2patients from the PR group (DS027 and DS046), 5 patientsfrom the SD group (DS011, DS047, DS050, DS055, andSG015), and 1 patient from the PD group (DS023). NF-kBactivation was measured by monitoring changes in nuclearp65/RelA localization, as a function ofmean pixel intensity,in pre- and posttreatment samples as shown in Fig. 1A.Minimal changes in nuclear RelA localization were detectedin 3 patient samples (DS027, DS050, and DS023). Down-regulationofnuclear p65/RelA after treatmentwasobservedin 3 patient posttreatment samples (DS011with 38% lowerexpression, DS047 with 31% lower expression, and DS055with 29% lower expression). Upregulation of nuclear RelAwas observed in 2 patients (DS046 with 44% higher expres-sion and SG015 with 149% higher expression).

Expression levels of Mcl-1, pJNK, PARP, and XIAP in thepre- and posttreatment CD138þ cells from 4 patients(DS047, DS050, DS055, and SG015) in the SD group weredetermined by Western blot analysis (Fig. 2). Althoughsome samples exhibited high magnitude changes in theposttreatment sample in comparison with the pretreatmentsample (e.g., an 81% decrease of PARP for patient DS055; a500% increase in XIAP for patient DS047), there was no

correlation of the changes with clinical activity as consistentincreases or decreases in expression were not observed forany of the proteins across all of the patients.

DiscussionIn the original study of the alvocidib/bortezomib regi-

men initiated inMarch 2004, 29 patients were first enrolledto the nonhybrid schedule at dose levels 1 to 7 withoutreaching theMTD. In2007, theprotocol underwent aCTEP-recommended amendment to proceed to a hybrid schedulebased on promising data in CLL (19). Results from thehybrid schedule trial have beenpublished (28). A total of 16patients were treated, consisting of 11 male and 5 femalepatients. Nine patients had NHL (6 patients had MCLsubtype), 6 had multiple myeloma, and 1 had an extra-medullary plasmacytoma. Treatment was on a 21-day cycle:bortezomib by intravenous push on days 1, 4, 8, and 11 andalvocidib on days 1 and 8 by 30-minute bolus infusionfollowed by a 4-hour continuous infusion. The MTD wasestablished as 1.3mg/m2 for bortezomib and 30mg/m2 foralvocidib (both the 30-minute bolus and4-hour infusions).There were 2 CRs (12%) and 5 PRs (31%) observed (overallresponse rate ¼ 44%). After determining the MTD for thehybrid schedule (28), data from the prehybrid schedulewere reevaluated and determined to demonstrate a similarresponse and toxicity profile. With the agreement of CTEP,the nonhybrid schema trial was reopened to patient enroll-ment in May of 2010.

The results of this study indicate that this schedule of thecombination of bortezomib and alvocidib can be admin-istered safely to patients with recurrent or refractory indo-lent B-cell neoplasms (including NHL and multiple mye-loma). The MTD was determined to be 1.3 mg/m2 ofbortezomib (intravenous push) followed by 40 mg/m2 ofalvocidib (1-hour infusion) on days 1, 4, 8, and 11 on a 21-

Table 4. Treatment response by diagnosisa

NHL Multiple myelomaWaldenstrom

macroglobulinemia Total

Diagnosis (n ¼ 15) (n ¼ 22) (n ¼ 2) (n ¼ 39)

CR 2b,c 1d 0 3PR 3 6 1 10Total CR þ PR (%) 5 (33%) 7 (32%) 1 (50%) 13 (33%)

SD 7 12 1 20PD 3e 3 0 6

aExcluded from the totals are 5 patients who were not evaluable for response: 3 patients came off treatment for adverse events beforethe disease assessment time point, 1 patient came off treatment in cycle 1 due to a second cancer (renal mass) and did not have aresponseassessment, and1patient cameoff treatment in cycle 1 for convenience (moveda far distance from the treatment center) anddid not have a response assessment.bIncludes 1 patient with prior autologous SCT.cIncludes 1 patient with MCL.dIncludes 1 patient with prior bortezomib.eIncludes 3 patients with MCL.

Holkova et al.




day cycle (Table 3). In a previous hybrid study of theseagents in the same patient population, the MTD was foundto be 1.3mg/m2 of bortezomib (intravenous push) on days1, 4, 8, and 11 followedby ahybrid schedule of 30mg/m2ofalvocidib (30-minute infusion) and then 30 mg/m2 ofalvocidib (4-hour infusion) on days 1 and 8 of a 21-daycycle (28).The most common hematologic toxicities for both the

current and previous study involved myelosuppression

(including grade 3 leukopenia and lymphopenia) andgrades 3 and 4 neutropenia and thrombocytopenia (Table2; ref. 28). Likewise, the nonhematologic toxicity profile forboth studies was very similar with grade 3 diarrhea, fatigue,and sensory neuropathy most frequently reported. One ofthe DLTs in the current regimen was grade 3 laboratory TLSin 1 patient with Waldenstrom macroglobulinemia at doselevel 7 (Table 2). The event was managed with intravenousfluids and laboratory abnormalities normalized within 12hours. This patient received a total of 6 cycles of thetreatment and achieved a PR. No TLS was observed withthe hybrid schedule (28). DLTs for both studies includedgrade 3 fatigue and febrile neutropenia, while the previousstudy also reported a single case of grade 3 aspartate ami-notransferase elevation. Two patients on the present studydeveloped herpes zoster (Table 2), but no additional epi-sodes were reported following prophylactic antiviral ther-apy. Similarly, no episodes of herpes zoster were reported inthe hybrid study, which included prophylactic antiviraltherapy. (28). Patients received a median of 5 cycles oftreatment on the current study (Table 1) compared with amedian of 4 cycles of treatment on the previous study (28).Thus, both study regimens displayed a similar safety profile.

Although the primary objective of this phase I study wasnot to determine the efficacy of the treatment regimen, 3CRs (NHL¼ 2,multiple myeloma¼ 1) and 10 PRs (NHL¼3, multiple myeloma ¼ 6, Waldenstrom macroglobuline-mia¼ 1) were observed for an overall CRþPR response rateof 13 patients out of 39 evaluable patients (33%; Table 4).This compares to an overall CRþPR response rate of 7patients of 16 evaluable patients (total ¼ 44%, NHL ¼33%, multiple myeloma ¼ 57%) in the previous study ofthe hybrid regimen (28). As the multiple myeloma patientin the present study who achieved a CR had previously

A

0

20

40

60

80

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120

DS027 DS046 DS011 DS047 DS050 DS055 SG015 DS023

PR SD PD

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mea

n R

elA

DS050 post

DS011 pre DS011 post

DS050 pre

DS015 postDS015 pre

B

Figure 1. Expression of p65/RelA in plasma cells obtained from patients with multiple myeloma (MM). A, p65/RelA fluorescent microscopy of plasma cellsisolated from 3 patients with SD. Bonemarrow aspirateswere collected before treatment and 24 hours after treatment, and plasma cells were stained for RelA(green) and DAPI (blue). Relative to the pretreatment samples, sample DS011 displays a decrease in expression, sample DS050 displays no change inexpression, and sampleSG015displays an increase in expression. B, expressionof nuclear p65/RelAwas analyzedby immunofluorescence as inA. Themeanpixel intensity for RelA is shown (mean � SD).

Figure 2. Pharmacodynamic analysis of pretreatment and posttreatmentbone marrow samples. CD138þ cells were isolated at baseline(pretreatment) and 24 hours after the first dose of alvocidib andbortezomib treatment (posttreatment).Whole-cell lysateswere prepared,electrophoresed on a 4%–12% NuPAGE gel, and Western blotted withprimary antibodies to pJNK, Mcl-1, PARP, XIAP, and GAPDH. Followingdevelopment with an IRDye 680LT-conjugated (LI-COR) secondaryantibody, the fluorescent signals were quantitated on an OdysseyImager. Values below the figures represent the level of assayed proteinsdetermined by densitometric analysis normalized to GAPDH levels,where the normalized level of the pretreatment sample is defined as100%.





progressed on bortezomib, this raises the possibility thateither alvocidib exhibited single-agent activity, or, as pre-viously observed in in vitro studies (25), it may have coop-erated with bortezomib to lower the cell death threshold.Previously reported response rates for bortezomib as asingle agent in relapsed/refractory disease are approximate-ly 33% forMCL (7), 35%multiplemyeloma (3), and 13.3%for indolent NHL including follicular lymphoma, marginalzone lymphoma, and small lymphocytic lymphoma (33).Because this study was not powered to characterize efficacy,firm conclusions about the activity of the combination ofbortezomib and alvocidib in comparison with bortezomibas a single agent cannot be drawn. Nevertheless, theresponse rates in this phase I trial, which involved multi-ply-treated relapsed/refractory patients, are encouragingand warrant further study.

This nonhybrid schedule produced comparable pharma-cokinetic parameter values as the hybrid schedule (28).Furthermore, the pharmacokinetics of the nonhybridschedule were consistent with published results using 1-hour infusion schedule (34). Alvocidib pharmacokineticsin the absence of bortezomib were not evaluated in thisclinical trial. Consequently, direct comparison cannot bemade. However, pharmacokinetic parameters are in veryclose agreement with a previous study (34, 35) for dose-normalized CMAX, dose-normalized AUC, half-life, andclearance. Together, these results suggest that the adminis-tration of bortezomib immediately before the alvocidibinfusion does not affect its disposition.

On the basis of previous findings demonstrating thatbortezomib and alvocidib interact synergistically to induceapoptosis in human leukemia cells (25, 26), it was plausibleto postulate that coadministration of these agents in vivo topatients with B-cell malignancies might induce analogousperturbations in apoptotic-regulatory pathways in tumorcells as those previously observed in vitro. To assess thefeasibility of acquiring the samples, and to begin evaluatingpharmacodynamic markers of biologic effects of treatment,CD138þ plasma cells were obtained from the bonemarrowof the patients with multiple myeloma, both before and 24hours after treatment with bortezomib and alvocidib.Because bortezomib can inactivate NF-kB, which may acti-vate the JNK pathway (26) as well as downregulate XIAP(36), translocation of the p65/RelA subunit of NF-kB to thenucleus and the accumulation of pJNK and XIAP wereselected as candidate markers. Similarly, because alvocidibcan attenuate Mcl-1 cytoprotective effects by preventingproteasome inhibitor-mediated Mcl-1 accumulation (25),total Mcl-1 protein levels were monitored. Finally, PARPcleavage was used as an indicator of apoptosis.

Of 24 multiple myeloma patients in the study, immu-nofluorescence staining for p65/RelA was conducted on 8paired plasma cell samples and Western blot analysis forpJNK, Mcl-1, PARP, and XIAP was conducted on 4 pairedplasma cell samples. Among the samples assayed for nuclearp65/RelA expression, there was no clear pattern of expres-sion associated with disease status. Although a decrease inexpression was observed in 3 samples (all PRs), an increase

in expressionwas observed in 2 samples (1 PR and1 SD; Fig.1). Similarly, among the 4 paired samples (all SD) analyzedby Western blot analysis, and despite the fact that single-agent alvocidib has been shown to diminish Mcl-1 expres-sion (37), only one sample showed adecrease in expression.While this result was unanticipated, it is possible that in thisinstance, diminished Mcl-1 proteasomal degradation mayhave predominated. Although a major mode of action ofthe bortezomib/alvocidib regimen is presumed to be apo-ptosis induction, only sample SG015 (from a patient withSD) displayed enhanced posttreatment PARP cleavage.Whether the inconclusive results seen here reflect the smallsample size, differing drug doses, or the possibility thatthese molecules may not be robust markers of therapeuticactivity cannot be determined in the context of this phase Itrial. While bone marrow sampling was mandatory forpatients with myeloma, it proved logistically cumbersome,thereby limiting the number of samples obtained. Thesequestions could possibly be answered in a more highlypowered phase II trialwith larger patient numbers andmoreuniform drug dosing.

In conclusion, this phase I study has determined theMTDfor the combination of bortezomib and alvocidib admin-istered as a 1-hour infusion in patients with recurrent orrefractory indolent B-cell cancers. Both the observed hema-tologic and nonhematologic toxicities were similar to thoseobserved with bortezomib as a single agent combined witha hybrid dose schedule of alvocidib (28). The dosing reg-imen employed here resulted in 3 CRs and 10 PRs in aheavily pretreated patient population. The frequency ofthese CRs and PRs was encouraging and similar to thatobserved in a previous trial of these agents that employed amore cumbersome hybrid dosing schedule (28). Becausethe simpler regimen used in the current trial demonstratedcomparable activity to the hybrid dosing scheme, andbecause the nature and frequency of the toxicities associatedwith the regimens did not differ substantively, the resultsreported here argue for selection of the nonhybrid regimenfor successor combination trials of alvocidib. As the toxi-cities of the current regimen were not inconsequential,plans are under development for a new phase I trial ofalvocidib and a next-generation orally active proteasomeinhibitor, MLN9708, administered in conjunction with thenonhybrid schedule of alvocidib in patients with multiplemyeloma who have progressed following any proteasomeinhibitor treatment.

Disclosure of Potential Conflicts of InterestG.D. Roodman reports receiving a commercial research grant from Eli

Lilly and is a consultant/advisory board member for Amgen. No potentialconflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: B. Holkova, A.D. Colevas, W.D. Figg, J.D. Roberts,S. GrantDevelopment of methodology: B. Holkova, M. Kmieciak, V. Ramakrish-nan, C.J. Peer, L.A. Doyle, D. Coppola, J.D. Roberts, S. GrantAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): B. Holkova, M. Kmieciak, E.B. Perkins, R.C. Baz,

Holkova et al.




G.D. Roodman, R.K. Stuart, C.J. Peer, L. Kang, M.B. Tombes, E. Shrader,C. Weir-Wiggins, W.D. Figg, D. Coppola, J.D. Roberts, D. SullivanAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): B. Holkova, M. Kmieciak, R.C. Baz,W. Wan, C.J. Peer, J. Dawson, E. Shrader, C. Weir-Wiggins, H. Sankala, K.T. Hogan, A.D. Colevas, W.D. Figg, D. Coppola, S. GrantWriting, review, and/or revision of the manuscript: B. Holkova,M. Kmieciak, P. Bose, R.C. Baz, G.D. Roodman, R.K. Stuart, V. Ramakrish-nan, C.J. Peer, M.B. Tombes, E. Shrader, H. Sankala, K.T. Hogan, A.D.Colevas, L.A. Doyle, W.D. Figg, D. Coppola, D. Sullivan, S. GrantAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): B. Holkova, J. Dawson,C. Honeycutt, M.B. Tombes, E. Shrader, M. WellonsStudy supervision: B. Holkova, R.C. Baz, C. Honeycutt, C. Weir-Wiggins,A.D. Colevas, L.A. Doyle, J.D. Roberts, D. SullivanOther (helped in acquistion of image analysis data): L. KangOther (study implementation, subject, site, and data management):M.B. Tombes

AcknowledgmentsThe authors thank Sarah Kolla (Virginia Commonwealth University)

for assistance with the pharmacodynamic studies, Erin Gardner (NCI)for her assistance with the pharmacokinetic studies, and Joe Johnson(H. Lee Moffitt Cancer Center & Research Institute) for his valuable

assistance in the Analytical Microscopy Core Facility supported by NCIP30 CA76292. Services in support of the research project were gener-ated by the Virginia Commonwealth University Massey Cancer CenterBiostatistics Shared Resource, supported, in part, with funding fromNCI Cancer Center Support Grant P30 CA016059.

Grant SupportThe study was supported by grants from the NIH [NCI R01 CA93738,

NCI R01 CA100866, NCI R21 CA110953, NCI R01 CA167708, NCI P50CA130805 (Lymphoma SPORE), NCI P30 CA016059 (Cancer CenterSupport Grant to Massey Cancer Center), NCI P30 CA76292 (CancerCenter Support Grant to H. Lee Moffitt Cancer Center & ResearchInstitute), NCI N01 Contract HHSN261201100100C for the SoutheastPhase 2 Consortium, NCRR M01 RR000065 (GCRC), and NationalCenter for Advancing Translational Sciences KL2TR000057 (CTSA)] andan award from the Multiple Myeloma Research Foundation.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received April 2, 2014; revised July 17, 2014; accepted August 16, 2014;published OnlineFirst September 23, 2014.

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2014;20:5652-5662. Published OnlineFirst September 23, 2014.Clin Cancer Res Beata Holkova, Maciej Kmieciak, E. Brent Perkins, et al. in Patients with Recurrent or Refractory Indolent B-cell Neoplasms(Bolus) Infusion Schedule of Alvocidib (Flavopiridol; NSC 649890) Phase I Trial of Bortezomib (PS-341; NSC 681239) and ''Nonhybrid''

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