Leukemia Research 23 (1999) 177–183

Safe mobilization of normal progenitors in advanced chronicmyeloid leukemia with intensive chemotherapy and

granulocyte-colony stimulating factor

James Morton a, Peter Mollee a, Kerry Taylor a,*, Andrew Grigg b, Simon Durrant c,Diana Moore a, Robyn Rodwell a, Paul Eliadis g, Cheryl Hutchins c, Brett Williams a,

Greg Seeley a, Susan Wright a, Cathryn Kelly a, Andrea Rentoul a, Harry Iland d,Kerry Atkinson e, Henry Januszewicz f, Ian Bunce g, John Bashford g,

Carolyn Stewart h, Debra Taylor a

a Mater Hospital, Raymond Terrace, South Brisbane, Q4101, Australiab Royal Melbourne Hospital, Melbourne, Australia

c Royal Brisbane Hospital, Brisbane, Australiad Royal Prince Alfred Hospital, Sydney, Australia

e St Vincent’s Hospital, Sydney, Australiaf Peter McCallum Institute, Melbourne, Australia

g Wesley Hospital, Brisbane, Australiah AMGEN, Australia

Received 3 March 1998; accepted 8 April 1998

Abstract

Twenty-one patients with advanced chronic myeloid leukemia (late chronic phase (n=8), accelerated phase (n=11) and blastcrisis (n=2)) were treated with idarubicin, cytarabine, and etoposide followed by G-CSF and subsequent collection of peripheralblood progenitor cells in the early recovery phase. Treatment was reasonably well tolerated with no deaths or intensive careadmissions. Despite the advanced phase of disease and heavy pretreatment with cytotoxics and interferon-alfa, 11 of 21 patients(52%) achieved a cytogenetic response. Of the nine major cytogenetic responses (complete (n=3) and partial (n=6)), sevenachieved adequate progenitor collections for consideration for autologous transplantation. The only predictor of response wasdisease duration (P=0.02). With a median follow-up of 1171 days from treatment it appears unlikely that G-CSF contributed todisease progression. Survival post-IcE was predicted by disease stage (P=0.0001). Intensive chemotherapy followed by G-CSFallowed adequate yields of predominantly Philadelphia chromosome negative progenitor cells to be obtained from one-third ofpatients with advanced CML. © 1999 Elsevier Science Ltd. All rights reserved.

Keywords: Chronic myeloid leukemia; Intensive chemotherapy; Autologous stem cell transplant

1. Introduction

Chronic myeloid leukemia (CML) is a clonal neo-plastic disorder of the primitive hematopoietic stem cellcharacterized by the presence of the Philadelphia (Ph)chromosome. The median survival from diagnosis is 4years with a predictable triphasic course [1]. Conven-tional chemotherapy affords hematological control ofthe chronic phase of CML but does little to alterdisease progression [2]. Interferon-alfa (a-IFN) pro-

Abbre6iations: a-IFN, interferon-alfa; aBMT, autologous bonemarrow transplantation; ANC, absolute neutrophil count; CML,chronic myeloid leukaemia; G-CSF, granulocyte-colony stimulatingfactor; IcE, idarubicin, cytarabine, etoposide; PBPC, peripheral bloodprogenitor cells; Ph+, Philadelphia positive; Ph−, Philadelphia nega-tive; RT-PCR, reverse transcriptase nested polymerase chain reaction.

* Corresponding author.

0145-2126/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.

PII: S0145-2126(98)00143-X

J. Morton et al. / Leukemia Research 23 (1999) 177–183178

duces durable major cytogenetic responses in earlychronic phase CML and may be associated with sur-vival advantage [3–5], with additional benefit fromthe addition of cytarabine [6]. Allogeneic stem celltransplantation is the only current curative therapy,however donor availability and age constraints limitthis modality to only 20% of CML patients. Newtreatment options are thus required for patients whofail a-IFN therapy and are not eligible for allogeneictransplantation.

High dose chemotherapy has been used to suppressPhiladelphia chromosome positive (Ph+) hemopoiesis,and may be associated with survival advantage, par-ticularly in those achieving a major cytogenetic re-sponse [7–10]. Furthermore, autologous bone marrowtransplantation (aBMT), even with 100% Ph+ pro-genitor cells, has resulted in Philadelphia chromosomenegative (Ph−) remissions of transient duration[11,12]. The outcome of autologous transplantationmay be improved by the use of better myeloablativeprotocols and infusion of predominantly Ph− progen-itor cells. Of particular interest, therefore, is the de-tection of Ph− peripheral blood progenitor cells(PBPC) in CML patients during the early recoveryphase following acute myeloid leukemia inductionchemotherapy [13]. The use of granulocyte colonystimulating factor (G-CSF) may minimize the toxicityassociated with this treatment, with the potential ad-ditional benefit of enrichment with normal progenitorcells, particularly in chronic phase disease [14–16].Limited data suggest that G-CSF can be safely ad-ministered to patients with CML, with no evidencefor acceleration of leukemic growth or disease pro-gression [17].

This study of patients with advanced CML evalu-ates the tolerability of intensive chemotherapy withG-CSF (Filgrastim) and its utility to mobilize suffi-cient numbers of Ph− progenitor cells for autologoustransplantation.

2. Materials and methods

Patients with Ph+ CML were eligible for study ifthey: had advanced disease stage (late chronicphase\12 months from diagnosis, accelerated phase[18] or blast crisis [19]); were 15–65 years of age; andhad an ECOG performance score between 0 and 2.Exclusion criteria included current major a-IFNresponse, previous myeloid growth factor exposure,and recent chemotherapy other than hydroxyurea.

Patients received induction chemotherapy (IcE)consisting of cytarabine 100 mg/m2/day by continuousi.v. infusion (days 1–7), idarubicin 12 mg/m2/day i.v.push (days 1–3), and etoposide 75 mg/m2/day i.v.over 1 h (days 1–7). G-CSF was commenced on day

+9 at 5 mg/kg/day and continued until the absoluteneutrophil count (ANC) exceeded 1.0×109/l on threeconsecutive days or 10×109/l on one day. A 30-minPBPC collection for cytogenetic analysis wasperformed once the white cell count exceeded0.4×109/l with recognizable myeloid recovery. Inthose obtaining a major cytogenetic response, orwhere PBPC analysis was not evaluable, consecutivedaily PBPC collections were performed aiming tocollect \1×106 CD34+ cells/kg. The majority ofPBPC collections were performed on a Cobe spectracell separator machine through a Hickmanplasmapheresis catheter (processing 1.5–2.0 totalblood volumes). A Baxter CS3000 was utilized forone patient. Repeat bone marrow and cytogeneticstudies were performed on day 28 with furtherchemotherapy permissible in patients achieving amajor cytogenetic response with adequate hemopoieticrecovery at day 43 (defined as ANC\1.5×109/lhaving completed G-CSF therapy,platelets\80×109/l, and no evidence of blast crisis).Three monthly follow-up included a bone marrowexamination with cytogenetic analysis.

2.1. Definitions of cytogenetic responses

Cytogenetic response to therapy was classified ascomplete (absence of Ph+ positive metaphases), par-tial (1–34% Ph+ metaphases), minor (35–95% Ph+

metaphases), or no response (96–100% Ph+

metaphases). A major cytogenetic response includedboth partial and complete cytogenetic responses. Re-verse transcriptase nested polymerase chain reaction(RT-PCR) for the BCR-ABL transcript was per-formed for patients in complete cytogenetic remission[20].

2.2. Statistical methods

Study endpoints included major cytogeneticresponse in the day 28 bone marrow examination andPBPC collections, and survival. Variables analyzedincluded disease duration, previous a-IFN majorcytogenetic response, age, and disease stage. Inaddition, bone marrow cytogenetic response followingIcE therapy was evaluated for its impact on survival.Influence of variables on cytogenetic response wasassessed by the Wilcoxon rank sum or chi squarestatistics. Survival was estimated by theKaplan–Meier method. The log-rank statistic wasused to test for the influence of predicting variableson survival. Statistical methods were performed usingthe STATA (University of Texas) statistical package,with the last follow-up assessment on 1 October 1997.Summary statistics use median values with ranges inbrackets.

J. Morton et al. / Leukemia Research 23 (1999) 177–183 179

Table 1Patient characteristics

21No. of patientsAge (years) 48 (range, 30–60)

14Sex (male)Disease stage

8Late chronic phaseAccelerated phase 11

2Blast phase26 (range, 7–120)Disease duration (months)

Previous treatmentHydroxyurea 20

15aa-IFN5Busulfan

a Four responders (discontinued due to toxicity), 11 non-responders.

significant (Kendall’s rank correlation efficient tau-a0.55, tau-b 0.69, P=0.0006). Major cytogenetic re-sponse was predicted by shorter disease duration (re-sponders median disease duration 24 months,non-responders 41 months, P=0.02) but not previousa-IFN response, disease stage, or age. One patient(accelerated phase) showed 43% blasts in his day 28bone marrow study (G-CSF ceased day 27). A subse-quent bone marrow study was not performed butcounts were controlled for 12 months with hydroxyureatherapy only. In the three patients who received asecond cycle of chemotherapy, cytogenetic response wassimilar to that observed at the completion of the firstcycle.

3.3. Leukapheresis collections

The initial leukapheresis collection was performed onday 25 (18–38 days). In total, 56 collections wereperformed on 14 patients (two to seven collections perpatient). Seven of nine patients achieving a major cyto-genetic response collected \1×106 CD34+ cells/kg(Table 3). All Ph− collections had evidence of theBCR-ABL transcript by RT-PCR analysis.

3.4. Toxicity (Table 4)

Non-hematological toxicity was predominantly gas-trointestinal (WHO grade 3 mucositis (n=5), grade 3nausea/vomiting (n=6), grade 3/4 colitis/diarrhoea(n=6)). Other toxicities included a subarachnoid hem-orrhage (conservative management with no sequelae),three central venous catheter complications, and oneepisode of anthracycline cardiotoxicity. Hematologicaltoxicity included neutropenic fever in all patients, witha median of five febrile days treated with 22 days ofsystemic antibiotics. Infectious complications includedbacteremia (n=10), oropharyngeal candidiasis (n=2)and herpes simplex virus mucositis (n=2). The time toneutrophil recovery (ANC\500×106/l) was 25 (19–38) days and to platelet recovery (unsupported plateletcount\20×109/l) was 27 (15–46) days. The medianduration of hospitalization was 31 days. No patientdied or required intensive care admission during thetreatment phase.

3. Results

3.1. Study population

Twenty-one patients were evaluated between January1993 and August 1995. Patient demographics are shownin Table 1. Twenty of 21 received the first cycle of IcE;one patient developed early central venous catheterthrombosis necessitating cessation of therapy after 3days (subsequent non-responder). Three patients re-ceived a second cycle of chemotherapy (two full dose,one reduced dose). Failure to complete further cycleswas due to inadequate response (n=11), patient refusal(n=5), cardiomyopathy (n=1) and delayed hemopoi-etic recovery (n=1).

3.2. Response to therapy

Day 28 bone marrow cytogenetics were available for18 patients, and cytogenetic analysis on PBPC collec-tions was used to assess treatment response for theother three patients (no bone marrow evaluation per-formed in two, one-dry tap). One patient with a day 28bone marrow study showing complete cytogenetic re-mission had 5–10% Ph+ metaphases on PBPC analysisand was reclassified as a major cytogenetic response.Overall nine of 21 (43%) achieved a major cytogeneticresponse to IcE therapy. Cytogenetic response accord-ing to disease stage is described in Table 2. Correlationbetween day 28 bone marrow and initial 30 min PBPCcytogenetic evaluation as shown in Fig. 1 was highly

Table 2Cytogenetic response according to disease stage

Late chronic phase (n=8)Day 28 bone marrow cytogenetic response Blast phase (n=2)Accelerated phase (n=11)

3 0 0CompletePartial 41 1

1 1 0Minor163No response

J. Morton et al. / Leukemia Research 23 (1999) 177–183180

Fig. 1. Correlation of day 28 bone marrow with 30-min peripheral blood progenitor cell (PBPC) cytogenetic analysis. No significant disparitybetween PBPC and bone marrow cytogenetics (Kendall’s rank correlation efficient tau-a 0.55, tau-b 0.69, P=0.0006).

3.5. Follow-up

Of the three complete responders, two (patients 6and 16) underwent autologous PBPC transplantationwhile in continued complete cytogenetic remission at41 days and 100 days respectively following IcE treat-ment. Both are currently alive and maintaining a ma-jor cytogenetic response. The third becameprogressively Ph+, reaching 100% Ph chromosomepositivity 780 days post-IcE; there was no subsequentresponse to homoharringtonine and the patient isawaiting transplantation. All six partial respondersbecame 100% Ph+ a median of 200 (112–240) dayspost-IcE. Two of these six underwent autologoustransplantation: one was transplanted in blast crisis250 days post IcE and died in a motor vehicle acci-dent while in complete cytogenetic remission; and thesecond was transplanted in chronic phase 120 dayspost IcE attaining a minor cytogenetic response. Twonon-responders underwent allogeneic transplantationin accelerated phase from an unrelated donor. Twelvepatients have died (11 transformed to blast phase,including the two who were allografted) at a medianfollow-up of 1171 (586–1570) days post IcE. Mortal-ity was associated with disease stage prior to IcEtherapy (P=0.0001). Previous a-IFN response, pa-tient age, disease duration, or IcE response did notpredict for mortality.

4. Discussion

Patients with advanced CML represent an ongoingtherapeutic challenge. They have a poor prognosisand have often been heavily pretreated with a-IFNand other drugs. Where allogeneic transplantation isnot possible, autografting represents a promising ex-perimental approach. As predominantly Ph− PBPCcollections have been shown to be important for theachievement of complete cytogenetic remissions posttransplant [21], we evaluated the role of intensivechemotherapy with G-CSF in mobilizing sufficientnumbers of Ph− PBPCs from patients with advancedCML.

One-third of our patients collected adequate yieldsof predominantly Ph− PBPCs with intensivechemotherapy using the IcE regimen. Higher responserates to IcE were observed for patients with shorterdisease duration and were independent of diseasestage at the time of IcE chemotherapy or previousa-IFN sensitivity. The treatment was reasonably tol-erated with no apparent toxicity attributable to G-CSF.

Our experience with IcE and G-CSF is comparableto other studies using similar agents. Carella et al.[14] administered ICE chemotherapy (higher dose cy-tarabine at 600–800 mg/m2 given as a 2-h infusionand lower dose of idarubicin at 6–8 mg/m2, both for5 days) resulting in Ph− PBPC collections in four of

J. Morton et al. / Leukemia Research 23 (1999) 177–183 181

Table 3Mobilization results in nine cytogenetic responders to IcE

CD34+ (×106/kg)Pre-IcE BM (% PBPC collections (%Patient Stage Days post IcE Day 28 BM (%No. of collec-Ph+)Ph)tions Ph)

0 1.504 70LCP 33 475 0 06 LCP 24 5.54

9.72–42010 100LCP 21 313 0–2511 AP 24 4 100 2.220 30–33a12a BC 21 7 100 0.4

0–4nd 2914 100AP 18 30 016 LCP 21 4 3.6100

0.420–252419 100AP 23 3100 0 5–1021 AP 23 5.84

Abbreviations: IcE, idarubicin, cytarabine, etoposide chemotherapy; BM, bone marrow; PBPC, peripheral blood progenitor cell; LCP late chronicphase; AP, accelerated phase; BC, blast phase; Ph, Philadelphia chromosome.a Collections 2, 3, 5 only (other collections\35% Ph positive).

13 (30%) patients with a disease duration of \2 years.This trend to higher cytogenetic response rate com-pared to the present study may reflect the shorterdisease duration in the Carella study or may be adose-dependent effect of cytarabine which has beenshown in vitro [22] and vivo [6] to have an importantand selective advantage in CML. The MD Andersongroup [16] reported their experience using a number ofmobilizing protocols, including high dose cytarabine, in55 patients with CML. Thirteen of 30 (43%) latechronic phase patients, three of 17 (17%) acceleratedphase patients and 0 of 8 (0%) blast crisis patientsachieved a major cytogenetic response. In contrast totheir experience and Carella’s report, we did not findsignificant differences in the percentage of Ph−

metaphases in the peripheral blood compared withbone marrow. Thus, the hypothesis that there is a‘therapeutic window’ in early myeloid recovery which

allows collection of Ph− PBPCs was not supported byour results.

As sufficient Ph− cells can be obtained in no morethan one-third of patients with advanced CML, anargument can be made to attempt to collect these cellsearlier in the disease course. Other investigators havereported high proportions of Ph− PBPC collectionsfollowing intensive chemotherapy in early chronicphase [14,15,23]. Less toxic mobilizing regimens in earlychronic phase, for example with single agent, intermedi-ate dose cyclophosphamide, are better tolerated butresult in a lower proportion of Ph− collections [24].

An important issue is whether G-CSF may adverselyaffect disease outcome. One case from this study mayhave demonstrated transient blast cell proliferation.With follow-up of over 3 years for this poor risk groupof patients, the incidence of blastic transformation andoverall survival is comparable to that expected in asimilar patient population. Hence it appears unlikelythat G-CSF caused acceleration of disease progression.This parallels our experience using G-CSF in CMLpatients to mobilize PBPCs following a-IFN response[25].

The benefits of autologous transplantation for CMLremain to be determined [26]. Historical comparisons ofselected patients undergoing aBMT with unmodifiedchronic phase cells with patients receiving conventionaltherapy [27] suggest a survival advantage followingaBMT, especially where a cytogenetic response occurspost aBMT. A beneficial effect may result from areduction in the total Ph+ stem cell burden of thepatient and a resultant reduced risk of random muta-tions and subsequent transformation [28]. The clinicaladvantage to transplantation with substantially Ph−

progenitor cells compared with unmodified progenitorcells is unknown. While post aBMT Ph chromosomenegativity has correlated with Ph chromosome negativ-ity of the infused product [21], cytogenetic responseshave been observed in up to 80% of chronic phase

Table 4Toxicity of IcE therapy

No. of patients 21Non-hematological toxicity

Gastrointestinala

5Grade 3 mucositisGrade 3 nausea/vomiting 6Grade 3/4 colitis/diarrhoea 6

OtherCentral line complications 3Anthracycline cardiotoxicity 1Subarachnoid hemorrhage 1

Hematological toxicityNeutropenic fever 21Documented bacteremia 10Median time to ANC\500×106/l (days) 25 (range, 19–38)Median time to platelet count\20×109/l 27 (range, 15–46)(days)

31 (range, 4–43)Median duration of hospitalization (days)

Abbreviations: IcE, idarubicin, cytarabine, etoposide chemotherapy;ANC, absolute neutrophil count.a WHO toxicity criteria.

J. Morton et al. / Leukemia Research 23 (1999) 177–183182

[29–31] and 30% of accelerated phase [32] patientstreated with single or double aBMTs using unmodifiedchronic phase peripheral blood or bone marrow pro-genitors. Occasional prolonged responses have beenobserved [29]. Virtually all patients autografted withpurged marrow or PBPCs [33,34] have become progres-sively Ph+ from 6 months post aBMT. Moreover,absence of detectable Ph+ cells from PBPC collectionsmay represent a reduction of relatively mature CMLprogenitor cells, rather than CML stem cells which arelikely to have low mitogenic activity and reduced ex-pression of BCR-ABL transcript [14]. Furthermore, theprocess of freezing and thawing progenitor cells resultsin a marked reduction in Ph+ long-term culture-initiat-ing cells in the absence of any other manipulation [35].

While patients with advanced CML should be con-sidered for mobilization of PBPCs following intensivechemotherapy and G-CSF with a view to subsequentautologous transplantation, uncertainty exists whetherPh negativity of PBPCs influences subsequent autograftoutcome or represents a disease with an inherentlybetter natural history. The value of collecting PBPCsfor autografting needs to be evaluated in prospectivetrials comparing reinfusion of predominantly Ph−

PBPCs with reinfusion of steady state Ph+ cells. Fur-thermore, while contributing substantially to toxicity,the role of additional agents (i.e. idarubicin andetoposide) in Ph chromosome suppression is unclearand requires further investigation.

Acknowledgements

Research support was from AMGEN, Australia.

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Contributions

S. Durrant, P. Eliadis, G. Seeley, S. Wright, H.Lland, K. Atkinson, H. Januszewicz, I. Buna, J. Bash-ford, provided study materials. C. Kelly and A. Ren-tould collected and assembled the data. D. Moore, C.Hutchins, B. Williams and D. Taylor provided techni-cal support. C. Stewart provided funding. J. Mortoninterpreted the data and provided statistical support. P.Mollee drafted the article. K. Taylor assisted withconcept and design. A. Grigg and R. Rodwell providedcritical revision of the article.

.