A novel triple purge strategy for eliminating chronic myelogenous ...

Bone Marrow Transplantation (2006) 37, 575–582 & 2006 Nature Publishing Group All rights reserved 0268-3369/06 $30.00

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ORIGINAL ARTICLE

A novel triple purge strategy for eliminating chronic myelogenous leukemia (CML) cells from autografts H Yang1, C Eaves2, M de Lima1, MS Lee3, RE Champlin1, JD McMannis1, SN Robinson1, T Niu1, WK Decker1, D Xing1, J Ng1, S Li1, X Yao1, AC Eaves2, R Jones1, BS Andersson1 and EJ Shpall1 1 Department of Blood and Marrow Transplantation, University of Texas MD Anderson Cancer Center, Houston, TX, USA; 2Terry Fox Laboratories, BC Cancer Research Center, Vancouver, BC, Canada and 3Department of Hematopathology, University of Texas MD Anderson Cancer Center, Houston, TX, USA

Imatinib-refractory chronic myelogenous leukemia (CML) patients can experience long-term disease-free survival with myeloablative therapy and allogeneic hematopoietic cell transplantation; however, associated complications carry a significant risk of mortality. Transplantation of autologous hematopoietic cells has a reduced risk of complications, but residual tumor cells in the autograft may contribute to relapse. Development of methods for purging tumor cells that do not compromise the engraftment potential of the normal hematopoietic cells in the autograft has been a long-standing goal. Since primitive CML cells differentiate more rapidly in vitro than their normal counterparts and are also preferentially killed by mafosfamide and imatinib, we examined the purging effectiveness on CD34 þ CML cells using a strategy that combines a brief exposure to imatinib (0.5– 1.0 lM for 72 h) and then mafosfamide (30–90 lg/ml for 30 min) followed by 2 weeks in culture with cytokines (100 ng/ml each of stem cell factor, granulocyte colonystimulating factor and thrombopoietin). Treatment with 1.0 lM imatinib, 60 lg/ml mafosfamide and 14 days of culture with cytokines eliminated BCR-ABL þ cells from chronic phase CML patient aphereses, while preserving normal progenitors. This novel purging strategy may offer a new approach to improving the effectiveness of autologous transplantation in imatinib-refractory CML patients. Bone Marrow Transplantation (2006) 37, 575–582. doi:10.1038/sj.bmt.1705284; published online 23 January 2006 Keywords: chronic myelogenous leukemia (CML); purging; imatinib; mafosfamide; imatinib refractory; CD34 þ

Correspondence: Dr H Yang, Department of Blood and Marrow Transplantation, University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030-4009, USA. E-mail: [email protected] Received 17 October 2005; revised 2 December 2005; accepted 5 December 2005; published online 23 January 2006

Introduction Imatinib has shown impressive activity in the treatment of chronic myelogenous leukemia (CML), delaying disease progression in the majority of patients.1–5 However, increasing experience suggests that the majority of CML patients are not cured with this drug.6–9 While the use of allotransplantation for the treatment of CML does provide a curative alternative, it is associated with substantial morbidity and mortality.10,11 Although the use of autologous transplantation is associated with considerably less morbidity and mortality, residual tumor cells in the autograft can likely contribute to disease relapse after transplantation.12 A method to successfully eradicate malignant cells in the graft would thus make autologous transplantation a more attractive therapeutic option if this could be achieved without impairing the repopulating potential of the graft. Mafosfamide and 4-hydroperoxycyclophosphamide (4HC) are metabolically active derivatives of cyclophosphamide that have been used clinically for more than a decade to purge autologous grafts for a variety of malignancies including CML.13–16 In more recent preclinical studies the combination of imatinib and mafosfamide has been shown to exert impressive synergistic antitumor activity against CML cells.17 This stimulated our interest in using this drug combination in an ex vivo autograft-purging regimen. Early mafosfamide/4-HC purging trials were characterized by delayed engraftment likely due to cytotoxic effects on short term repopulating cells.18 Autografts pre-treated with a combination of mafosfamide and imatinib would therefore also be expected to give the same result. However, if the numbers of remaining drugtreated normal progenitors could be increased ex vivo prior to transplantation, an improved recovery might be achieved. Further, since CML cells differentiate more rapidly in vitro than their normal counterparts, maintaining CML autografts in vitro with cytokines would further eliminate the malignant stem cells while expanding normal progenitors.19–23 The feasibility of this approach was established some years ago by early clinical studies that used short-term culture alone to purge CML autografts. This treatment was also associated with delayed

Novel triple-step CML autograft purging H Yang et al

576

hematologic recovery in some patients,24,25 but these studies also predated the availability of recombinant growth factors now known to support substantial progenitor cell expansion in vitro.26,27 There are reports that imatinib does not affect quiescent CML stem cells.28 We added the alkylating agent for the widely reported antitumor synergy between imatinib and mafosfamide. Even if the malignant stem cells are not killed immediately with the imatinib–mafosfamide treatment, we postulated that the culture would further stress the cells with alkylated DNA, and cause them to apoptose. Here we report the development of a novel ex vivo purging strategy that combines imatinib and mafosfamide treatment with a subsequent 2-week period of culture of the drug-treated cells with cytokines. Treatment conditions were optimized to maximize the eradication of residual contaminating CML cells from autologous peripheral blood progenitor cell (PBPC) samples from CML patients, while preserving normal progenitors.

Materials and methods Hematopoietic cell sources Purging experiments were conducted using two established Ph þ CML cell lines (K562 29 and KBM-7/B530). K562 cells were cultured in RPMI medium (Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) (Hyclone, Logan, UT, USA). KBM-7/B5 cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) (Gibco, Grand Island, NY, USA) supplemented with 20% (v/v) FBS. Both cell lines were maintained at 371C, 5% CO2 in air, fully humidified atmosphere. A series of apheresis products were obtained from GCSF mobilized normal donors (N ¼ 5) and chronic phase CML patients (N ¼ 4) under MD Anderson Institutional Review Board protocol Lab02-630. The apheresis products from CML patients #1 and 2 had been cryopreserved in the early 1990s, prior to the availability of imatinib, while they were receiving interferon for cytogenetically detectable disease. The other two products (patients #3 and 4) were collected more recently from imatinib-refractory CML patients. Patient #3 had experienced a transient hematologic response, but despite increasing doses of imatinb to 800 mg/day, had progressive cytogenetic and molecular disease when the pheresis product was donated. Patient #4 was experiencing molecular progression of disease on 800 mg imatinib/day at the time the apheresis product was donated for research. Positive selection procedure CD34 þ cells were isolated from thawed pheresis products from normal donors and CML patients, by magneticactivated cell sorting (MACS) according to the manufacturer’s instructions using the MidiMACS device (Miltenyi Biotech, Auburn, CA, USA) and CliniMACS CD34 MicroBeads (Miltenyi Biotech, Auburn, CA, USA).31 Isolated CD34 þ cells were cultured overnight in ex vivo expansion medium: aMEM containing 10% (v/v) FBS and supplemented with 100 ng/ml each of stem cell factor (SCF) Bone Marrow Transplantation

and thrombopoietin (TPO) (both from R&D Systems Inc., Minneapolis, MN, USA), and granulocyte colony-stimulating factor (G-CSF, Neupogen Filgrastim, Amgen Inc., Thousand Oaks, CA, USA) prior to use. The efficiency of the MACS process was confirmed by flow cytometric analysis of pre- and post-selection fractions.

Culture conditions CD34 þ PBPCs were cultured for 14 days in ex vivo expansion medium at 371C, 5% CO2. On day 7, sufficient additional ex vivo expansion medium was added to double the total culture volume. On day 14, BCR-ABL message levels were determined in the cultured CML PBPCs by quantitative real-time polymerase chain reaction (Q-RTPCR) (see below). Imatinib treatment CD34 þ cells from the normal donor and CML patient samples were treated with a range of imatinib (Novartis Pharmaceuticals Corp., Basel, Switzerland) concentrations (0.5–1.0 mM) for 3 days, then washed and cultured in ex vivo expansion medium. The presence of BCR-ABL þ cells after imatinib treatment and culture was evaluated by Q-RTPCR. Combination treatment CD34 þ cells from normal donors and CML patient samples were treated with a range of imatinib concentrations (0.5–1.0 mM) for 3 days and with a range of mafosfamide (Baxter Oncology, Frankfurt, Germany) concentrations (30–90 mg/ml) for the final 30 min of culture. The cells were then washed free of drug and BCR-ABL þ transcript levels evaluated by Q-RT-PCR. To assess the additional effects of subsequent culture with cytokines (as described above), the cells were then washed 3 times and incubated at 1–2 105 cells/ml in ex vivo expansion medium for 14 days and then tested for BCR-ABL þ transcripts and CFC content (Figure 1). Viability assays Cell viability was measured using the MTS ([3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2h-tetrazolium]) assay (Promega, Madison, WI, USA).32 Briefly, viability is measured by following the conversion of MTS into a formazan dye by the reductase system of living cells. The cell lines (K562 and KBM7/B5) and human CD34 þ cells from normal PBPC samples were cultured in triplicate in flat-bottomed 96-well plates at 5 103 cells/well. Normal PBPC CD34 þ cells were cultured in ex vivo expansion medium. For a single drug treatment, cells were incubated with different concentrations (0.1–5 mM) of imatinib for 24, 48, 72 and 96 h, or 30–90 mg/ml mafosfamide for 30 min, at 371C, 5% CO2 and in fully humidified atmosphere. For combination treatments, cells were treated with imatinib for 72 h, and mafosfamide was added for the final 30 min of culture. MTS cell viability assays were performed in triplicate on post-treatment samples.


577 Step 1

Step 2

Step 3

CD34 selection of PBPC

Incubation with imatinib for 72 hr followed by mafosfamide for 30 min

Wash cells Culture for 14 days in ex vivo expansion medium

RT-PCR (BCR/ABL) CFU-GM

RT-PCR ( BCR/ABL) CFU-GM/FACS

Figure 1 Experimental Design. CD34 þ cells were isolated from thawed normal donor or CML patient aphereses, using magnetic activated cell sorting (MACS). CD34 þ cells were cultured overnight in ex vivo expansion medium, which contained aMEM plus SCF, G-CSF and TPO. The following day, CD34 þ cells were treated with imatinib (3 days) and mafosfamide (added for the final 30 min of culture). Cells were then washed three times and cultured in ex vivo expansion medium for 2 weeks. After treatment and culture, the ability of cells to form colonies in semisolid methylcellulose was assessed and the percentage of CD34 þ cells was determined by flow cytometric analysis. For the CML patient samples, the levels of BCR-ABL-positive cells were evaluated by Q-RT-PCR assessment of BCR-ABL transcript levels.

Colorimetric changes in individual wells were determined by measurements at an absorbance of 490 nm (OD490) 2–3 h after addition of MTS reagent using a universal microplate reader (MRX Revelation, DYNEX Technologies Inc., Chantilly, VA, USA). Background absorbance (OD490) was determined with a media plus MTS reagent control. The mean optical density of the triplicate wells for each imatinib7mafosfamide dose was expressed as percentage of control (cells cultured without drugs). Percent cell growth inhibition was calculated as follows: Percent ð%Þ growth inhibition ¼ ½1 ðOD490 experimental OD490 controlÞ 100:

Flow cytometry assay The percentage of CD34 þ cells was determined using antibodies labeled either with allophycocyanin, or phycoerythrin (Becton-Dickinson, Mountainview, CA, USA). Flow cytometric analysis was performed using a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA, USA). Briefly 1 106 unselected cells, 0.1–0.5 106 CD34 þ -enriched cells, or cells after 14 days of culture, were incubated for 20 min at room temperature with saturating levels of fluorescently-labeled antibodies, washed and fixed (with 1.6% (v/v) paraformaldehyde). A minimum of 10 000 events were acquired per sample. Data acquisition and analysis was performed using CellQuest software (BD). Real-time polymerase chain reaction (Q-RT-PCR) assay for quantification of BCR-ABL transcripts Q-RT-PCR was used to measure changes in the level of BCR-ABL transcripts relative to retinoic acid receptoralpha (RARa) as an internal control to assess the effectiveness of CML cell purging. Approximately 104–105 imatinib/mafosfamide-treated cells (approximately 80%

CD34 þ ) were analyzed from each sample of drug-treated cells. For cells subjected to the 2-week culture procedure, 8 105–1 106 cells were analyzed. The Q-RT-PCR methodology for the detection of Ph-positive cells has been previously described.33 Briefly, RNA was extracted using Trizol (Invitrogen, Gaithersburg, MD, USA) followed by reverse transcription using random hexamers and Superscript II Reverse Transcriptase (Invitrogen, Gaithersburg, MD, USA). Specific primer sets and double-labeled fluorogenic probes for c-ABL and RARa, respectively, were then used to amplify and detect BCR-ABL and RARa transcript cDNAs. The c-abl probe was 50 -labeled with 6carboxy-fluorescein (6-FAM) and 30 -labeled with 6-carboxytetramethyl rhodamine (TAMRA). The RARa probe was labeled with VIC at the 50 end and TAMRA at the 30 end. PCR was performed using the ABI PRISMt 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) for 45 cycles with the following cycle profile: 951C/30 s and 651C/1 min. During PCR, fluorescence acquisition was monitored cycle by cycle to measure the accumulation of the reporter dyes, 6-FAM and VIC, respectively. Based on the cycle threshold method (according to manufacturer’s instructions), the measurements of BCR-ABL and RARa transcripts in the tested samples were calculated against the standard curve plots of serially diluted samples prepared from K562 cells. Results were expressed as the ratio of BCR-ABL/RARa transcripts 100. The sensitivity limit of our PCR was approximately 1–5 leukemia cells whose amplification curves typically crisscrossed the predetermined threshold at 36–38 cycles. Co-amplification of BCR-ABL transcripts cDNAs and the internal standard RARa cDNA helped identify false negatives as a result of poor PCR efficiencies or degraded RNA. An assay was deemed suboptimal if the cycle threshold of RARa amplification in the test sample was less than 25. Three negative controls and one reagent control were also routinely included in each assay to assure no false positives due to cross-contamination were present.

Colony-forming cell (CFC) assays After treatment and ex vivo culture, cells were assayed for their ability to form colonies in semisolid methylcellulose. Briefly, cells were plated at 0.1–1.0 104 cells in 1 ml of methylcellulose medium (Methocult H4435, Stem Cell Technologies Inc., Vancouver, BC, Canada) containing SCF (50 ng/ml), GM-CSF (20 ng/ml), IL-3 (20 ng/ml), IL-6 (20 ng/ml), G-CSF (20 ng/ml) and erythropoietin (3 U/ml) in duplicate 35 mm dishes (Falcon). Cultures were incubated in a humidified atmosphere at 37 C with 5% CO2 for a period of 2 weeks, after which time colonies were scored. Colonies were identified as clusters of X20 translucent cells using an inverted microscope.34

Results Effect of imatinib as a single agent Initially, the growth inhibition dose–response of two CML cell lines, K562 and KBM7/B5, to imatinib was tested. When K562 cells were treated with X1.0 mM imatinib for Bone Marrow Transplantation


Effect of combined imatinib plus mafosfamide treatment on CML cell lines A maximum dose of 1 mM imatinib was considered optimal for combination drug testing since this dose produced 490% growth inhibition of K562 cells. K562 and KBM-7/ B5 (imatinib resistant) cells were then treated with imatinib alone (0.25–1.0 mM), or in combination with mafosfamide (30, 60 and 90 mg/ml), in a total of 12 different drug combinations. In the more imatinib-sensitive K562 cell line, the addition of mafosfamide did not add to the effect of imatinib. However, with the imatinib-resistant KBM-7/B5 cells, a significant additive effect was seen especially at 0.75 or 1.0 mM imatinib plus 60 mg/ml mafosfamide (Po0.001). As previously described, optimal treatment with 1 mM imatinib alone inhibited the growth of the imatinibresistant KBM7/B5 cells by only 70%. However, this was increased to 85% when KBM7/B5 cells were treated with 1 mM imatinib and 60 mg/ml mafosfamide. Effect of combined imatinib plus mafosfamide treatment and subsequent culture on normal CD34 þ progenitor cells In order to evaluate the effect of the combined imatinib plus mafosfamide treatment on normal progenitors, normal CD34 þ PBPCs from five different donors were treated with imatinib for 72 h alone or in combination with mafosfamide during the final 30 min of incubation. In addition, aliquots of the drug-treated cells were washed and cultured for an additional 14 days in ex vivo expansion medium to examine the effect of imatinib7mafosfamide on the subsequent ex vivo expansion of normal hematopoietic progenitors. In these experiments the ‘Input Control’ refers to the number of cells, CD34 þ cells or CFC in the CD34 þ fraction. These cells were not treated with imatinib/mafosfamide, but cultured directly following positive selection. The ‘Output Control’ refers to that same fraction following 17 (3 þ 14) days of ex vivo culture with the SCF-G-CSF-TPO cytokine regimen described above. Total nucleated cell results The total nucleated cells increased from 1.0 106 in the untreated ‘Input Control,’ to 4374 106 in the untreated ‘Output Control’ compared with recovery of 2071.1 106 in the sample treated with 1.0 mM imatinib þ 60 mg/ml mafosfamide followed by ex vivo culture (Figure 2). Immediately after treatment with 1.0 mM imatinib plus Bone Marrow Transplantation

a

b

Absolute number of total nucleated cells (×106)

3 days, a 96% inhibition of cell output was observed; this effect was not further increased when the duration of exposure, or imatinib concentration, was increased (data not shown). The same exposure duration and dose of imatinib induced less growth inhibition (70%) in cultures of the more imatinib-resistant KBM7/B5 cells with 10% of KBM7/B5 cells surviving even a 4-day exposure (data not shown). After incubation of CML cells (KBM7/B5 and K562) with 1 mM imatinib for 3 days, 70–96% growth inhibition was obtained. Similarly treated, normal CD34 þ PBPCs showed only 40% growth inhibition. These data confirmed the greater sensitivity of primitive CML cells to imatinib when compared to their normal counterparts.

1.2

Absolute number of total nucleated cells (× 106)

578

50

Output control

M 60 g/ml ± Im

M 30 g/ml ± Im

M 90 g/ml ± lm

1.0

Im only

0.8 0.6 0.4 0.2 0.0

Input control

0

0.5 1 0.75 M Imatinib

1.0

40 30 20 10 0

Input control

0

0.5

0.75 1 M Imatinib

1.0

Figure 2 Effect of imatinib plus mafosfamide with or without subsequent culture for 2 weeks on total nucleated cell (TNC) output in cultures of normal CD34 þ PBPCs. (a Prior to ex vivo expansion) TNC number immediately after exposure to imatinib alone (3 days), mafosfamide alone (30 min), or the combination of imatinib (3 days) plus mafosfamide (final 30 min). The solid bar (’) is the TNC number (control output) present after 3 days in expansion medium only (no drug). With increasing doses of imatinib plus mafosfamide, TNC numbers (initially starting at 1.0 106 cells) decreased. Immediately after treatment with 1.0 mM imatinib plus 60 mg/ml mafosfamide, cell numbers decreased to approximately 60% of the pretreatment ‘Input Control’. (b Following ex vivo expansion) the same cells as shown in (a) (treated with imatinib for 3 days and mafosfamide for 30 min), were cultured in ex vivo expansion medium for 2 weeks and TNC numbers again determined. The solid bar (’) represents the TNC number (control output) present after a total of 17 days in expansion medium (3 days no drug þ 2 weeks in culture). Total nucleated cells increased from 1.0 106 in the untreated ‘Input Control,’ to 4374 106 in the ‘Control Output’ and 2071.1 106 in the sample treated with 1.0 mM imatinib þ 60 mg/ml mafosfamide followed by ex vivo culture (data as mean7 s.e.m., n ¼ 5 replicate experiments).

60 mg/ml mafosfamide, cell numbers decreased to approximately 60% of the pretreatment input number (Figure 2a). After an additional 2 weeks in culture with cytokines, these numbers increased significantly (20 fold, Po0.001) (Figure 2b).

CFC results The number of CFC increased from 49.478.7 103 in the untreated ‘Input Control,’ to 7557155 103 in the ‘Output Control’ compared with 222744 103 in the sample treated with 1.0 mM imatinib þ 60 mg/ml mafosfamide followed by ex vivo culture (Figure 3). Assessment of CFC numbers in the same cultures showed that the higher concentration of imatinib (1.0 mM) plus mafosfamide (60 mg/ ml) initially reduced CFC by up to 9.2-fold (Figure 3a) but their numbers then increased to give a 4.5-fold (Po0.01) overall net expansion relative to the input value (Figure 3b). This increase was only threefold less than the CFC expansion obtained from normal untreated cells (Figure 3b).


579

Absolute number of CFU-GM (×103)

Im 0.75 M 70

Im 1.0 M

60

Im 0.75 M + M 60 g/ml Im 1.0 M + M 60 g/ml

50 40 30 20 10 0 Input control

Absolute number of CFU-GM (×103)

b

1000

Output after Im ± M

×15

Im 1.0 M × 1.9

2.0

M 60 g/ml × 1..5

1.6

Im1.0 + M60 × 1.3 × 1.1

1.2 0.8 0.4 0.0

Input control

Output

Figure 4 Effect of imatinib plus mafosfamide on CD34 þ cell output in

800 600 ×7 400

Output control

M 60 g/ml

Absolute number of CD34 + cells (× 10 6)

a

×6 ×4 × 4.5 × 4.5

200

cultures of CD34 þ normal PBPCs with subsequent 2 weeks of culture. After 3 days (imatinib alone) or mafosfamide alone (30 min), or the combination of imatinib (3 days) plus mafosfamide (30 min) and 2 weeks ex vivo culture, the percentage of human CD34 þ cells was determined by flow cytometry. Fold increases in absolute numbers of CD34 þ cells relative to the input value are shown. The absolute number of CD34 þ cells was preserved following the combination of imatinib (3 days) plus mafosfamide (final 30 min of culture) and 2 weeks ex vivo culture (data as mean7s.e.m., n ¼ 5 replicate experiments).

0 Input control

Output after Im ± M + culture

Figure 3 Effect of imatinib plus mafosfamide with or without subsequent culture for 2 weeks on CFC outputs in cultures of CD34 þ normal PBPCs. (Data are from the same experiments shown in Figure 2.) (a Prior to ex vivo expansion) CFC numbers measured immediately after treatment with imatinib alone for 3 days, mafosfamide alone for 30 min or the combination of imatinib (3 days) plus mafosfamide (30 min). With increasing doses of imatinib7mafosfamide, there was a reduction in absolute numbers of CFC. Imatinib (1.0 mM) plus mafosfamide (60 mg/ml) initially reduced CFC by up to 90% compared to untreated ‘Input Control.’ (b Following ex vivo expansion) CFC numbers after treatment and culture are shown. In both panels, the input value is the number of CFCs present at culture initiation. The solid bar (’) represents the number of CFCs present after 17 days of culture in ex vivo expansion medium (3 days no drug þ 2 weeks in culture). Net fold increases in absolute numbers of CFC relative to input are shown (Po0.01) (data as mean7s.e.m., n ¼ 5 replicate experiments).

product of patient #2 by simply culturing the cells for the same total period as used for the drug treatment (3 days) plus the subsequent 2 weeks. The product from patient #1 became BCR-ABL negative after X0.75 mM imatinib þ X60 mg/ml mafosfamide treatment. Of note, neither culture alone, nor the combined drug treatment alone, eliminated BCR-ABL-positive cells from the samples obtained from the two patients with imatinib-refractory disease (patients #3 and 4). However, BCR-ABL-positive cells were eliminated from those two products when the drug treatment and 2-week culture were combined (X0.75 mM imatinib and X60 mg/ml mafosfamide plus culture for 2 weeks (Tables 1 and 2).

Discussion CD34 þ cell results The number of CD34 þ cells increased from 0.93 106 in the untreated ‘Input Control,’ to 1.770.2 106 in the ‘Output Control’ compared with recovery of 1.070.1 106 in the sample treated with 1.0 mM imatinib þ 60 mg/ml mafosfamide followed by ex vivo culture (Figure 4). Parallel measurements of the number of viable CD34 þ cells present immediately after drug treatment and subsequently after 2 weeks in culture with cytokines showed that the absolute number of CD34 þ cells was preserved at the end of the procedure (Figure 4). Effect of combined imatinib plus mafosfamide treatment and subsequent culture on CML patient’s products CD34 þ cells were isolated from CML phereses as described above and treated with imatinib7mafosfamide and cultured for 2 weeks. Q-RT-PCR assay was used to quantify BCR-ABL-positive cells in samples obtained immediately after drug treatment and subsequently at the end of the 2week culture period. The results, (summarized in Table 1) showed that BCR-ABL-positive cells were eliminated in the

Transplantation of autologous hematopoietic progenitor cells may be an important therapeutic option for imatinibrefractory CML patients who are not candidates for allogeneic transplantation, but only if recovery of hematopoiesis without risk of increased relapse could be assured. Here we describe a novel multi-step strategy for treating autologous CML patient PBPCs that attempts to maximize the depletion of CML cells while maintaining the content of normal progenitors. We demonstrated that exposure of CD34 þ cells from CML patient apheresis products first to imatinib (0.75–1.0 mM) for 3 days, then to mafosfamide (60 mg/ml) for 30 min followed by a further incubation for 2 weeks (in the absence of drugs) in medium containing a growth factor cocktail designed to expand normal progenitors, reduced the level of BCR-ABL þ cells by at least 10 000-fold in four of four cultures initiated with cells from four different chronic phase CML patients. Under the same conditions normal donor PBPC products revealed a 20-fold expansion of total nucleated cells, a 4.5-fold increase of CFC numbers and 1.1-fold expansion (maintenance) of CD34 þ progenitors. Bone Marrow Transplantation


580 Summary of Q-RT-PCR data for treated CML samples

Table 1 Patient Chronic Chronic Chronic Chronic

phase phase phase phase

(patient (patient (patient (patient

#2) #1) #3) #4)

imatinib imatinib imatinib imatinib

Culture alone

Imatinib+mafosfamidea alone

Imatinib+mafosfamidea+culture

0.00 1.72 5.86 3.63

0.00 0.00 1.29 1.69

0.00 0.00 0.00 0.00

naive naive refractory refractory

Q-RT-PCR data show that in one sample (patient #2, imatinib naive) BCR-ABL-positive cells were eliminated by culture alone. BCR-ABL-positive cells were eliminated from samples from patient #1 (imatinib naive) following imatinib and mafosfamide treatment only, although imatinib and mafosfamide treatment and ex vivo culture was required to eliminate BCR-ABL-positive cells from samples from patients #3 and 4 (imatinib refractory). a 0.75 mM imatinib plus 60 mg/ml mafosfamide. The values shown are the ratios of BCR-ABL/RARa transcripts 100.

Table 2 Representative Q-RT-PCR results of treated cells from an imatinib-refractory CML patient (patient #3) CML patient#3 Treatment

Q-RT-PCR After drug treatment

After 14 days of culture

136.09 (no drug treatment)

3.86

Imatinib 0.5 mM +Mafosfamide 30 mg/ml +Mafosfamide 60 mg/ml +Mafosfamide 90 mg/ml

274.78 30.74 3.88 0.14

5.14 3.09 1.09 0.00


162.95 6.27 1.29 0.11

5.63 1.63 0.00 0.00


107.48 10.51 0.39 0.01

3.20 1.79 0.00 0.00

Culture alone

All Q-RT-PCR results were positive immediately after treatment, demonstrating that neither imatinib alone, nor the combination of imatinib and mafosfamide, was able to eliminate all BCR-ABL+ cells. However, BCR-ABL+ cells were not detectable after 0.5 mM imatinib, 90 mg/ml mafosfamide and 2 weeks of culture, or 0.75–1 mM imatinib, 60–90 mg/ml mafosfamide and 2 weeks culture. The level of RT-PCR positivity was estimated as the ratios of BCR-ABL/RARa transcript 100.

These data suggest that for chronic phase CML, such a novel purging protocol might be effective at preparing a useful autograft free of contaminating CML cells. Imatinib induces apoptosis in sensitive BCR-ABL þ cells17,35–37 and, in preliminary studies, we have confirmed that cells treated with mafosfamide in addition to imatinib showed enhanced staining with Annexin V and PI indicative of an additive effect (data not shown). Nevertheless, exposure to both imatinib and mafosfamide was insufficient to eliminate all BCR-ABL þ cells. However, addition of a further 2 weeks in culture after removal of the imatinib and the mafosfamide successfully eliminated all BCRABL þ cells. In early CML purging studies, it was shown that CML stem cells, measured as leukemic long-term culture-initiating cells (LTC-ICs) were selectively purged by exposure to 4-HC- and Ph-negative cells were maintained in vitro.22,38 These results may be explained, in part, by two abnormal Bone Marrow Transplantation

characteristics of CML LTC-ICs: they possess an intrinsically determined increased proliferative activity,39 and reduced self-renewal capacity.23 The latter is manifested as a 450-fold decline in LTC-IC activity in vitro within 10 days under conditions where normal LTC-IC numbers are maintained or increase.20,40 Additional studies are in progress to determine whether this treatment maintains sufficient numbers of repopulating cells, and to investigate whether in vivo recovery of transplanted patients would be achieved. It would also be of interest to determine whether the addition of Flt3-ligand to the expansion medium would improve the normal hematopoietic activity of the treated cells since this cytokine has been demonstrated to improve their amplification in other studies.41–43 The promising results of the combined purging strategy presented here indicate that further development of this approach is warranted as a step towards devising a protocol that can be used clinically. Typically, apheresis products containing X2 106 CD34 þ cells/kg produce prompt engraftment in autograft recipients.44 We have shown that the CD34 þ cell numbers are maintained following exposure to imatinib–mafosfamide and 2 weeks of culture. For a clinical trial, CML patient products will be required to contain at least 2 106 CD34 þ cells/kg in the CD34-selected fraction, prior to the triple purging procedure. Procurement of such a dose will be more difficult from CML patients than normal donors. Nevertheless, with multiple phereses, such doses have been collected on a prophylactic hematopoietic cell storage protocol from chronic phase CML patients in cytogenetic remission (MD Anderson protocol 03-0710). Thus, there will be a subset of CML patients for whom this purging procedure could likely be performed safely. Such a clinical trial is being developed for chronic phase CML patients with progressive disease on imatinib or similar tyrosine kinase receptor inhibitors. Those patients will be mobilized with cyclophosphamide plus G-CSF and GM-CSF. Sufficient aphereses will be performed to collect at least 4 106 CD34 þ cells/kg; 2 106 CD34 þ cells/kg will be frozen as an unmanipulated back up fraction and the remaining cells will be treated with imatinib and mafosfamide followed by ex vivo culture. Patients will then receive high-dose therapy with the infusion of the manipulated graft and additional G-CSF and GM-CSF. The primary objectives of the study include feasibility of the purging procedures and time to engraftment. If effective, a larger study to assess the efficacy of this approach will be designed.


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Acknowledgements The authors gratefully acknowledge the support of Mr Michael Thomas, Department of Blood and Marrow Transplantation, MD Anderson Cancer Center, Novartis Pharmaceuticals Corp., Basel, Switzerland for providing the imatinib, and Baxter Oncology, Frankfurt, Germany for providing the mafosfamide. Financial Support: This work was funded in part by NCI #2PO1CA49696-15 (RC/EJS) and NCI R21 CA108137-01 (MdeL).

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