Abstract

Resistance to imatinib mesylate is an emerging problem in the treatment of chronic myeloid leukemia (CML), often associated with point mutations in the Bcr-Abl kinase domain. Outcome of patients with such mutations after allogeneic stem cell transplantation (Allo-SCT) is unknown. Ten imatinib-resistant patients with Bcr-Abl kinase mutations received a transplant: 9 had CML (3 in chronic phase, 4 in accelerated phase, and 2 in blast phase) and 1 had Philadelphia-positive acute lymphocytic leukemia (ALL). Patients harbored 9 different protein kinase mutations (T315I mutation, n = 2). Preparative regimens were ablative (n = 7) and nonablative (n = 3). All patients engrafted; there were no treatment-related deaths. Disease response was complete molecular (CMR; n = 7), major molecular (n = 2), and no response (n = 1). Three patients (mutations Q252H, E255K, and T315I) died of relapse after Allo-SCT. Seven patients are alive (6 in CMR) for a median of 19 months. Allo-SCT remains an important salvage option for patients who develop resistance to imatinib through Bcr-Abl mutations.

Introduction

Imatinib mesylate is a potent and selective tyrosine kinase inhibitor that has become standard therapy for patients with chronic myeloid leukemia (CML).1,2  Most newly diagnosed CML patients with chronic-phase (CP) disease treated with imatinib achieve durable responses. However, a small percentage of CP patients and most advanced-phase subjects relapse on therapy.3-5 

Resistance to imatinib occurs at a rate of approximately 4% per year among patients treated in early CP and at higher rates in those treated in advanced stages. Resistance is most frequently mediated by mutations within the kinase domain of Bcr-Abl and, to a lesser extent, by amplification of the Bcr-Abl genomic locus and other mechanisms.6,7  Point mutations have been reported in 30% to 90% of resistant patients.

Allogeneic stem cell transplantation (Allo-SCT) is a potentially curative treatment for CML.8  However, there is little data in patients with imatinib resistance–associated mutations. In order to address this problem, we analyzed the outcome of patients with Bcr-Abl kinase mutations treated with related or unrelated donor hematopoietic stem cell transplantation (HSCT) at our institution.

Study design

Between July 2002 and October 2005, 10 patients with imatinib-refractory CML (n = 9) or Philadelphia-positive (Ph-positive) acute lymphocytic leukemia (ALL; n = 1) received allo-SC transplants from human leukocyte antigen (HLA)–matched related (n = 5) or unrelated (n = 5) donors. Failure to imatinib was defined as (1) loss of a cytogenetic or complete hematologic response (CHR) while on therapy; (2) failure to achieve a CHR (for patients in CP) or any hematologic response (for patients in accelerated phase [AP] or blast phase [BP]) after 3 months of imatinib therapy; or (3) persistence of 100% Ph-positive metaphases after 6 months or greater than 35% after 12 months of therapy.

Preparative regimens are described in Table 1. Graft-versus-host disease (GVHD) prophylaxis consisted of tacrolimus and methotrexate 5 mg/m2 intravenously on days 1, 3, 6, and 11 after transplantation. All patients received granulocyte colony-stimulating factor 5 μg/kg daily from day 7 until neutrophil engraftment. Antithymocyte globulin was given to recipients of unrelated donor transplants. Informed consent was obtained in all cases. The M. D. Anderson Cancer Center Institutional Review Board approved this retrospective study.

Table 1.

Patient and transplantation characteristics


UPIN

Age, y

Sex

Stage at start of imatinib

Time on imatinib, mo

Best response to imatinib

Mutation type

Semiquantitative assessment of mutation*

Stage at mutation's detection

Status at Allo-SCT

Preparative regimen

Donor type

Source of stem cells
1   46   M   CE   24   CGCR   F486S   100%   AP   AP   BuCy   MUD   BM  
2   50   M   AP   24   CHR   F317L   Mostly   CP   CP   BuCy   MRD   BM  
3   48   F   CE   7   CGCR   L248V   Low level   CP   CP   BuCy   MRD   BM  
4   26   F   CE   3   CHR   Y253H   Mixed   BP   CHR   BuCy   MRD   PB  
5   54   M   Ph+ ALL   8   CGCR   Q252H   Low level   Ph+ ALL   MMR   FM   MUD   BM  
6   41   M   CE   24   CGCR   T315I   100%   AP   AP   BuCy   MUD   BM  
7   63   M   CP   21   MCR   T315I   100%   CP   MCR   FM   MUD   BM  
8   37   M   CE   14   MinorCG   G250E   100%   AP   AP   BuCy   MUD   BM  
9   31   M   AP   14   CHR   F359V   100%   AP   AP   FM   MRD   PB  
10
 
46
 
M
 
AP
 
5
 
MinorCG
 
E255K
 
100%
 
BP
 
CHR
 
BuFlu
 
MRD
 
PB
 

UPIN

Age, y

Sex

Stage at start of imatinib

Time on imatinib, mo

Best response to imatinib

Mutation type

Semiquantitative assessment of mutation*

Stage at mutation's detection

Status at Allo-SCT

Preparative regimen

Donor type

Source of stem cells
1   46   M   CE   24   CGCR   F486S   100%   AP   AP   BuCy   MUD   BM  
2   50   M   AP   24   CHR   F317L   Mostly   CP   CP   BuCy   MRD   BM  
3   48   F   CE   7   CGCR   L248V   Low level   CP   CP   BuCy   MRD   BM  
4   26   F   CE   3   CHR   Y253H   Mixed   BP   CHR   BuCy   MRD   PB  
5   54   M   Ph+ ALL   8   CGCR   Q252H   Low level   Ph+ ALL   MMR   FM   MUD   BM  
6   41   M   CE   24   CGCR   T315I   100%   AP   AP   BuCy   MUD   BM  
7   63   M   CP   21   MCR   T315I   100%   CP   MCR   FM   MUD   BM  
8   37   M   CE   14   MinorCG   G250E   100%   AP   AP   BuCy   MUD   BM  
9   31   M   AP   14   CHR   F359V   100%   AP   AP   FM   MRD   PB  
10
 
46
 
M
 
AP
 
5
 
MinorCG
 
E255K
 
100%
 
BP
 
CHR
 
BuFlu
 
MRD
 
PB
 

UPIN indicates patient unique identifier; M, male; CE, early chronic phase; CGCR, complete cytogenetic response; AP, accelerated phase; BuCy, intravenous busulfan 0.8 mg/kg every 6 hours for 16 doses and cyclophosphamide 60 mg/kg for 2 days; MUD, matched unrelated donor; BM, bone marrow; CHR, complete hematologic response; CP, chronic phase; MRD, matched related donor; F, female; BP, blast phase; PB, peripheral blood; Ph+ ALL, Philadelphia-positive acute lymphocytic leukemia; MMR, major molecular response; FM, fludarabine 30 mg/m2 for 4 days and melphalan 140 mg/m2; MCR, major cytogenetic response; MinorCG, minor cytogenetic response; and BuFlu, fludarabine 40 mg/m2 for 4 days, intravenous busulfan 130 mg/m2 for 2 days, and antithymocyte globulin.

*

Estimate of proportion of mutation-bearing cells.

Standard criteria were used to score CML response.9  A complete molecular response (CMR) was defined as an undetectable Bcr-Abl transcript level. A major molecular response (MMR) was defined as Bcr-Abl/Abl ratio less than 0.05%.

Bcr-Abl transcripts were quantitated in a single-tube multiplex real-time reverse transcription–polymerase chain reaction (RT-PCR) assay using TaqMan chemistry (Applied Biosystems, Foster City, CA).10  Total RNA was isolated from peripheral blood or bone marrow samples by Trizol solubilization (Gibco-BRL, Gaithersburg, MD) and cDNA was synthesized by reverse transcriptase (Superscript II; Invitrogen, Carlsbad, CA). The resulting cDNA was subjected to PCR to amplify b2a2, b3a2, and e1a2 Bcr-Abl fusion transcripts, and quantitative Bcr-Abl levels were normalized to total abl transcript levels as described previously.10 

For mutational analysis, the entire kinase domain of the Bcr-Abl fusion transcript was sequenced using a nested PCR method. The Bcr-Abl fusion transcript was first amplified followed by 2 separate PCR reactions that cover exons 221 to 390 and codons 350 to 500 of the Abl kinase domain, respectively, essentially as described.11  Standard dideoxy chain termination cycle sequencing was done using a 3100 genetic analyzer (Applied Biosystems) with analysis using Seqscape v2.0 software (Applied Biosystems). All mutations were confirmed by sequencing of forward and reverse strands, with approximate sensitivity of 15% mutation-bearing cells.11 

Hematopoietic chimerism was evaluated on peripheral blood or bone marrow by multiplex PCR-based DNA microsatellite polymorphism analysis using PCR D6S264, D3S1282, D18S62, and D3S1300 fluorescence-labeled primer sets, followed by separation by capillary electrophoresis and analysis using GeneScan software (Applied Biosystems). Mixed chimerism was defined as the presence of any detectable (1%) recipient DNA.

Toxicity was graded according to National Cancer Institute criteria. GVHD was graded according to consensus criteria.12 

Results and discussion

A total of 10 patients (3 in CP, 4 in AP, 2 in BP, and 1 with Ph-positive ALL) received a transplant. The median age was 44 years (range, 26-63 years). Nine different protein kinase mutations were detected. Five patients harbored a P-loop mutation. At the time of Allo-SCT, 1 patient (patient unique identifier [UPIN] 5) was in MMR (Ph-positive ALL, mutation Q252H), 1 (UPIN 7) was in major cytogenetic response, and 2 (UPIN 4 and 10) were in complete hematologic response. The 6 remaining patients were in CP (n = 2) and AP (n = 4) with no hematologic response. Patients' characteristics are summarized in Table 1.

All patients engrafted at a median of 13 days (range, 10-17 days). There were no major toxicities and no treatment-related deaths. Chimerism studies at days 30 and 100 after HSCT were 100% of donor type in all but 1 patient. Nine patients achieved a complete cytogenetic response 1 month after transplantation. All patients were assessed for molecular response more than once: 7 achieved a CMR at a median of 2 months (range, 1-6 months) after transplantation; 2 achieved an MMR (mutations F359V and T315I) by 3 and 4 months after transplantation, with transcript levels of 0.012% and 0.04%, respectively (the first one [UPIN 9] is still responding with the last transcript level of 0.004%, while the second relapsed [UPIN 6] after 6 months). Three patients (ALL with Q252H [UPIN 5], AP with T315I [UPIN 6], and BP with E255K [UPIN 10]) progressed and died of their disease, respectively, 7, 11, and 4 months after Allo-SCT. Patients UPIN 5 and 6 had achieved a CMR and an MMR, whereas patient UPIN 10 did not respond to Allo-SCT. Seven patients are alive (6 in CMR and 1 in MMR) after a median follow-up of 19 months (range, 13-24 months). Outcomes of treatment are summarized in Table 2.

Table 2.

Transplantation outcomes


UPIN

Days to ANC greater than 0.5 × 109/L

Days to platelet count greater than 20 × 109/L

Donor chimerism on transplantation, day +30/+100

Bcr-Abl/Abl ratio, day + 100

Toxicity (grade)

Infections

aGVHD (grade)

cGVHD

Best response

Relapse

DFS, mo

Status at last follow-up

Survival, mo
1   13   19   100%   NA   None   None   None   None   CMR   No   13+   CMR   13+  
2   14   54   100%   0.0471   NV (1)   Hemorrhagic cystitis   Skin (2)   Skin   CMR   No   19+   CMR   19+  
3   17   19   100%   0.008   D (1), NV (2), M (2)   None   None   None   CMR   No   21+   CMR   21+  
4   15   17   100%   0   None  C difficile, hemorrhagic cystitis   None   Skin   CMR   No   24+   CMR   24+  
5   13   43   100%   0.01   NV (2)   CMV   None   Skin   CMR   Yes   6   Dead   7  
6   12   14   100%   0.046   NV (1), D (1)   Bacterial pneumonia, CMV   Ocular (1)   Liver   MMR   Yes   6   Dead   11  
7   10   12   100%   0   None   CMV   Skin (2)   None   CMR   No   17+   CMR   17+  
8   12   27   100%   0   LFT (1), NV (2), D (2)   None   Skin (2)   None   CMR   No   20+   CMR   20+  
9   11   24   100%   0   None   None   GI (1)   None   MMR   No   22+   MMR   22+  
10
 
13
 
NA
 
Mixed
 
NA
 
M (2), D (2)
 
Enterococcus pneumonia
 
Skin (2), Liver (1)
 
NA
 
PD
 
Yes
 
0
 
Dead
 
4
 

UPIN

Days to ANC greater than 0.5 × 109/L

Days to platelet count greater than 20 × 109/L

Donor chimerism on transplantation, day +30/+100

Bcr-Abl/Abl ratio, day + 100

Toxicity (grade)

Infections

aGVHD (grade)

cGVHD

Best response

Relapse

DFS, mo

Status at last follow-up

Survival, mo
1   13   19   100%   NA   None   None   None   None   CMR   No   13+   CMR   13+  
2   14   54   100%   0.0471   NV (1)   Hemorrhagic cystitis   Skin (2)   Skin   CMR   No   19+   CMR   19+  
3   17   19   100%   0.008   D (1), NV (2), M (2)   None   None   None   CMR   No   21+   CMR   21+  
4   15   17   100%   0   None  C difficile, hemorrhagic cystitis   None   Skin   CMR   No   24+   CMR   24+  
5   13   43   100%   0.01   NV (2)   CMV   None   Skin   CMR   Yes   6   Dead   7  
6   12   14   100%   0.046   NV (1), D (1)   Bacterial pneumonia, CMV   Ocular (1)   Liver   MMR   Yes   6   Dead   11  
7   10   12   100%   0   None   CMV   Skin (2)   None   CMR   No   17+   CMR   17+  
8   12   27   100%   0   LFT (1), NV (2), D (2)   None   Skin (2)   None   CMR   No   20+   CMR   20+  
9   11   24   100%   0   None   None   GI (1)   None   MMR   No   22+   MMR   22+  
10
 
13
 
NA
 
Mixed
 
NA
 
M (2), D (2)
 
Enterococcus pneumonia
 
Skin (2), Liver (1)
 
NA
 
PD
 
Yes
 
0
 
Dead
 
4
 

ANC indicates absolute neutrophil count; aGVHD, acute graft-versus-host disease; cGVHD, chronic graft-versus-host disease; DFS, disease-free survival; NA, not applicable; CMR, complete molecular response; N, nausea; V, vomiting; D, diarrhea; M, mucositis; C difficile, Clostridium difficile; CMV, cytomegalovirus; MMR, major molecular response; LFT, increase of the liver functional tests; GI, gastrointestinal; and PD, progressive disease.

This is the first report on the outcome of imatinib mesylate–refractory patients with kinase mutations undergoing Allo-SCT. Specifically, we showed that in such cases, Allo-SCT is feasible, safe, and frequently effective. A high rate of complete clinical and molecular responses was achieved, despite the fact that most patients received a transplant with imatinib-resistant advanced disease. In this small series, we could detect no difference in outcome between P-loop versus non–P-loop mutations.9  Interestingly, 1 of the 2 patients with the T315I mutation responded and remains in molecular remission after 17 months of follow-up, whereas the other relapsed 6 months after transplantation and died shortly after that. This mutation confers resistance to imatinib and to the novel tyrosine kinase inhibitors (NTKIs).13,14 

The NTKIs dasatinib (BMS-354825) and AMN-107, currently being evaluated in clinical trials, have shown promising results in patients with CML resistant to imatinib.15-17  Both drugs are active in patients with mutant Bcr-Abl kinase except when the disease is driven by the T315I mutation. In advanced-stage patients such as ours, consideration should be given to maintenance use of NTKI or other agents after transplantation.

These preliminary results support the use of Allo-SCT as a salvage option for patients developing resistance to imatinib through Bcr-Abl mutations. This subset of patients is likely to benefit from early transplantation, should a suitable related or unrelated donor be identified. Long-term follow-up of a larger number of patients will be necessary to fully document the efficacy and benefit of allogeneic transplantation in this setting.

Prepublished online as Blood First Edition Paper, April 6, 2006; DOI 10.1182/blood-2006-02-001933.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

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