Sensitive Detection of Pre-Existing BCR-ABL Kinase Domain Mutations in CD34+ Cells of Newly Diagnosed Chronic-Phase Chronic Myeloid Leukemia Patients Is Associated with Imatinib Resistance: Implications in the Post-Imatinib Era

Background BCR-ABL kinase domain mutations are infrequently detected in newly diagnosed chronic-phase chronic myeloid leukemia (CML) patients. Recent studies indicate the presence of pre-existing BCR-ABL mutations in a higher percentage of CML patients when CD34+ stem/progenitor cells are investigated using sensitive techniques, and these mutations are associated with imatinib resistance and disease progression. However, such studies were limited to smaller number of patients. Methods We investigated BCR-ABL kinase domain mutations in CD34+ cells from 100 chronic-phase CML patients by multiplex allele-specific PCR and sequencing at diagnosis. Mutations were re-investigated upon manifestation of imatinib resistance using allele-specific PCR and direct sequencing of BCR-ABL kinase domain. Results Pre-existing BCR-ABL mutations were detected in 32/100 patients and included F311L, M351T, and T315I. After a median follow-up of 30 months (range 8–48), all patients with pre-existing BCR-ABL mutations exhibited imatinib resistance. Of the 68 patients without pre-existing BCR-ABL mutations, 24 developed imatinib resistance; allele-specific PCR and BCR-ABL kinase domain sequencing detected mutations in 22 of these patients. All 32 patients with pre-existing BCR-ABL mutations had the same mutations after manifestation of imatinib-resistance. In imatinib-resistant patients without pre-existing BCR-ABL mutations, we detected F311L, M351T, Y253F, and T315I mutations. All imatinib-resistant patients except T315I and Y253F mutations responded to imatinib dose escalation. Conclusion Pre-existing BCR-ABL mutations can be detected in a substantial number of chronic-phase CML patients by sensitive allele-specific PCR technique using CD34+ cells. These mutations are associated with imatinib resistance if affecting drug binding directly or indirectly. After the recent approval of nilotinib, dasatinib, bosutinib and ponatinib for treatment of chronic myeloid leukemia along with imatinib, all of which vary in their effectiveness against mutated BCR-ABL forms, detection of pre-existing BCR-ABL mutations can help in selection of appropriate first-line drug therapy. Thus, mutation testing using CD34+ cells may facilitate improved, patient-tailored treatment.

[1]  J. Radich,et al.  Polymerase chain reaction detection of the BCR-ABL fusion transcript after allogeneic marrow transplantation for chronic myeloid leukemia: results and implications in 346 patients. , 1995, Blood.

[2]  M. Baccarani,et al.  Contribution of ABL Kinase Domain Mutations to Imatinib Resistance in Different Subsets of Philadelphia-Positive Patients: By the GIMEMA Working Party on Chronic Myeloid Leukemia , 2006, Clinical Cancer Research.

[3]  C. Sawyers,et al.  Detection of BCR-ABL kinase mutations in CD34+ cells from chronic myelogenous leukemia patients in complete cytogenetic remission on imatinib mesylate treatment. , 2005, Blood.

[4]  J. Melo,et al.  Chronic myeloid leukemia--advances in biology and new approaches to treatment. , 2003, The New England journal of medicine.

[5]  B. Dörken,et al.  Preexistence and evolution of imatinib mesylate-resistant clones in chronic myelogenous leukemia detected by a PNA-based PCR clamping technique , 2003, Annals of Hematology.

[6]  E. Dolgin.  As leukemia options grow, drugs jockey to be first-line therapies , 2013, Nature Medicine.

[7]  M. Gasparetto,et al.  Chronic myeloid leukemia stem cells possess multiple unique features of resistance to BCR-ABL targeted therapies , 2007, Leukemia.

[8]  H. Kantarjian,et al.  BCR-ABL tyrosine kinase inhibitors in chronic myeloid leukemia: using guidelines to make rational treatment choices. , 2008, Journal of the National Comprehensive Cancer Network : JNCCN.

[9]  B. Druker,et al.  Mutations of the BCR-ABL-kinase domain occur in a minority of patients with stable complete cytogenetic response to imatinib , 2007, Leukemia.

[10]  R. Bhatia,et al.  Persistence of leukemia stem cells in chronic myelogenous leukemia patients in prolonged remission with imatinib treatment. , 2011, Blood.

[11]  J. Cayuela,et al.  Mutation status and clinical outcome of 89 imatinib mesylate-resistant chronic myelogenous leukemia patients: a retrospective analysis from the French intergroup of CML (Fi(ϕ)-LMC GROUP) , 2006, Leukemia.

[12]  K. Lewandowski,et al.  Dasatinib treatment can overcome imatinib and nilotinib resistance in CML patient carrying F359I mutation of BCR-ABL oncogene , 2010, Journal of Applied Genetics.

[13]  T. Hughes,et al.  Detection of BCR-ABL mutations and resistance to imatinib mesylate. , 2006, Methods in molecular medicine.

[14]  C. Eaves,et al.  Instability of BCR-ABL gene in primary and cultured chronic myeloid leukemia stem cells. , 2007, Journal of the National Cancer Institute.

[15]  C. Auewarakul,et al.  BCR-ABL kinase domain mutations in tyrosine kinase inhibitors-naïve and -exposed Southeast Asian chronic myeloid leukemia patients. , 2012, Experimental and molecular pathology.

[16]  D. Marin,et al.  Adherence is the critical factor for achieving molecular responses in patients with chronic myeloid leukemia who achieve complete cytogenetic responses on imatinib. , 2010, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[17]  R. Clark,et al.  A population study of imatinib in chronic myeloid leukaemia demonstrates lower efficacy than in clinical trials , 2008, Leukemia.

[18]  L. Barsky,et al.  In Vitro Identification of Single CD34+CD38− Cells With Both Lymphoid and Myeloid Potential , 1998 .

[19]  J. Apperley.  Part I: mechanisms of resistance to imatinib in chronic myeloid leukaemia. , 2007, The Lancet. Oncology.

[20]  M. Guillier,et al.  Evidence of ABL-kinase domain mutations in highly purified primitive stem cell populations of patients with chronic myelogenous leukemia. , 2004, Biochemical and biophysical research communications.

[21]  D. Marin,et al.  Finding of kinase domain mutations in patients with chronic phase chronic myeloid leukemia responding to imatinib may identify those at high risk of disease progression. , 2008, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[22]  M. Gordon.  Finding of Kinase Domain Mutations in Patients With Chronic Phase Chronic Myeloid Leukemia Responding to Imatinib May Identify Those at High Risk of Disease Progression , 2009 .

[23]  T. Clackson,et al.  AP24534, a pan-BCR-ABL inhibitor for chronic myeloid leukemia, potently inhibits the T315I mutant and overcomes mutation-based resistance. , 2009, Cancer cell.

[24]  C. Preudhomme,et al.  A mutation conferring resistance to imatinib at the time of diagnosis of chronic myelogenous leukemia. , 2003, The New England journal of medicine.

[25]  R. Herrmann,et al.  Real-time quantitative PCR analysis can be used as a primary screen to identify patients with CML treated with imatinib who have BCR-ABL kinase domain mutations. , 2004, Blood.

[26]  L. Barsky,et al.  In vitro identification of single CD34+CD38- cells with both lymphoid and myeloid potential. , 1998, Blood.

[27]  R. Foà,et al.  Philadelphia-positive acute lymphoblastic leukemia patients already harbor BCR-ABL kinase domain mutations at low levels at the time of diagnosis , 2011, Haematologica.

[28]  T. Holyoake,et al.  Primitive, quiescent, Philadelphia-positive stem cells from patients with chronic myeloid leukemia are insensitive to STI571 in vitro. , 2002, Blood.

[29]  J. Goldman.  How I treat chronic myeloid leukemia in the imatinib era. , 2007, Blood.

[30]  H. Kantarjian,et al.  Dose escalation of imatinib mesylate can overcome resistance to standard-dose therapy in patients with chronic myelogenous leukemia. , 2003, Blood.

[31]  Francisco Cervantes,et al.  Evolving concepts in the management of chronic myeloid leukemia: recommendations from an expert panel on behalf of the European LeukemiaNet. , 2006, Blood.

[32]  Z. Iqbal,et al.  Case study of primary imatinib resistance and correlation of BCR-ABL multiple mutations in chronic myeloid leukemia , 2004 .

[33]  P. N. Rao,et al.  Clinical Resistance to STI-571 Cancer Therapy Caused by BCR-ABL Gene Mutation or Amplification , 2001, Science.

[34]  Martin C. Müller,et al.  Impact of baseline BCR-ABL mutations on response to nilotinib in patients with chronic myeloid leukemia in chronic phase. , 2009, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[35]  C. Eaves,et al.  Properties of CD34+ CML stem/progenitor cells that correlate with different clinical responses to imatinib mesylate. , 2010, Blood.

[36]  J. Kuriyan,et al.  Multiple BCR-ABL kinase domain mutations confer polyclonal resistance to the tyrosine kinase inhibitor imatinib (STI571) in chronic phase and blast crisis chronic myeloid leukemia. , 2002, Cancer cell.

[37]  J. Oldenburg,et al.  Kinase domain mutations of BCR-ABL frequently precede imatinib-based therapy and give rise to relapse in patients with de novo Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL). , 2007, Blood.

[38]  J. Radich,et al.  NCCN Task Force report: tyrosine kinase inhibitor therapy selection in the management of patients with chronic myelogenous leukemia. , 2011, Journal of the National Comprehensive Cancer Network : JNCCN.

[39]  M. Pirmohamed,et al.  Expression of the Uptake Drug Transporter hOCT1 is an Important Clinical Determinant of the Response to Imatinib in Chronic Myeloid Leukemia , 2008, Clinical pharmacology and therapeutics.

[40]  C. Eaves,et al.  Primitive quiescent leukemic cells from patients with chronic myeloid leukemia spontaneously initiate factor-independent growth in vitro in association with up-regulation of expression of interleukin-3. , 2001, Blood.

[41]  Claude Preudhomme,et al.  Several types of mutations of the Abl gene can be found in chronic myeloid leukemia patients resistant to STI571, and they can pre-exist to the onset of treatment. , 2002, Blood.

[42]  M. Copland,et al.  Dasatinib (BMS-354825) targets an earlier progenitor population than imatinib in primary CML but does not eliminate the quiescent fraction. , 2006, Blood.

[43]  A. Ballestrero,et al.  Kinase domain mutations of BCR-ABL identified at diagnosis before imatinib-based therapy are associated with progression in patients with high Sokal risk chronic phase chronic myeloid leukemia , 2010, Leukemia & lymphoma.

[44]  Su-Hyun Kim,et al.  Comparison of allele specific oligonucleotide-polymerase chain reaction and direct sequencing for high throughput screening of ABL kinase domain mutations in chronic myeloid leukemia resistant to imatinib. , 2006, Haematologica.

[45]  S. McWeeney,et al.  High-sensitivity detection of BCR-ABL kinase domain mutations in imatinib-naive patients: correlation with clonal cytogenetic evolution but not response to therapy. , 2005, Blood.

[46]  M. Krajinovic,et al.  Multidrug resistance gene (MDR1) polymorphisms are associated with major molecular responses to standard-dose imatinib in chronic myeloid leukemia. , 2008, Blood.

[47]  Franziska Michor,et al.  Fitness Conferred by BCR-ABL Kinase Domain Mutations Determines the Risk of Pre-Existing Resistance in Chronic Myeloid Leukemia , 2011, PloS one.

[48]  H. Kantarjian,et al.  Beyond dose escalation: clinical options for relapse or resistance in chronic myelogenous leukemia. , 2008, Journal of the National Comprehensive Cancer Network : JNCCN.