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Instability of BCR–ABL Gene in Primary and Cultured Chronic Myeloid Leukemia Stem Cells
Affiliations of authors: Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC, Canada (XJ, KMS, AE, CE); Departments of Medical Genetics (XJ, CE), Medicine (AE, CE), and Pathology and Laboratory Medicine (AE, CE), University of British Columbia, Vancouver, BC, Canada
Correspondence to: Xiaoyan Jiang, MD, PhD, Terry Fox Laboratory, 675 West 10th Ave, Vancouver, BC, V5Z 1L3, Canada (e-mail: xjiang{at}bccrc.ca).
Background: Imatinib mesylate treatment causes remissions in a majority of patients with chronic myeloid leukemia (CML), but relapses are an increasing problem. We hypothesized that imatinib-resistant leukemic cells emerge from CML stem cells that acquire BCR–ABL gene mutations even before exposure to BCR–ABL–targeted agents such as imatinib.
Methods: Lineage-negative (i.e., immature) CD34+CD38– CML stem cell–enriched populations were isolated from five patients with chronic phase CML samples by fluorescence-activated cell sorting. To identify BCR–ABL gene mutations, complementary DNAs (cDNAs) prepared from purified CML stem cells were subjected to allele-specific amplification using primers corresponding to 16 kinase domain mutations, with normal bone marrow cells serving as negative controls. We also cloned and directly sequenced BCR–ABL cDNAs prepared from freshly isolated CML stem cells and from their progeny generated after 3–5 weeks of culture.
Results: In 20%–33% of cDNA preparations from freshly isolated CML stem cell–enriched populations, both allele-specific amplification and direct sequencing methods revealed mutations in sequences corresponding to the BCR–ABL kinase domain. Mutations were not observed in cDNA sequences encoding the c-ABL kinase domain that were obtained from similar types of primitive normal cells. More than 70 different BCR–ABL mutations (including frameshift mutations and premature stop codons) were identified in the progeny of cultured CML stem cells. Analysis of individual clones derived from the cultured cells demonstrated that new BCR–ABL mutations were produced.
Conclusions: Primary CML stem cells display instability of the BCR–ABL fusion gene both in vivo and in vitro. Thus, patients may possess leukemic stem cells with BCR–ABL kinase mutations before initiation of BCR–ABL–targeted therapies and would likely be predisposed to develop resistance to these agents.
| CONTEXT AND CAVEATS Prior knowledge While treatment with imatinib causes remissions in the majority of patients with chronic myeloid leukemia, relapses often occur. The mechanisms by which resistance to imatinib develops are not well characterized. Study design Chonic myeloid leukemia stem cells from patients were isolated and genetically characterized for mutations in the oncogene (BCR–ABL) that causes the disease. Contribution These chronic myeloid leukemia stem cells were found to be genetically unstable, with a high frequency of mutation, leading to alterations in the BCR–ABL protein, the target of imatinib. Implications Resistance to imatinib may ultimately derive from mutations in a population of chronic myeloid leukemia stem cells that occur before exposure to chemotherapy. Further investigation of how mutations develop in these particular cells is needed. Limitations More research will be needed to translate these insights into improved therapeutics for patients.
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Manuscript received August 14, 2006; revised February 13, 2007; accepted March 19, 2007.
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J Natl Cancer Inst 2007 99: 662-663.
J Natl Cancer Inst 2007 99: 657.
J Natl Cancer Inst 2007 99: 657.
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