Instability of BCR-ABL gene in primary and cultured chronic myeloid leukemia stem cells.
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.
Jiang
Xiaoyan
X
Terry Fox Laboratory, British Columbia Cancer Agency, 675 West 10th Ave, Vancouver, BC, V5Z 1L3, Canada. xjiang@bccrc.ca
Saw
Kyi Min
KM
Eaves
Allen
A
Eaves
Connie
C
D001549:Benzamides; D003114:Colony-Forming Units Assay; D004252:DNA Mutational Analysis; D017931:DNA Primers; D016313:Genes, abl / Q000187:drug effects; D042822:Genomic Instability; D006801:Humans; D000068877:Imatinib Mesylate; D018189:Immunomagnetic Separation; D017404:In Situ Hybridization, Fluorescence; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000097:blood / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D007958:Leukocyte Count; D009154:Mutation; D010879:Piperazines / Q000627:therapeutic use; D047428:Protein Kinase Inhibitors / Q000627:therapeutic use; D011743:Pyrimidines / Q000627:therapeutic use; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000502:physiology*; D014407:Tumor Cells, Cultured
D001549:Benzamides; D017931:DNA Primers; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D000068877:Imatinib Mesylate