title: Identification of e6a2 BCR-ABL fusion in a Philadelphia-positive CML with marked basophilia: implications for treatment strategy.
abstract: OBJECTIVE
  This is a case report of a 51 year old male with marked splenomegaly, basophilia, severe thrombocytopenia, anemia and high SFKL phosphorylation downstream of Bcr-Abl, investigated for association of the e6a2 BCR-ABL fusion gene and marked basophilia. The treatment strategy implications in patients with Philadelphia positive CML are described.
  
  
  METHODS
  RT-PCR and sequencing were carried out on the peripheral blood leukocytes to detect the type of BCR-ABL transcript. The BCR-ABL mutational status was assessed using sequencing of the RT-PCR products. The in vitro test of sensitivity to TKIs was based on detecting inhibited phosphorylation of the Crkl and Phospho-Src family kinases (SFK, Tyr416) using immunodetection.
  
  
  RESULTS
  The cytogenetics revealed 90% of Ph+ (Philadelphia) cells in the bone marrow aspirate with no additional clonal chromosomal abnormalities at diagnosis. This correlated with an accelerated phase of the CML. Sequencing analysis of reverse transcribed and PCR amplified BCR-ABL transcript revealed a rare e6a2 fusion, with no evidence for Bcr-Abl kinase domain mutation. Western blot analysis showed high phosphorylation (activation) of Crkl and the Src family of kinases (P-SFK). In vitro test of sensitivity of the patients' leukemic cells to imatinib demonstrated sensitivity of Bcr-Abl tyrosine kinase to imatinib, as assessed by a decrease in phosphorylated Crkl and the disappearance of P-SFK, suggesting that P-Src reflects only the Bcr-Abl-dependent Src activity. The initial treatment strategy was reduced imatinib and search for an unrelated hematopoietic stem cell donor (according to the ELN recommendations). The patient was allografted with peripheral stem cells from an HLA- identical male donor but on day +70 graft failure occurred. He was allografted again with the peripheral stem cells from an HLA-identical female donor, engrafted on day +15 and showed 100% donor chimerism with no evidence of the e6a2 BCR-ABL fusion transcript on day +30.
  
  
  CONCLUSIONS
  The clinical disease course in patients with the rare e6a2 BCR-ABL transcript variant is aggressive. This may be the result of increased kinase activity due to partial loss of the guanine exchange factor/dbl-like domain which mediates the interaction with several Ras-like G-proteins involved in cell proliferation, signal transduction, and cytoskeletal organization. For the above reasons, these patients should receive stem cell transplant immediately after a short course of treatment with imatinib/ dual Src/Abl kinase inhibitor or they should be registered in clinical trials with experimental agents.
authors.affiliation: Department of Hemato-Oncology, University Hospital Olomouc and Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech Republic. peter.rohon@centrum.cz
authors.forename: Peter
authors.initials: P
authors.lastname: Rohon
authors.forename: Martina
authors.initials: M
authors.lastname: Divoka
authors.forename: Lenka
authors.initials: L
authors.lastname: Calabkova
authors.forename: Renata
authors.initials: R
authors.lastname: Mojzikova
authors.forename: Beata
authors.initials: B
authors.lastname: Katrincsakova
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Rusinakova
authors.forename: Anna
authors.initials: A
authors.lastname: Lapcikova
authors.forename: Ludek
authors.initials: L
authors.lastname: Raida
authors.forename: Edgar
authors.initials: E
authors.lastname: Faber
authors.forename: Marie
authors.initials: M
authors.lastname: Jarosova
authors.forename: Vladimir
authors.initials: V
authors.lastname: Divoky
authors.forename: Karel
authors.initials: K
authors.lastname: Indrak
mesh_terms: D001491:Basophils; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000097:blood / Q000235:genetics* / Q000628:therapy; D007958:Leukocyte Count; D008297:Male; D008875:Middle Aged; D050596:Oncogene Fusion; D010677:Philadelphia Chromosome; D033581:Stem Cell Transplantation