Identification of E6a2 BCR-Abl Fusion in a Philadelphia-Positive CML With Marked Basophilia: Implications for Treatment Strategy

Abstract

Aim. 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. Conclusion. 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.

Establishment of quantitative nested-PCR of Abelson interactor 1 transcript variant-11

“…RT-PCR is still the most rapid and sensitive method that can accurately quantify the expression of TSVs [ 23 , 24 , 25 , 26 , 27 ]. For ABI1-TSV-11, we established a rapid and sensitive quantitative nested-PCR detection technique based on quantitative nested-PCR strategy composed of pre-amplification and exon-exon junction (EEJ)-specific primers (SupplementaryFigure 1 and Figure 1 ) [ 15 , 16 ].…”

“…For systematic and high-throughput detection and multivariable analyses, expressed sequence tag (EST) sequences [ 12 ], exon microarrays [ 13 ], and next-generation sequencing (NGS) are valid qualitative and quantitative methods for evaluating TSV expression, cancer mechanisms, and prognoses of patients. However, there is lack of quick and accurate method to detect the expression of ABI-TSV-11 mRNA [ 14 , 15 , 16 ]. Regular PCR using spanning exon-exon junction (EEJ) sequences as primers can effectively amplify TSVs specifically, quickly and accurately [ 15 , 16 ].…”

“…However, there is lack of quick and accurate method to detect the expression of ABI-TSV-11 mRNA [ 14 , 15 , 16 ]. Regular PCR using spanning exon-exon junction (EEJ) sequences as primers can effectively amplify TSVs specifically, quickly and accurately [ 15 , 16 ]. However, this reaction may generate false-positive results in the presence of non-specific amplification.…”

A novel <em>BCR-ABL1</em> mutation in a patient with Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia

“…The remaining Ph+ ALL cases are associated with P210 BCR-ABL1 . The transforming potential of the P190 isoform is believed to result from the partial loss of BCR domains, namely, the guanine exchange factor/dbl-like domain that mediates the communication with several Ras proteins, which are crucial for the regulation of signaling pathways and processes, such as cell proliferation, differentiation, adhesion, apoptosis and migration 4–7. More recently, Reckel et al8 demonstrated the existence of strong differences in the interactome and tyrosine phosphoproteome between P190 and P210 signaling pathways.…”

Chronic Myeloid Leukemia with an e6a2BCR-ABL1Fusion Transcript: Cooperating Mutations at Blast Crisis and Molecular Monitoring

“…Although responses to imatinib have been reported [ 10 , 11 ], several cases of ABL1 kinase domain mutation-associated imatinib resistant, e6a2 BCR-ABL1 CML have been documented [ 12 , 13 ] with limited information on the efficacy of front line second-generation TKIs in this genotype. As e6a2 BCR-ABL1 CML is associated with aggressive disease, allogeneic stem cell transplantation (ASCT) remains the only curative option and in such circumstances conventional AML therapy has been combined with a second-generation TKI as a bridge to ASCT [ 14 – 16 ].…”