Technical aspects and clinical applications of measuring BCR‐ABL1 transcripts number in chronic myeloid leukemia

Abstract

Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by a triphasic clinical course, the morphologic expansion of a terminally differentiated myeloid cell and the presence of the BCR‐ABL1 fusion gene, the hallmark of CML. The fusion gene is usually, but not always, associated with a Philadelphia chromosome, the result of a reciprocal exchange of genetic material between chromosome 22 and chromosome 9, which leads to the production of the activated BCR‐ABL1 gene and oncoprotein. The breakpoint in the BCR gene occurs commonly downstream of exons e13 or e14 (M‐BCR) and less frequently downstream of exons e1 and e2 (m‐BCR). Less than 1% of cases carry a breakpoint downstream of exon 6 or 8 (“variant fusion genes”) or exon 19 (μ‐BCR). Breakpoints in the ABL1 gene cluster upstream of exon a2 (or of exon a3 in less than 5% of patients with CML). Conventional cytogenetic, fluorescence in situ hybridization, and molecular testing for the BCR‐ABL1 fusion gene are key investigations for the diagnosis and monitoring of CML. Treatment using tyrosine kinase inhibitors has revolutionized the management of CML with hematologic and cytogenetic response within 12–18 months observed in >85% of patients. Nevertheless, between 15 and 20% of patients may evolve to blastic phase. Measurement of low level or “minimal” residual disease using molecular tests is becoming the gold‐standard approach to measure response to therapy due to its higher sensitivity compared to other routine techniques. The technical aspects and clinical applications of molecular monitoring will be the main focus of this article. Am. J. Hematol., 2009. © 2009 Wiley‐Liss, Inc.

Chronic myeloid leukemia: mechanisms of blastic transformation

“…Indeed, it appears today that continued use of TKIs to treat CML-CP may prevent BP in a large proportion of patients, but 15%-20% of patients, most of whom will have been classified as nonresponders, may progress to BP (11). Indeed, the GIMEMA Working Party (Italian Group for Adult Hematologic Diseases) reported that the detection of TKI-resistant BCR-ABL1 mutations in CML-CP is associated with a greater likelihood of disease progression (12).…”

RT-qPCR and RT-Digital PCR: A Comparison of Different Platforms for the Evaluation of Residual Disease in Chronic Myeloid Leukemia

“…All cDNA samples were stored at Ϫ80°C at the Hammersmith Hospital (for a median of 2 years, range 1-7 years) following the processing of peripheral blood samples as they arrived at the laboratory according to a previously described protocol (15)(16). Briefly, 20 -30 mL of peripheral blood was washed in red cell lysis buffer and the total white blood cell pellet was lysed in 1.5 mL RLT buffer (Qiagen) and stored at Ϫ80°C.…”

Duplex quantitative PCR for molecular monitoring of BCR‐ABL1‐associated hematological malignancies

“…In total, 92 patient samples were collected and processed according to standard procedure and in accordance with the EAC recommendations [2,6]. Briefly, all samples were peripheral blood in EDTA and processed within 72 hr of being drawn.…”

“…Endogenous control genes, used to normalize the target gene expression, were evaluated, and ABL1 was judged to be the most appropriate [3]. Particular effort has been expended recently to optimize and standardize the quantification of BCR-ABL1 transcripts especially following to the worldwide use of first-and second-generation tyrosine kinase inhibitor-based therapies [1][2][3][4][5][6].Because of a high throughput of MRD samples, both in-house and referral, a routine assay was initially developed for use in our laboratory that quantitated BCR-ABL1 and ABL1 transcripts in separate qPCR runs (simplex tests). This assay was based on the EAC protocol but utilized different ABL1 primers and probes, hence referred to as the ''Hammersmith'' set, because of the presence of a G6PD sequence insert (part of an earlier competitive PCR assay) in the ABL1 exon 3 [7].…”

“…Endogenous control genes, used to normalize the target gene expression, were evaluated, and ABL1 was judged to be the most appropriate [3]. Particular effort has been expended recently to optimize and standardize the quantification of BCR-ABL1 transcripts especially following to the worldwide use of first-and second-generation tyrosine kinase inhibitor-based therapies [1][2][3][4][5][6].…”