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Evaluating patients with chronic myeloid leukemia and Philadelphia chromosome positive B-cell acute lymphoblastic leukemia receiving tyrosine kinase inhibitor (TKI), therapy, who are apparently failing treatment
This is the preferred initial test to identify the presence of acquired BCR-ABL mutations associated with TKI-resistance.
ChroChronic myeloid leukemia (CML) is characterized by the presence of the t(9:22) BCR-ABL abnormality, resulting in formation of a fusion BCR-ABL mRNA and protein. The ABL component of this oncoprotein contains tyrosine kinase activity and is thought to play a central role in the proliferative phenotype of this leukemia.
Recent advances have resulted in a number of therapeutic drugs that inhibit the ABL tyrosine kinase, as well as other protein tyrosine kinases. Imatinib mesylate (Gleevec, Novartis) is the prototype of these tyrosine kinase inhibitors (TKIs), which are capable of inducing durable hematologic and (in most patients) cytogenetic remissions. Unfortunately, a significant subset of patients can develop functional resistance to TKIs, due in a large number of tumors to the acquisition of point mutations in the kinase domain (KD) of the chimeric ABL gene. To date, over 50 distinct mutations have been described, although 15 of these account for more than 80% of the mutations encountered and have well documented in vitro or clinical resistance to TKIs.
Recognition of TKI resistance is important in CML, as the effect of some mutations can be overcome by increasing imatinib dosage, whereas others require switching to either a different (second-generation) TKI, or alternative therapy. The common T315I KD mutation is particularly important, given that this alteration confers pan-resistance to all currently employed TKIs. Typically, TKI resistance is suspected in a CML patient who shows loss of initial therapeutic response (eg, cytogenetic relapse), or a significant and sustained increase in molecular BCR-ABL quantitative levels. Similar considerations are also present in patients with Philadelphia chromosome positive B-cell acute lymphoblastic leukemia, who can also be treated using TKI therapy.
Point mutations in ABL are typically detected by direct sequencing of PCR products, following RT-PCR amplification of the BCR-ABL mRNA transcript from a peripheral blood specimen. However, direct sequencing has limited analytic sensitivity (approximately 20%-30% mutant alleles). In contrast, this test utilizes a novel strategy to detect 15 of the most common ABL KD mutations accounting for >80% of the most common and clinically important mutations. The sensitivity of this ASPE/Luminex approach is enhanced, with a better lower limit of detection in the range of 5% to 8% mutant alleles, with very high specificity.
An interpretive report will be provided.
The presence of 1 or more point mutations in the translocated portion of the ABL region of the BCR-ABL fusion mRNA is considered a positive result, indicating tyrosine kinase inhibitor (eg, imatinib) resistance
This assay does not detect all possible kinase domain (KD) mutations; thus, a negative result by this assay does not exclude the presence of a rare, less well characterized or unknown mutation that could be associated with some degree of tyrosine kinase inhibitor resistance. The clinical significance of such rarely occurring mutations is, however, uncertain.
The quantitative level of BCR-ABL transcript is critical for a successful assay mutation analysis. If the BCR-ABL quantitative PCR level is too low, RT-PCR amplification of BCR-ABL may be unsuccessful. Although laboratory standards are yet to be developed, a BCR-ABL/ABL quantitative level above 0.1% is generally considered to be required in order to detect KD mutations by this assay.
EDTA blood specimens are preferred for testing. Bone marrow specimens are acceptable; there occasionally are specimen failures from bone marrow RNA, for reasons that are not completely understood. Heparin anticoagulant cannot be used because of PCR inhibition.
Assay precision does not appear to be significantly affected by specimen transport or moderate delays in processing. However, in specimen with lower levels of BCR-ABL, these conditions may cause sufficient RNA degradation to produce false-negative results. Thus, specimens should be shipped as quickly as possible and specimens >3 days old at the time of receipt are unacceptable.
1. Hughes T, Deininger M, Hochhaus A, et al: Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and recommendations for harmonizing current methodology for detecting BCR-ABL transcripts and kinase domain mutations and for expressing results. Blood 2006;108:28-37
2. Jabbour E, Kantarjian H, Jones D, et al: Frequency and clinical significance of BCR-ABL mutations in patients with chronic myeloid leukemia treated with imatinib mesylate. Leukemia 2006;20:1767-1773
3. Oncology NPGi. Chronic myelogenous leukemia: National Comprehensive Cancer Network; 2008 Available from URL: nccn.org/professionals/physician_gls/PDF/cml.pdf
4. Baccarani M, Saglio G, Goldman J, et al: Evolving concepts in the management of chronic myeloid leukemia: recommendations from an expert panel on behalf of the European Leukemia Net. Blood 2006;108:1809-1820
5. Jones D, Kamel-Reid S, Bahler D, et al: Laboratory practice guidelines for detecting and reporting BCR-ABL drug resistance mutations in chronic myelogenous leukemia and acute lymphoblastic leukemia A Report of the Association for Molecular Pathology. J Mol Diagn 2009;11:4-11