BCR/ABL1, Tyrosine Kinase Inhibitor Resistance, Kinase Domain Mutation Screen, Sanger Sequencing
Clinical Information Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test
Chronic myelogenous leukemia (CML) is characterized by the presence of the t(9:22) BCR-ABL1 abnormality, resulting in formation of a fusion BCR-ABL1 mRNA and protein. The ABL1 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 ABL1 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 cases (approximately 50%) to the acquisition of point mutations in the kinase domain (KD) of the chimeric ABL1 gene. To date, over 50 distinct mutations have been described, although a smaller subset of these (<20) account for the majority of patients with clinical resistance to TKIs, or have well documented in vitro data in the published literature.
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 except ponatinib. 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-ABL1 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 the oncogenic BCR-ABL1 are typically detected by direct sequencing of PCR products, following RT-PCR amplification of the BCR-ABL mRNA transcript from a peripheral blood specimen. This approach ensures comprehensive screening of the clinically relevant KD region. Because this technique requires inclusion of a longer region of ABL1 in the BCR-ABL1 RT-PCR product, low levels of the BCR-ABL1 mRNA transcript (below 0.01% normalized BCR-ABL1 on the International Scale, IS) may not be efficiently amplified (in contrast to similar amplicons generated by quantitative RT-PCR for diagnosis or monitoring).
Evaluating patients with chronic myelogenous leukemia and Philadelphia chromosome positive B-cell acute lymphoblastic leukemia receiving tyrosine kinase inhibitor (TKI) therapy, who are apparently failing treatment
Preferred initial test to identify the presence of acquired BCR-ABL1 mutations associated with TKI-resistance
The presence of one or more point mutations in the translocated portion of the ABL1 region of the BCR-ABL1 fusion mRNA is considered a positive result, indicating tyrosine kinase inhibitor (TKI) resistance. The specific type of mutation may influence the sensitivity to a specific TKI, and could be useful in guiding therapeutic options for an individual patient.
Cautions Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances
This assay is comprehensive for detecting BCR-ABL1 KD mutations, but does not detect all possible mutations in ABL1; this, 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-ABL1 transcript is critical for a successful assay mutation analysis, because the amplification efficiency for a longer mRNA template is decreased with a low abundance of target. If the BCR-ABL1 quantitative PCR level is too low, RT-PCR amplification of BCR-ABL1 may be unsuccessful to yield product for sequencing. Although laboratory standards are yet to be developed, a BCR-ABL1/ABL1 quantitative level above 0.1% is generally considered to be required in order to detect KD mutations by this assay.
Subclonal mutations may be difficult to identify by Sanger sequencing method, even if the BCR-ABL1 mRNA amplification was successful. This is due to the inherit sensitivity level limit of sequencing, which is typically around 15% to 20% mutant allele in a wild-type background.
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. Ambient specimens over 3 days old and refrigerate specimens over 5 days old at the time of receipt are unacceptable.
Reference Values Describes reference intervals and additional information for interpretation of test results. May include intervals based on age and sex when appropriate. Intervals are Mayo-derived, unless otherwise designated. If an interpretive report is provided, the reference value field will state this.
An interpretive report will be provided.
Clinical References Provides recommendations for further in-depth reading of a clinical nature
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. Press RD, Kamel-Reid S, Ang D: BCR-ABL1 RT-qPCR for Monitoring the Molecular Response to Tyrosine Kinase Inhibitors in Chronic Myeloid Leukemia. J Mol Diagn 2013;15:565-576
3. Baccarani M, Deininger MW, Rosti G, et al: European LeukemiaNet recommendations for the management of chronic myeloid leukemia: 2013. Blood 2013;122:872-884
4. 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