BCR/ABL, Tyrosine Kinase Inhibitor Resistance, Kinase Domain Mutation Screen
NY State Approved Indicates the status of NY State approval and if the test is orderable for NY State clients.
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.
Testing Algorithm Delineates situation(s) when tests are added to the initial order. This includes reflex and additional tests.
If BCR/ABL fusion type (p210, p190, p185 or p230) are not provided, BADX/89006 BCR/ABL, mRNA Detection, Reverse Transcription-PCR (RT-PCR), Qualitative, Diagnostic Assay will be performed at an additional charge.
Special Instructions and Forms Describes specimen collection and preparation information, test algorithms, and other information pertinent to test. Also includes pertinent information and consent forms to be used when requesting a particular test
Reverse Transcription-Polymerase Chain Reaction (RT-PCR) with Fluorescent-Bead Array Analysis Allele-Specific Primer Extension (ASPE) and Detection by Luminex Bead Array
(PCR is utilized pursuant to a license agreement with Roche Molecular Systems, Inc.)
Reporting Name A shorter/abbreviated version of the Published Name for a test; an abbreviated test name
BCR/ABL Mutation, ASPE
Specimen Type Describes the specimen type needed for testing
Specimen Required Defines the optimal specimen. This field describes the type of specimen required to perform the test and the preferred volume to complete testing. The volume allows automated processing, fastest throughput and, when indicated, repeat or reflex testing.
The following information is required:
1. Patient's fusion type (p210, p190, p185 or p230)
2. Pertinent clinical history
3. Clinical or morphologic suspicion
4. Date of collection
5. Specimen source (blood or bone marrow)
1. Hematopathology Patient Information Sheet (Supply T676) in Special Instructions
2. If not ordering electronically, submit a Hematopathology/Molecular Oncology Request Form (Supply T241) with the specimen.
Specimen must arrive within 72 hours of collection.
Note: If BCR/ABL fusion type (p210, p190, p185 or p230) are not provided, BADX/89006 BCR/ABL, mRNA Detection, Reverse Transcription-PCR (RT-PCR), Qualitative, Diagnostic Assay will be performed at an additional charge.
Submit only 1 of the following specimens:
Specimen Type: Whole blood
Container/Tube: Lavender top (EDTA)
Specimen Volume: 3 mL
1. Invert several times to mix blood.
2. Send specimen in original tube.
3. Label specimen as blood.
Specimen Type: Bone marrow
Container/Tube: Lavender top (EDTA)
Specimen Volume: 2 mL
1. Invert several times to mix bone marrow.
2. Send specimen in original tube.
3. Label specimen as bone marrow.
Specimen Minimum Volume Defines the amount of specimen required to perform an assay once, including instrument and container dead space. Submitting the minimum specimen volume makes it impossible to repeat the test or perform confirmatory or perform reflex testing. In some situations, a minimum specimen volume may result in a QNS (quantity not sufficient) result, requiring a second specimen to be collected.
Mild reject; Gross reject
Specimen Stability Information Provides a description of the temperatures required to transport a specimen to the laboratory. Alternate acceptable temperature(s) are also included.
|Varies||Ambient (preferred)||72 hours|
Clinical Information Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test
Chronic 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 chmeric 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 (Ph+) B-cell acute lymphoblastic leukemia (ALL), 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.
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.
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 TKI (eg, imatinib) resistance.
Cautions Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances
This assay does not detect all possible 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 TKI 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.
Clinical Reference 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 tyrosone 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
Method Description Describes how the test is performed and provides a method-specific reference
Total RNA is extracted from the sample using the miRNeasy extraction kit (Qiagen). cDNA is made using the High Capacity cDNA Kit (Applied Biosystems). A first-round PCR is performed using primers directed against BCR and ABL regions to generate a PCR product representing the translocated allele only (p210, p190, p185 or p230 transcript types). A second (nested) PCR is next performed to amplify the ABL KD region using template from the first-round PCR product. Aliquots of the ABL PCR are subjected to multiplex allele-specific primer extension (ASPE) reactions using primers specific for the targeted mutation sites (M244V, G250E, Q252H [G->C, G->T], Y253F, Y253H, E255K, T315I, F317L [C->G, C->A, T->C], M351T, E255G, F359V, and H396R). The ASPE reactions which have biotin-dCTP incorporated, are incubated with microsphere beads containing specific recognition tag sequences (FlexMap, Luminex) and streptavidin-phycoerythrin is added to label any bound ASPE sequences. The target mutations are recognized following flow cytometry by specific bead address and fluorescence intensity.(Unpublished Mayo method)
Day(s) and Time(s) Test Performed Outlines the days and times the test is performed. This field reflects the day and time the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time required before the test is performed. Some tests are listed as continuously performed, which means assays are performed several times during the day.
Monday through Friday
Analytic Time Defines the amount of time it takes the laboratory to setup and perform the test. This is defined in number of days. The shortest interval of time expressed is "same day/1 day," which means the results may be available the same day that the sample is received in the testing laboratory. One day means results are available 1 day after the sample is received in the laboratory.
Specimen Retention Time Outlines the length of time after testing that a specimen is kept in the laboratory before it is discarded
Performing Laboratory Location The location of the laboratory that performs the test
Test Classification Provides information regarding the medical device classification for laboratory test kits and reagents. Tests may be classified as cleared or approved by the US Food and Drug Administration (FDA) and used per manufacturer's instructions, or as products that do not undergo full FDA review and approval, and are then labeled as an Analyte Specific Reagent (ASR), Investigation Use Only (IUO) product, or a Research Use Only (RUO) product.
This test was developed and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. This test has not been cleared or approved by the U.S. Food and Drug Administration.
CPT Code Information Provides guidance in determining the appropriate Current Procedural Terminology (CPT) code(s) information for each test or profile. The listed CPT codes reflect Mayo Medical Laboratories interpretation of CPT coding requirements. It is the responsibility of each laboratory to determine correct CPT codes to use for billing.
81403-ABL1 (c-abl oncogene 1, receptor tyrosine kinase) (eg, acquired imatinib tyrosine kinase inhibitor resistance), variants in the kinase domain
LOINC® Code Information Provides guidance in determining the Logical Observation Identifiers Names and Codes (LOINC) values for the result codes returned for this test or profile.
|Result ID||Reporting Name||LOINC Code|
|MP004||Specimen Type||In Process|
|MOFF||BCRABL Fusion Form||N/A|