Label Name: ABL1 KD
Lab Discipline: Molecular Diagnostics
Institution:  Duke University Health System 
EAP ID:  LAB6013 
Last Review:  3/26/2017 11:06:52 PM
Container & Volume
  Age Group   Container   Volume  
  0  - 18 Years LAVENDER TOP TUBE 1  ML
Label Reminders
  Be sure to include the patient's name, history #, date and time of collection, and collector's initials.
Collection Notes
  • Peripheral blood: One lavender-top EDTA tube (minimum of 1 ml) is required from a patient with a normal WBC count. Four lavender-top tubes are required for a patient with a low WBC count. Send unprocessed peripheral blood to the laboratory promptly at ambient temperature.

    Bone Marrow: One lavender-top EDTA tube (minimum of 1 ml) is required. Send unprocessed bone marrow to the laboratory promptly at ambient temperature.


  Deliver to laboratory at ambient temperature. If there is a delay of more than 24 hours in delivery, please refrigerate the sample. Samples must arrive within 48 hours of collection. DO NOT FREEZE.

Turn Around Time -  Routine: 14 days   Stat: N/A
Reference Values
  This assay uses PCR followed by Sanger DNA sequencing to identify TKI-resistant mutations in the kinase domain of the BCR/ABL1 fusion protein. Total cellular RNA is extracted from peripheral blood or bone marrow samples and cDNA is generated using reverse transcriptase. PCR is then performed using this cDNA and oligonucleotide primers that specifically target the BCR/ABL1 fusion gene located on the Philadelphia chromosome. One PCR reaction amplifies the e1a2 (m-BCR) transcript, and a second PCR reaction amplifies both the b2a2 and b3a2 (M-BCR) transcripts. The resulting PCR products are purified with an exonuclease/phosphatase mixture (ExoSAP-IT) and sequenced using the Big Dye Terminator v3.1 Cycle Sequencing Kit (ABI) and sequencing primers that flank the ABL1 kinase domain. The products of the completed sequencing reactions are purified using the Big Dye XTerminator Purification Kit (ABI) and resolved using the ABI 3130xl Genetic Analyzer. Data is analyzed using SeqScape Software v2.6 and compared to the reference sequence (GenBank NM_005157) for the ABL1 gene from c.703-1230 (p.235-410).

This test was developed and its performance characteristics determined by the DUHS Clinical Molecular Diagnostics Laboratory. It has not been cleared or approved by the U.S. Food and Drug Administration. This test is used for clinical purposes. It should not be regarded as investigational or for research. This laboratory is certified under the Clinical Laboratory Improvement Amendments of 1988 ("CLIA") as qualified to perform high complexity clinical testing.
Clinical Significance and Interpretive Data

All cases of chronic myelogenous leukemia (within the WHO leukemia subtype CML, BCR-ABL1 positive), about 20-35% of adult acute lymphoblastic leukemia (ALL) and 3-5% of pediatric ALL are associated with a translocation between chromosomes 9 and 22 (t(9;22)q34;q11.2). This results in a small derivative chromosome known as the Philadelphia chromosome (Ph). On this derivative 22, the ABL1 proto-oncogene on chromosome 9 is fused to the BCR gene on chromosome 22. For CML patients, the fusion joins the b2 or b3 exons of BCR to the a2 exon of ABL1 and gives rise to the p210 BCR/ABL1 chimeric fusion protein. Cells expressing the BCR/ABL1 oncoprotein have constitutively upregulated ABL1 tyrosine kinase activity leading to uncontrolled cell proliferation, reduced apoptosis, defective adhesion, and genomic instability. Thus, BCR/ABL1 expression confers a proliferative and survival advantage over normal hematopoietic cells.

Tyrosine kinase inhibitors (TKIs) which target the ABL1 kinase domain of the BCR/ABL1 fusion protein are currently the treatments of choice for CML patients. The TKI Imatinib (Gleevec, ST1571) is effective as a first line therapy in most CML patients at the time of diagnosis. Patients treated with Imatinib usually show a reduction in disease burden, and many patients on Imatinib achieve a complete molecular response (ie. BCR/ABL1 expressing CML cells become undetectable). However, a proportion of patients treated with Imatinib relapse due to the development of Imatinib resistant CML clones. Primary Imatinib resistance, which is defined as a failure to achieve an adequate response to initial therapy within 12-18 months, occurs in approximately 15-25% of patients in the chronic phase of CML. Primary resistance usually results from molecular mechanisms that are both BCR/ABL1 dependent and BCR/ABL1 independent. Secondary resistance to Imatinib, which is defined as a loss of hematologic or cytogenetic response following an initial response to treatment, occurs in 10-15% of CML patients. Secondary resistance usually results from acquired mutations in the ABL1 kinase domain (KD) of the BCR/ABL1 gene that render it insensitive to Imatinib. The incidence of BCR/ABL1 mutations is greater in the more advanced phases of CML, with patients progressing to the blast phase on TKI therapy having a much higher rate of BCR/ABL1 mutations than those maintained in the chronic phase of CML.

Most patients (about 60%) who develop secondary Imatinib resistance harbor mutations in the kinase domain of BCR/ABL1. TKI resistant mutations in occur in four major regions of the ABL1 kinase domain: the P-loop, the imatinib binding domain, the catalytic domain, and the activation loop. Direct sequencing of amino acids 235-410 of ABL1 from the BCR-ABL1 transcript can detect mutations within these four regions. Seven specific mutations (M244V, G250E, Y253F/H, E255K/V, T315I, M351T, and F359V) make up 85% of all kinase domain mutations that are detected in TKI resistant patients. Over 90 different point mutations have been reported to confer either complete or partial resistance to one or more TKIs.


In the context of a rising BCR/ABL1 transcript level, the detection of a BCR/ABL1 kinase domain mutation suggests imatinib resistance. A patient with Imatinib resistant CML may respond to a second-generation TKI such as Dasatinib or Nilotinib. These FDA approved TKIs are affective in most patients who develop Imatinib resistance or who become Imatinib intolerant. However, the most common acquired BCR/ABL1 mutation, T315I, is completely resistant to all three of these TKIs. Patients who harbor the T315I mutation should consider for alternative (non-TKI based) therapies.

Some BCR/ABL1 kinase domain mutations are only partial resistant to Imatinib. For these partially resistant mutations, dose-escalation from the standard 400 mg/day to 600-800 mg/day performs moderately well in achieving a major cytogenetic response. Dose escalation is not recommended for highly resistant mutations.

In some patients, ABL1 tyrosine kinase domain mutations can be found at very low levels even at the time of diagnosis. These mutations are not induced by imatinib (or other TKIs), but instead are present as part of the normal genetic heterogeineity that can occur within a population of neoplastic CML cells. With Imatinib treatment, the proportion cells with these pre-existing mutations can increase. However, this does not occur in all patients. Some will still achieve a complete molecular response to TKI therapy. Thus, mutations detected at low levels at the time of diagnosis may not have clinical significance in a patient with a stable or declining BCR-ABL1 transcript level. Testing in this setting is not recommended. However, the presence of KD mutations in the context of rising disease-burden is a good indicator of TKI resistance.


• BCR/ABL1 sequence changes that have been previously documented in the literature and confer TKI resistance will be reported and interpreted as mutations. Mutations will be characterized as fully resistant, partially resistant or sensitive to imatinib, dasatinib and nilotinib.

• Sequence changes that have been documented previously but do not confer TKI resistance will be reported and interpreted as TKI sensitive.

• Sequence changes that have not been documented previously but are unlikely to change the function BCR/ABL1 (e.g. silent mutations) will be reported and interpreted as TKI sensitive.

• Sequence changes that have not been previously described but may change the function of BCR/ABL1 will be reported but speculation of TKI sensitivity will not be provided.


Druker BJ et al. (2006) Five-year follow-up of patients receiving imatinib for chronic myeloid leukemia.N Engl J Med. 355: 2408-17.

Hochhaus A, et. al. (2008) Favorable long-term follow-up results over 6 years for response, survival, and safety with imatinib mesylate therapy in chronic-phase chronic myeloid leukemia after failure of interferon-alpha treatment. Blood. 111(3): 1039-43.

Hughes T, et. al. (2006) 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 expression results. Blood. 108: 28-37.

Jabbour E, et. al. (2006) Frequency and clinical significance of BCR-ABL mutations in patients with chronic myeloid leukemia treated with imatinib mesylate. Leukemia. 20(10): 1767-73.

National Comprehensive Cancer Network. NCCN Clinical Practive Guidelines in Oncology: Chronic Myelogenous Leukemia. V.1.2008. Available at www.nccn.org

O’Brien SG, et al. (2003) Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med. 348: 994–1004.

O'Hare T, et. Al. (2007) Bcr-Abl kinase domain mutations, drug resistance, and the road to a cure for chronic myeloid leukemia. Blood. 110(7): 2242-9.

Ou Joyce, Vergilio JA, Bagg A. (2008) Molecular diagnosis and monitoring in the clinical management of patients with chronic myelogenous leukemia treated with tyrosine kinase inhibitors. Am. J. Hematol. 83(4): 296-302.

Schiffer CA. et al. (2007) BCR-ABL tyrosine kinase inhibitors for chronic myelogenous leukemia. N Engl J Med. 357: 258-65.

Ramirez P. and DiPersio JF. (2008) Therapy Options in Imatinib Failures. The Oncologist. 12: 424-434.

Shah NP, et al. (2004) Overriding imatinib resistance with a novel ABL kinase inhibitor. Science. 305(5682): 399-401.

Soverini S. et. al. (2006). Contribution of ABL kinase domain mutations to imatinib resistance in different subsets of Philadelphia-positive patients: By the GIMEMA Working Party on Chronic Myeloid Leukemia. Clin Cancer Res. 12: 7374-9.

Willis SG, et. al. (2005) High-sensitivity detection of BCR-ABL kinase domain mutations in imatinib-naive patients: correlation with clonal cytogenetic evolution but not response to therapy. Blood. 106(6): 2128-37.
    BCR/ABL Mutation analysis is indicated in the following settings:

• A CML patient who progresses to the accelerated phase or blast phase of CML while treated with Imatinib (or other TKI).

• A CML patient with a hematologic relapse, a loss of complete cytogenetic response, or a 1 log or greater increase in the BCR-ABL1 transcript levels while treated with Imatinib (or other TKI)

• A patient in the chronic phase of CML who has a suboptimal initial response to imatinib (or other TKI) as defined as a failure to achieve a complete hematologic response at 3 months, a failure to achieve a minimal cytogenetic response at 6 months, or a failure to achieve a major cytogenetic response at 12 months.

• A CML patients with a previously detected BCR/ABL1 kinase domain mutation. In this setting, testing can be performed after three months to determine the response to a change in therapy.
    • A BCR/ABL1 transcript expression level of >0.05% as determined by Duke Molecular Diagnostic Laboratory qPCR assay is recommended for ABL1 kinase domain sequencing. A result may not be obtained for samples with expression below 0.05%.

• The sensitivity of DNA sequencing is high for the detection of nucleotide base changes and small deletions and insertions in the region analyzed.

• This assay may not detect an acquired mutation which is present below the 15% detection limit (i.e., mutant cell population of < 15%).

• In samples co-expressing b2a2 and b3a2 transcripts, PCR for this assay is unable to differentiate b2a2 and b3a2. Preferential amplification of one transcript may occur, with subsequent sequence analysis including only one transcript population.

• Only amino acids 235-410 of the ABL1 gene from BCR/ABL1 are examined. Changes outside of this region will not be detected. The non-rearranged ABL1 gene is not examined.

• The presence of a mutant population containing a large deletion, duplication, insertion, aberrant splicing, or sequence alteration adversely affecting primer binding may not be identified using these methods.
Test Synonyms
  Synonym(s): BCR/ABL1
Synonym(s): BCR/ABL1 Kinase domain mutation analysis
Synonym(s): Dasatinib
Synonym(s): Imatinib
Synonym(s): Imatinib Sensitivity Testing
Synonym(s): Nilotinib
Molecular Diagnostics Laboratory

Medical Director:
 Michael Datto, M.D., Ph.D.
 Phone: 919-684-6965
 Email: michael.datto@duke.edu
Lab Director:
 Catherine Rehder Ph.D, FACMG
 Phone: 919-613-8434
 Email: catherine.rehder@duke.edu
Lab Director:
 Siby Sebastian Ph.D., DABMG
 Phone: 919-613-8432
 Email: siby.s@duke.edu

 Wadsworth Bldg, Cytogenetics, Rm 0220
 2351 Erwin Rd
 Durham,  NC  27705
 Phone: 919-684-2698
 FAX: 919-668-5424

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