BCR/ABL1 QUANTITATIVE PCR - INITIAL TESTING
Label Name: BCR/ABL1 QNT
Lab Discipline: Molecular Diagnostics
Institution:  Duke University Health System 
EAP ID:  LAB6083 
Last Review:  3/26/2017 11:11:06 PM
Specimen Type
  Whole Blood (EDTA)
Container & Volume
  Age Group   Container   Volume  
  0  - 18 Years LAVENDER TOP TUBE 3  ML
Label Reminders
  Be sure to include the patient's name, history #, date and time of collection, and collector's initials.

Collection Notes
  All:
  • 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.

    PERIPHERAL BLOOD AND BONE MARROW SPECIMENS MUST ARRIVE AT THE LABORATORY WITHIN 48 HOURS OF COLLECTION. THE SPECIMEN CANNOT BE FROZEN. GREEN-TOP (HEPARIN) TUBES ARE NOT ACCEPTABLE FOR TESTING.


 
Transport
  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: 10 days   Stat: N/A
Reference Values
BCR/ABL1 QNT
BCR/ABL1 transcript not detected.
Methodology
  This assay uses the Taqman Real-Time Quantitative PCR (qPCR) technique to detect BCR/ABL1 mRNA expression levels in peripheral blood and bone marrow. This assay is performed using total cellular RNA that is extracted from fresh/unfixed EDTA anticoagulated samples. cDNA is generated from this RNA using reverse transcriptase. qPCR is then performed on an aliquot of this cDNA using oligonucleotide primers and FAM-TAMRA dual-labeled Taqman probes. For each cDNA sample, four unique probe/primer sets are used. These target the three BCR/ABL1 transcripts (e1a2, b2a2 and b3a2) and an endogenous reference transcript (BCR). A threshold cycle value (Ct) is measured for each PCR reaction. This represents the PCR cycle at which amplified product is first detected. For each sample, a delta Ct value is calculated as the difference in Ct value between the target BCR/ABL1 transcript (b2a2, b3a2 or e1a2) and the reference BCR transcript. A small delta Ct represents a high expression level of BCR/ABL1. Positive control standard curves are generated in parallel for each primer/probe set. Positive control samples are derived from three different cell lines (IVS-0032 SupB15, IVS-0003 BV173 and IVS-0011 K562), each expressing a specific (b2a2, b3a2 or e1a2) BCR/ABL1 fusion transcript. The standard curves cover 4 orders of magnitude with the lower detection limit set at 0.01% BCR/ABL1 expressing cells. Using these standard curves, the delta Ct value is converted into a percent of cells expressing BCR/ABL1.

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
    BACKGROUND:

Most cases (99%) of chronic myeloid leukemia (CML), 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. In the majority of ALL patients, the fusion joins the e1 exon of BCR to the a2 exon of ABL1 and gives rise to the p190 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 advantage over normal hematopoietic cells, and BCR/ABL1 kinase activity is necessary and sufficient for the development and progression of CML.

CLINICAL SIGNIFICANCE AND UTILITY:

The detection of the BCR/ABL fusion transcript is a key component in the diagnosis CML and can provide important prognostic and treatment information for patients with both CML and ALL. In addition to determining the presence or absence of BCR/ABL, current technologies now allow us to determine the approximate proportion of cells expressing this transcript in a clinical sample. This quantitative measure of BCR/ABL expression provides important prognostic information and serves as a reliable means of monitoring minimal residual disease, treatment efficacy and early signs of recurrence.

INTERPRETATION:

Any BCR/ABL1 transcripts detected above the level detected in the 0.01% cell line positive control is reported as p210/ p190 detected (transcript detected) along with the delta Ct value and fold change from the previous sample for the patient. Detection at a level below this value is reported, but an accurate quantity of transcript can not be provided.

A 10-fold or greater increase in the expression of BCR/ABL1 is interpreted as a significant change and likely represents failure of the patient's current therapy. Repeat testing in one month is recommended to confirm recurrence, or treatment failure.

In this assay, the delta CT for a sample is reported. A small delta Ct represents a high level of BCR/ABL1 expression (the average delta Ct for cells that do not express BCR/ABL1 is greater than 19). Although the delta Ct is an abstract measure of transcript level it can be used to determined relative changes in BCR/ABL1 transcript levels over time. An approximation of the proportion of cells expressing BCR/ABL1 is also provided based on cell line based standard curves.

The results of this test should be interpreted in conjunction with the patient's clinical findings and other laboratory test results. Please contact the Molecular Diagnostics Laboratory (684-2698) for additional help or questions.

REFERENCES:

Druker et al (2006) Five-Year Follow-up of Patients Receiving Imatinib for Chronic Myeloid Leukemia. NEJM 355(23) 2408-2417.

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

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.
   
Indications
    Clinical Suspicion of CML: To detect the BCR/ABL1 p210 transcript in bone marrow or peripheral blood samples to diagnose CML and to establish a baseline expression value for future testing.

Diagnosis of ALL: To detect the p190 transcripts in bone marrow or peripheral blood samples to help determine prognosis and to establish a baseline expression value for future testing.

Minimal residual disease (MRD) monitoring of BCR/ABL1 fusion gene transcript levels in patients who have undergone treatment (imatinib or bone marrow transplantation) for CML or ALL. Current NCCN guidelines recommend testing every 3 months.

Confirmation of Recurrent disease or treatment failure: Repeat testing of a patient's BCR/ABL1 expression level is indicated to confirm a 10 fold (1 log) increase in expression. This should be performed within 1 month of previous testing.

   
Limitations
    This assay cannot detect translocations whose breakpoints fall outside the probe or PCR primer regions.

This result does not rule out the presence of extremely low levels of BCR/ABL1 transcript that are below the detection limit of this assay (0.01% of cells expressing the BCR/ABL1 transcript).

Since this test uses cell lines as the quantitative controls to generate a standard curve and each clonal cell population may express different amounts of BCR/ABL1 transcript, the calculated percentage of BCR/ABL1 transcripts in a positive patient sample may differ from the actual percentage of BCR/ABL1 expressing cells in the specimen.

Poor RNA template quality or quantity and the presence of PCR inhibitors can affect the results of this assay. If present, these factors usually lead to an uninterpretable result rather than an inaccurate result. Rare mutations, translocations or polymorphisms within the BCR/ABL1 fusion gene could affect the binding of PCR primers or probes leading to a false negative result or an inaccurate measurement of transcript abundance. The results of this test should be interpreted in conjunction with the patient's clinical findings and other laboratory test results. This test is intended for use as an aid in determining patient prognosis and making individualized treatment decisions. It is not a substitute for a physician's judgment and clinical experience. Please contact the DUHS Clinical Molecular Diagnostics Laboratory (919-684-2698) for additional information or for help interpreting the results of this test.

   
Test Synonyms
  Synonym(s): BCR/ABL1
Synonym(s): CML
Synonym(s): Imatinib
Molecular Diagnostics Laboratory
(MDX)

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

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

Performing Times: