Download Quantitative PCR for the Detection of BCR

Administrative Office
St. Joseph's Hospital Site, L301-10
50 Charlton Avenue East
HAMILTON, Ontario, CANADA L8N 4A6
PHONE: (905) 521-6141
FAX: (905) 521-6142
http://www.fhs.mcmaster.ca/hrlmp/
Issue No. 78
QUARTERLY NEWSLETTER
December 2004
Quantitative PCR for the Detection
of BCR-ABL p210 Transcripts
Chromosomal translocations of BCR-ABL genes are characteristic of Chronic Myeloid Leukemia (CML) in
95% of cases (1-4). The BCR-ABL fusion gene results from a reciprocal translocation between the long
arms of chromosome 9 and 22, causing transposition of the 3’ segment of the ABL gene from 9q34 to the
5’ segment of the BCR gene on 22q11 (5). The rearranged BCR-ABL gene is transcribed into a chimeric
BCR-ABL mRNA. The majority of CML patients (95%) exhibit transcripts with major BCR-ABL
breakpoints of b2/a2 or b3/a2 that result from the fusion of either BCR exon b2 or b3 to the ABL a2 (6-8).
Translation of both b2a2 and b3a2 transcripts yields a 210-kD fusion protein (p210). A number of
prognostic factors have been used to predict patient outcome, including, chromosomal abnormalities,
fusion genes, as well as response to initial therapy and minimal residual disease (MRD).
Currently the Hamilton Laboratory is offering the following molecular tests for the detection of rearranged
BCR-ABL gene:

Fluorescence in situ hybridization (FISH) using fluorescent-labeled probes to BCR and
ABL
(detects translocation and additional deletions of 9q or 22q)

Reverse Transcription and PCR amplification (RT-PCR)

Nested RT-PCR.
Nested RT-PCR assays can achieve sensitivity of more than 10-4, thus detecting MRD. However, this
type of assay does not allow quantification of MRD levels. Recently, the clinical value of Real-Time
Quantitative PCR has been demonstrated (9, 10). Many studies have confirmed that sequential analysis
of residual disease by Q-PCR for BCR-ABL transcripts has the advantage of monitoring the dynamics of
CML. Performed at regular intervals, Q-PCR can indicate the activity of a malignant clone, thus predicting
the impeding clinical relapse in patients who are still in hematological and cytogenetic remission. This can
be used as the basis for improved management. Patients with high quantitative levels of BCR-ABL RNA,
or patients who convert from PCR-negative to PCR-positive, or who show an increasing number of
transcripts, are likely to relapse after bone marrow transplant (10, 11). In addition, quantification of the
BCR-ABL transcripts was shown to be useful in monitoring response to a-interferon and imatinib-treated
(Gleevec) patients.
Patients on Gleevec often achieve a three-log reduction in the level of BCR-ABL, and some patients
achieve undetectable levels (2, 3, 4). Levels are typically monitored every few months to assess the
efficacy of therapy and risk of relapse. Following transplant, BCR-ABL RT-PCR is useful for predicting the
risk of relapse. Testing is recommended every 4 weeks after transplant, then every 3 months once levels
fall below detectable, and then every 6 months after the first year.
The Hamilton Laboratory has developed a Quantitative Real-Time RT-PCR (Q-PCR) assay for the
quantification of the Major BCR-ABL (b2/a2 or b3/a2) translocation breakpoints. The assay allows the
detection and quantification of low copy number MRD. The characteristics of the molecular methods
offered are summarized in the table below.
Method
FISH
Nested RT-PCR
Real-time RT-PCR
Limit of resolution
Advantages
Disadvantages
1—5% BCR-ABL
Semi-quantitative
False-Positive
>10-4 BCR-ABL
Very Sensitive
Not quantitative
(RNA dilution)
>10-4 BCR-ABL
Very Sensitive
(RNA dilution)
Quantitative High Precision
The preferred sample is EDTA anticoagulated blood (purple-top). RNA is extracted and converted to
cDNA. The cDNA is then subjected to PCR amplification using primers flanking the translocation
breakpoints and a fluorescent probe, which allows measurement of the product in real time (9,12). Q-PCR
testing is performed on all diagnostic samples that are positive for the p210 BCR-ABL translocation based
on RT-PCR, in order to establish levels of BCR-ABL transcripts prior to treatment. Thereafter, monitoring
of patients is performed using Q-PCR on peripheral blood. Levels of BCR-ABL transcripts are expressed
as a log reduction if baseline levels of expression are known for the patient. In summary,
Patients appropriate for Q-PCR molecular testing include:

Patients diagnosed with CML prior to initiation of treatment

Patients monitored for relapse and treatment efficacy.

Post-transplant testing every 4 weeks after transplant, then every 3 months once levels
fall below detectable, and then every 6 months after the first year.
Requirements and Shipping:
Samples: 5-10 ml whole blood collected in one lavender top (EDTA). Send blood at room temperature.
Do not refrigerate or freeze. Enclose a completed Cancer Genetics requisition with all samples and
follow instructions for submission of the specimen. For information or copies of the requisition please
contact the laboratory at 905-521-5085.
References
1. Sawyers C. Chronic Myeloid Leukemia. N Engl J Med 1999;340:1330-40.
2. Cross NC, Lin F, Chase A, Bungey J, Hughes TP, Goldman JM. Competitive polymerase chain
reaction to estimate the number of BCR-ABL transcripts in chronic myeloid leukemia patients
after bone marrow transcription. Blood 1993;82:1929-36.
3. Hochhaus A, Lin F, Reiter A, et al. Quantification of residual disease in chronic myelogenous
leukemia patients on interferon-a therapy by competitive polymerase chain reaction. Blood
1996;87:1549-55.
4. Kreipe H, Felgner J, Jaquet K, et al. DNA analysis to aid in the diagnosis of chronic
myeloproliferative disorders. Am J Clin Path 1992;98:46-54.
5. Groffen J, Heisterkamp N. The BCR/ABL hybrid gene. Baillieres Clin Haematol 1987;243:290-3.
6. Bernards A, Rubin CM, Westbrook CA, Paskind M, Baltimore D. The first intron in human c-abl
gene is at least 200kb long and is a target for translocations in chronic myelogenous leukemia.
Mol Cell Biol 1989;7:3231-6.
7. Cross NC. Detection of BCR-ABL in hematological malignancies by RT-PCR. In: Methods in
Molecular Medicine, Molecular Diagnosis of Cancer, edited by: FE Cotter Humana Press Inc.
Totowa, NJ. 1996.
8. van der Velden V, Hochhaus A, Cazzanigia G, Szczepanski T, Gabert J., van Dongen J.
Detection of minimal residual disease in hematopoietic malignancies by real-time quantitative
PCR:principles, approaches, and laboratory aspects. Leukemia 2003: 17: 1013-1034.
9. Gabert J. Detection of recurrent translocations using real time PCR; assessment of the technique
for diagnosis and detection of minimal residual disease. Hematologica 1999; 84: 107-109
10. Lion T, Izraeli S, Henn T, Gaiger A, Mor W, Gadner H. Monitoring of residual disease in chronic
myelogenous leukemia by quantitative polymerase chain reaction. Leukemia 1992;6:495.
11. Delage R, Soiffer RJ, Dear K, Ritz J. Clinical significance of bcr-abl gene rearrangement detected
by polymerase chain reaction after allogeneic bone marrow transplantation in chronic
myelogenous leukemia. Blood 1991;78:2759-67.
12. Gabert J, Beillard E, Van Der Velden VH, et al. Standardization and quality control studies of
‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts
for residual disease detection in leukemia –A Europe Against Cancer Program. Leukemia 2003;
17: 2318-2357.
Dr. R. Carter
Discipline Director - Genetics
Hamilton regional Laboratory Medicine Program