OVERVIEW
Quantitative reverse-transcription polymerase chain reaction (QRT-PCR) is the most sensitive assay available for the measurement of BCR::ABL1 mRNA. A major advantage of QRT-PCR over other methods of measuring molecular burden is that the results are quantitative in nature. This allows for monitoring response to therapy over time. QRT-PCR from peripheral blood should be performed before initiation of tyrosine kinase inhibitor (TKI) therapy in order to confirm that the transcript variant present at diagnosis is amenable to monitoring via this method.
For patients harbouring the e1a2 (minor, p190) transcript variant, cumulative results over time are reported categorically as well as both a % ratio [BCR::ABL1]/[BCR] and a log ratio of [BCR::ABL1]/[BCR].
For patients harbouring either the e13a2 or e14a2 (major, p210) transcript variant, cumulatively results over time are reported categorically as well as both a standardized % ratio (IS burden %) and a log burden (log IS/100).
For patients harbouring the e1a3, e13a3, e14a3, e19a2, or e19a3 transcript variant, results are reported qualitatively as either positive or negative for the presence of the transcript variant in question.
INDICATION
1. Reflex test in patients with demonstrated t(9;22) positive disease (diagnosis either by FISH or karyotype)
2. Follow-up test for monitoring of minimal residual disease (or measurable residual disease).
The following testing interval applies to follow-up testing. Specimens received by the laboratory outside of these intervals, without prior consultation, will be discarded.
- Uncomplicated, chronic phase patients treated with Imatinib, 400mg daily
- Every three months until a stable (~ 1year) major molecular response (MMR) is achieved (molecular burden of 0.1% or -3.00).
- Subsequent to the achievement of a stable MMR testing transitions to every six months, indefinitely
- Patients treated with Imatinib, <>400mg daily
- Every three months, indefinitely
- Patients treated with anything other than Imatinib.
- Every three months, indefinitely
- Patients who discontinue treatment for a limited time due to planned (or unplanned) events
- Every month, until resumption of therapy (to a maximum of one year)
- After one year, monitoring transitions to every three months indefinitely
- Once treatment is re-initiated, monitoring transitions to the intervals outlined in 1, 2, and 3.
- Patients attempting treatment free remission
- Every month for one year
- After one year, monitoring transitions to every three months indefinitely
- In the event that treatment is re-initiated, monitoring transitions to the intervals outlined in 1, 2, and 3.
- Post-transplant patients not being treated with a TKI
- Monitoring is at the discretion of the treating physician.
- For any scenario not covered above, please contact the laboratory in order to come to a mutually agreeable testing interval.
REFERRAL
Any treating physician.
TEST REQUIREMENTS
- Completed CGL Myeloid Testing requisition form [link]
- One of the following specimens (please see our Guidelines/Policies for Samples and Transport)
Preferred:
- 20mL Peripheral Blood in EDTA tubes (purple top)
Alternative:
- 2.5mL Bone marrow aspirate in EDTA tubes (purple top)
- Peripheral blood derived white blood cells fixed in methanol/acetic acid (residual cytogenetic specimen)
- Bone marrow specimen fixed in methanol/acetic acid (residual cytogenetic specimen)
Note: Results reported against the e1a2 (minor, p190) or e13a2/e14a2 (major, p190) transcript variant are only quantitatively comparable to previous analyses should the specimen source and type be the same (i.e. fresh peripheral blood to fresh peripheral blood).
Figure 1: Select requisition entries for QRT-PCR testing
TRANSPORT
Click here for guidelines on transporting specimens.
METHOD
- e1a2 (minor, p190)
Total RNA is extracted from the submitted specimen and converted to cDNA (random primed reverse transcription – RT). The amount of BCR::ABL1 fusion transcript (e1a2) present in this sample is then quantified with respect to BCR transcript using multipoint standard curves for each via QPCR and expressed both as ratio (percent) and as a log ratio. In order to avoid misinterpretation, the results are also reported categorically.
- e13a2/e14a2 (major, p210)
Total RNA is extracted from the submitted specimen and converted to cDNA (random primed reverse transcription – RT). The amount of BCR::ABL1 fusion transcript (e13a2/e14a2) present in this sample is then quantified with respect to BCR transcript using multipoint standard curves for each via QPCR and expressed both as ratio (percent) and as a log ratio. Prior to reporting, these are normalized to the International Scale. In order to avoid misinterpretation, the results are also reported categorically.
- e1a3, e13a3, e14a3, e19a2, or e19a3
Total RNA is extracted from the submitted specimen and converted to cDNA (random primed reverse transcription – RT) for subsequent QPCR. Results are reported simply as positive or negative for the presence of the transcript variant in question
CLINICAL UTILITY
Up to 99% of CML cases will have a translocation involving BCR and ABL1 detectable via one of these methods.
At diagnosis, QRT-PCR positivity is equivalent to the identification of t(9;22) by FISH. Subsequent MRD monitoring will follow the breakpoint identified by this test.
TURN AROUND TIME
Results are reported within ten working days from receipt of specimen and completed requisition form.
SELECTED REFERENCES
Hughes T and Branford S (2003) Molecular monitoring of chronic myeloid leukemia. Sem Hematol 40(s2):62-68. (PMID: 12783378)
White HE et al (2010) Establishment of the first World Health Organization international reference panel for quantitation of BCR-ABL mRNA. Blood 116:e111. (PMID: 20720184)
White H et al (2015) A certified plasmid reference material for the standardization of BCR-ABL1 mRNA quantification by real-time quantitative PCR. Leukemia 29:369-376. (PMID: 25036192)
Baccarani et al (2019) The proportion of different BCR-BL1 transcipt types in chronic myeloid leukemia. An international overview. Leukemia 33:1173-1183. (PMID: 30675008)