The significance of major and stable molecular responses in chronic myeloid leukemia in the tyrosine kinase inhibitor era

Abstract

Tyrosine kinase inhibitors have changed the management and outcomes of chronic myeloid leukemia patients. Quantitative polymerase chain reaction is used to monitor molecular responses to tyrosine kinase inhibitors. Molecular monitoring represents the most sensitive tool to judge chronic myeloid leukemia disease course and allows early detection of relapse. Evidence of achieving molecular response is important for several reasons: 1. early molecular response is associated with major molecular response rates at 18-24 months; 2. patients achieving major molecular response are less likely to lose their complete cytogenetic response; 3. a durable, stable major molecular response is associated with increased progression-free survival. However, standardization of molecular techniques is still challenging.

Keywords: Cytogenetic; Leukemia, myelogenous, chronic, BCR-ABL positive; Monitoring; Mutation; Polymerase chain reaction.

Figure 1

Standardization and validation levels for proficient chronic myeloid leukemia molecular monitoring The work-flow for chronic myeloid leukemia (CML) molecular monitoring in our laboratory starts with the definition of BCR-ABL transcript type by qualitative multiplex assay in pre-treatment samples. A: Standardization of a stable and controlled absolute quantification assay according to Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines. Plasmid standards with a BCR-ABL insert are prepared twice annually by sub-cloning an original plasmid kindly provided by Dr. NCP Cross. Multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) with hydrolysis probes and primers for both e13a2 and e14a2 transcripts are run with ABL as the control gene. The same plasmid is used to quantify BCR-ABL and ABL, which should control any variation in plasmid quantification efficiency. Standard curves are prepared to cover a dynamic range of 10⁶-10¹, and the assay limit of detection is 10. Together with the standards, in each RT-qPCR assay a NTC (non-template control, with DNA sample), NAC (non-amplification control, with a negative BCR-ABL cDNA), Blank (only reaction buffer) and "low" and "high" copy quality controls are included. The quality control stocks are prepared with cell dilutions of K562 and Kasumi cell lines. The RT-qPCR results are considered reliable if the ABL copy number is ≥ 30,000 and RT-qPCR negativity is defined as undetectable BCR-ABL transcripts only when a sample meets acceptable criteria for the ABL copy number. Definition of a complete molecular response (CMR) requires an undetectable BCR-ABL transcript level by RT-qPCR confirmed by negative nested-polymerase chain reaction (PCR), whose lowest limit of detection is 10^-5. B: Conversion factor (CF) calculation and validation. Each BCR-ABL/ABL value is converted to the IS applying our derived laboratory-specific CF as validated by the Australian Molecular Laboratory at Adelaide. For that, an expressive number of samples, including patients in various disease phases, were tested in parallel at INCA laboratory and at the validating Center at Adelaide. Bias plot of the data generated at INCA for calculation of the CF (i) and the same data with converted validation data (ii). If there were no difference in the estimated mean bias, the dotted line would be at 0, thus, the CF was the antilog of the mean bias. The CF was validated by subsequent sample exchange. C: The plots show an example of how the application of our CF reduces coefficient of variation in 70 of our samples at the specific values of 0.01%, 0.1%, 1% and 10%.