The significance of major and stable molecular responses in chronic myeloid leukemia in the tyrosine kinase inhibitor era 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%. What is the importance of MMR after complete cytogenetic response? As most CML patients treated with a TKI will reach a CCyR, quantification of residual BCR-ABL transcripts by RT-qPCR is the only available tool to further monitor response kinetics. It is assumed that the amount of BCR-ABL transcripts mirrors the number of residual BCR-ABL Ph+ cells. A close agreement between the absolute value of BCR-ABL transcripts given as 1% in the IS and a CCyR can be demonstrated.(8) The ELN recently updated its recommendations to standardize treatment for patients with CML.(15) It recommends that a patient should be seen as an optimal responder to IM when a CCyR is achieved within the first 12 months of treatment. Attainment of a MMR within 18 months is associated with an improved probability of prolonged event-free survival. Conversely, failure to achieve MMR at 18 months is regarded as a suboptimal response. The time required for a patient to reach a CCyR and Rev Bras Hematol Hemoter. 2011;33(6):455-60 a MMR is considered an early predictor of therapeutic response with a direct correlation with clinical outcome. However, clinical trials do not necessarily reproduce "real" clinical practice. While most randomized clinical studies in the world were carried out with newly diagnosed CP-CML patients, our experience was built on the responses of patients treated with IM as second-line therapy after treatment with interferon-alpha because Brazil public health care centers were not allowed to use IM as front-line therapy until 2008. Thus, a late responder group, compared to the ELN recommendations for CP-CML patients treated with first-line IM, was identified. Late responder patients represented approximately 40% of the patients that did not achieve optimal responses. Attainment of CCyR at any time was associated with a better OS. The results of this study show that an early predictor marker able to discriminate between the competing possibilities of achieving an optimum response versus progression is still missing. 457