Renault IZ, Scholl V, Hassan R, Capelleti P, Lima M, Cortes J or intolerance. To date, their use in Brazil is limited mainly to these situations. The effectiveness of TKIs is evaluated by cytogenetic and molecular responses at defined time-points. In this scenario, tumor burden reflected by these responses is a predictive parameter of individual response to TKIs and is considered the main biomarker to test the efficacy of new drugs in clinical studies. The concepts that the therapeutic benefit of a TKI can be predicted by the speed of reduction in tumor burden (the number of Philadelphia+ or BCR-ABL+ cells) and that disease response is the major parameter for estimating the probability of progression-free survival (PFS) and overall survival (OS), are now well established. Conversely, acquisition of additional abnormalities is associated with resistance. Achievement of complete cytogenetic response (CCyR) has remained the gold standard for an optimal outcome in CML because it is associated with an improved probability of survival.(7) After achievement of CCyR, the main parameter for response assessment, is the molecular response (MR), measured by the reduction in BCR-ABL transcripts using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and classified according to a uniform scale referred to as the International Reporting Scale (IS) that is valid for any patient and attributes an absolute value of 0.1% to a 3 log reduction from a hypothetical 100% BCR-ABL value at diagnosis.(8) Molecular biology for BCR-ABL monitoring: measuring BCR-ABL transcripts The IS resulted from recent effort to create an international standard, where laboratory-specific values of the BCR-ABL/constitutive gene for each patient sample obtained by RT-qPCR are converted to an IS score applying a derived laboratory-specific correction factor validated by a reference laboratory.(8,9) According to the recommendations of the European Leukemia Net (ELN), optimal responders should achieve a major molecular response (MMR), a ≤ 0.1% value on the IS, by 18 months of therapy. Reaching a proficient approach to reliably monitor molecular responses in BCR-ABL+ patients requires several steps, including the standardization of a stable and controlled assay according to the Minimum Information for Publication of Quantitative Real-Time Polymerase Chain Reaction Experiments (MIQE) guidelines,(10) the internal validation in a clinical setting, and international validation with a reference laboratory, in order to obtain a correction factor to convert BCR-ABL levels according to the IS. This process, including the different levels of validation based on the experience of the Molecular Biology laboratory at the Instituto Nacional de Câncer (INCA), is shown in Figure 1. Since 2006, 661 patients with CML treated at INCA and 18 centers throughout Brazil were investigated and successfully monitored. 456 The work flow for CML molecular monitoring in our laboratory starts with the definition of the BCR-ABL transcript type by a qualitative multiplex assay in pre- treatment samples.(11) This procedure is performed to assure that RT-qPCR monitoring will target the correct fusion type and false negative results occurring due to the presence of atypical transcripts (fusions involving exon 6 BCR e6a2, e8a2, e19a2 and exon 3 ABL e13a3 or e14a3 fusions) not contemplated in the quantitative assay will be safely excluded.(12,13) If the fusion subtype is not identified by qualitative polymerase chain reaction (PCR) prior to starting treatment, qRT-PCR false negative results cannot be excluded. Molecular responses are assessed by absolute quantification via RT-qPCR assays at diagnosis and then approximately every 3 months, independently on the type of cytogenetic response of the patient. A multiplex RT-qPCR, including hydrolysis probes and primers for both e13a2 and e14a2 transcripts, is run with ABL as the control gene. For absolute quantification purposes, every RT-qPCR run includes standard curves prepared using plasmids, as well as "low" and "high" copy quality controls, as recommended (Figure 1).(14) Each BCR-ABL/ABL value is converted to the IS applying our laboratory-specific conversion factor which was validated by theAustralian Molecular Laboratory at Adelaide. For this, a significant number of samples, including patients in various disease phases, were tested at different times using different RT-qPCR runs, both in the INCA laboratory and at the validating center in Adelaide. BCR-ABL transcripts are undetectable with the reference molecular methodology at a sensibility of 4.5 log. Definition of a complete molecular response (CMR) requires an undetectable BCR-ABL transcript level by RT-qPCR confirmed by negative nested-PCR, where the lowest limit of detection is 10-5. Thus, molecular responses are reported according to BCR-ABL transcript levels as compared to a standardized baseline used as an IS. MMR is a 3-log reduction from the baseline or the absolute value of ≤ 0.1% on the IS. Accordingly, a 2-log reduction ranges from 1% to 0.1% in the IS; a 1-log reduction from 10% to 1% in the IS and non- significant reduction is equivalent to values of more than 10% in the IS. The adoption of these laboratory procedures has overcome the clinician's lack of confidence in molecular test interpretation related to differences in protocols that can result in greatly varying end-points. This was important for several reasons as it: (i) provides an early tool to predict patient response; (ii) allows the use of BCR-ABL/ABL values to guide clinical decisions; (iii) facilitates patient mobility between different clinical facilities and (iv) facilitates interpretation of clinical research among different reference centers in the world, since MMR is the primary end-point of various clinical trials ofABL-inhibitors. Rev Bras Hematol Hemoter. 2011;33(6):455-60