Programme for Harmonization to the International Scale in Latin America for BCR-ABL1 quantification in CML patients: findings and recommendations.

Objectives The quantitation of BCR-ABL1 mRNA is mandatory for chronic myeloid leukemia (CML) patients, and RT-qPCR is the most extensively used method in testing laboratories worldwide. Nevertheless, substantial variation in RT-qPCR results makes inter-laboratory comparability hard. To facilitate inter-laboratory comparative assessment, an international scale (IS) for BCR-ABL1 was proposed. Methods The laboratory-specific conversion factor (CF) to the IS can be derived from the World Health Organization (WHO) genetic reference panel; however, this material is limited to the manufacturers to produce and calibrate secondary reference reagents. Therefore, we developed secondary reference calibrators, as lyophilized cellular material, aligned to the IS. Our purpose was both to re-evaluate the CF in 18 previously harmonized laboratories and to propagate the IS to new laboratories. Results Our field trial including 30 laboratories across Latin America showed that, after correction of raw BCR-ABL1/ABL1 ratios using CF, the relative mean bias was significantly reduced. We also performed a follow-up of participating laboratories by annually revalidating the process; our results support the need for continuous revalidation of CFs. All participating laboratories also received a calibrator to determine the limit of quantification (LOQ); 90% of them could reproducibly detect BCR-ABL1, indicating that these laboratories can report a consistent deep molecular response. In addition, aiming to investigate the variability of BCR-ABL1 measurements across different RNA inputs, we calculated PCR efficiency for each individual assay by using different amounts of RNA. Conclusions In conclusion, for the first time in Latin America, we have successfully organized a harmonization platform for BCR-ABL1 measurement that could be of immediate clinical benefit for monitoring the molecular response of patients in low-resource regions.

Tratamiento con imatinib y el farmacogenotipo CYP3A4 en relación con la expansión clonal Ph(+) en leucemia mieloide crónica (LMC) / Imatinib treatment and pharmacogenotype CYP3A4 in relation with the clonal expansion Ph(+) in chronic myeloid leukemia (CML)

Introducción: imatinib es un inhibidor de la tirosina-kinasa BCR-ABL que revolucionó el tratamiento de pacientes con leucemia mieloide crónica (LMC) positivos para cromosoma Philadelphia (Ph+). Este medicamento se metaboliza principalmente por la enzima CYP3A4, cuyo gen presenta variaciones interindividuales tipo SNPs que pueden interferir con la efectividad del tratamiento, como son los polimorfismos CYP3A4*1B y CYP3A4*2 que han mostrado influencia significativa en la actividad metabólica de esta importante enzima farmacológica. Objetivos: Evaluar la frecuencia de polimorfismos de importancia farmacogenética en el gen CYP3A4 en una población de pacientes con LMC tratados con imatinib y en una población control de 164 personas. Correlacionar el genotipo con la evolución de la expansión clonal Ph(+) y la duración del tratamiento. Metodología: Genotipificación PCR-RFLP para los SNPs CYP3A4*1B y CYP3A4*2. Bandeo replicativo tipo RBHG para la evaluación citogenética de blastos espontßneos con o sin presencia del marcador Ph(+). Resultados: Los análisis citogenéticos revelaron una correlación directa entre el tiempo de tratamiento con imatinib y el porcentaje de reducción de blastos Philadelphia (+). Las genotipificaciones evidenciaron que la presencia del polimorfismo CYP3A4*1B no influye en la respuesta citogenética de los pacientes Ph+ tratados con imatinib, y que el polimorfismo fármaco relevante CYP3A4*2 estß ausente en esta población colombiana de controles y pacientes. Conclusiones: El farmacogenotipo CYP3A4*2 (exón 7) no afecta la respuesta citogenética positiva inducida por el imatinib en pacientes con LMC, en quienes la frecuencia de células Ph(+) por lo general se reduce en relación directa con la duración del tratamiento.

Introduction: Imatinib is an inhibitor of the BCR-ABL tyrosine-kinase that has dramatically changed the treatment of patient with Chronic myeloid leukemia (CML) positive for the Philadelphia chromosome (Ph+). This compound is mainly metabolized by the cytochrome CYP3A4 enzyme, coded by a gene with individual variations that could interfere with the effectiveness of the treatment, due to the fact that particular single nucleotide polymorphisms (SNPs), i.e., CYP3A4*1B y CYP3A4*2, have shown to exert a significant influence on the metabolic activity of this pharmacologically important enzyme.Objective: Evaluate the frequency of pharmacogenetically important polymorphisms in the CYP3A4 gen in a Colombian population of patients with CML being treated with this novel drug (Imatinib), in parallel with a control population of 164 healthy individuals. Correlate the evolution of the clonal expansion Ph(+) with the presence of these SNPs and the length of treatment.Methodology: PCR-RFLP genotyping for the CYP3A4* 1B y CYP3A4*2 SNPs. RBHG replication banding for the evaluation of the presence of the Ph(+) markers in spontaneous mitotic blasts.Results: A positive cytogenetic response and/or correlation was detected between the length of the imatinib treatment and a reduction in the percentage of Ph(+) blasts. Genotyping indicate that CYP3A4*1B polymorphism does no affect the cytogenetic response in imatinib treated Ph(+) patients, and that the pharmacorelevant CYP3A4*2 SNP is not present in this population of patients and controls (N=194). Conclusions: The pharmacogenotype CYP3A4*2 (exon 7) does not affect the induced positive cytogenetic response triggered by the imatinib treatment, that generally induces a reduction in Ph(+) blasts en relation with the duration of the treatment.