RESUMEN
Reverse transcription quantitative polymerase chain reaction (RTqPCR)is currently the most sensitive tool available for the routine monitoring of disease level in patients undergoing treatment for BCRABL1 associated malignancies. Considerable effort has been invested at both the local and international levels to standardise the methodology and reporting criteria used to assess this critical metric. In an effort to accommodate the demands of increasing sample throughput and greater standardization, we adapted the current best-practice guidelines to encompass automation platforms and improved multiplex RT-qPCR technology.
Asunto(s)
Proteínas de Fusión bcr-abl/sangre , Ensayos Analíticos de Alto Rendimiento , Automatización de Laboratorios , Biomarcadores , Difusión de Innovaciones , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Ensayos Analíticos de Alto Rendimiento/normas , Humanos , Cinética , Límite de Detección , Sondas Moleculares/metabolismo , Reacción en Cadena de la Polimerasa Multiplex , Proteínas de Neoplasias , Proteínas Proto-Oncogénicas c-abl/sangre , Proteínas Proto-Oncogénicas c-abl/genética , Proteínas Proto-Oncogénicas c-abl/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The introduction of tyrosine kinase inhibitors (TKIs), starting with imatinib and followed by second and third generation TKIs, has significantly changed the clinical management of patients with chronic myeloid leukemia (CML). Despite their unprecedented clinical success, a proportion of patients fail to achieve complete cytogenetic remission by 12 months of treatment (primary resistance) while others experience progressive resistance after an initial response (secondary resistance). BCR-ABL1 kinase domain (KD) mutations have been detected in a proportion of patients at the time of treatment failure, and therefore their identification and monitoring plays an important role in therapeutic decisions particularly when switching TKIs. When monitoring KD mutations in a clinical laboratory, the choice of method should take into account turnaround time, cost, sensitivity, specificity, and ability to accurately quantify the size of the mutant clone. In this article, we describe in a "manual" style the methods most widely used in our laboratory to monitor KD mutations in patients with CML including direct sequencing, D-HPLC, and pyrosequencing. Advantages, disadvantages, interpretation of results, and their clinical applications are reviewed for each method.
Asunto(s)
Análisis Mutacional de ADN/métodos , Proteínas de Fusión bcr-abl/genética , Genes abl , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Proteínas Tirosina Quinasas/genética , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Cromatografía Líquida de Alta Presión/métodos , Resistencia a Antineoplásicos/genética , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Mutación , Reacción en Cadena de la Polimerasa/métodos , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Estructura Terciaria de Proteína/genética , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Control de Calidad , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , ARN Neoplásico/genética , ARN Neoplásico/aislamiento & purificación , Análisis de Secuencia de ADN/métodos , Manejo de EspecímenesAsunto(s)
Proteínas de Fusión bcr-abl/análisis , Leucemia Mielógena Crónica BCR-ABL Positiva/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Proteínas de Fusión bcr-abl/genética , Ensayos Analíticos de Alto Rendimiento , Humanos , Neoplasia Residual/diagnóstico , Reacción en Cadena de la Polimerasa/normas , Sensibilidad y EspecificidadRESUMEN
Molecular monitoring of BCR-ABL1 expression in chronic myeloid leukaemia (CML) is well established. As the International Scale (IS) normalised BCR-ABL1/ABL1 ratio at 3 months post-treatment is now an important milestone in patients' treatment schedule, the reliable and reproducible measurement of BCR-ABL1 levels is therefore paramount. IS conversion factors (CF) are established via sample exchange and yearly ratification with an external reference laboratory. Since any change to an established IS CF could lead to discontinuity in longitudinal results, we wished to add an internal verification step as an additional safeguard. We used the Cepheid GeneXpert qPCR and IS calibrated Xpert BCR-ABL Monitor cartridge system, parallel to our in-house pipeline on 50 CML samples, over the period of one week to verify the CF for those samples and compare it to the externally provided CF. The median non-IS in-house BCR-ABL1/ABL1 values were significantly different than that from the IS GeneXpert, but they became non-significant when adjusted to CF provided by the CXM and by the current external CF, validating it. These metrics can help decide to accept or reject an updated CF value, especially where a significant change in CF might lead to a discontinuity in ongoing patient monitoring.