RESUMEN
External quality assurance (EQA) programs are vital to ensure high quality and standardized results in molecular diagnostics. It is important that EQA for quantitative analysis takes into account the variation in methodology. Results cannot be expected to be more accurate than limits of the technology used, and it is essential to recognize factors causing substantial outlier results. The present study aimed to identify parameters of specific importance for JAK2 V617F quantification by quantitative PCR, using different starting materials, assays, and technical platforms. Sixteen samples were issued to participating laboratories in two EQA rounds. In the first round, 19 laboratories from 11 European countries analyzing JAK2 V617F as part of their routine diagnostics returned results from in-house assays. In the second round, 25 laboratories from 17 countries participated. Despite variations in starting material, assay set-up and instrumentation the laboratories were generally well aligned in the EQA program. However, EQA based on a single technology appears to be a valuable tool to achieve standardization of the quantification of JAK2 V617F allelic burden.
Asunto(s)
Janus Quinasa 2/genética , Mutación Missense , Patología Molecular/normas , Garantía de la Calidad de Atención de Salud , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Sustitución de Aminoácidos , Femenino , Humanos , MasculinoRESUMEN
Since the discovery of the JAK2 V617F mutation in the majority of the myeloproliferative neoplasms (MPN) of polycythemia vera, essential thrombocythemia and primary myelofibrosis ten years ago, further MPN-specific mutational events, notably in JAK2 exon 12, MPL exon 10 and CALR exon 9 have been identified. These discoveries have been rapidly incorporated into evolving molecular diagnostic algorithms. Whilst many of these mutations appear to have prognostic implications, establishing MPN diagnosis is of immediate clinical importance with selection, implementation and the continual evaluation of the appropriate laboratory methodology to achieve this diagnosis similarly vital. The advantages and limitations of these approaches in identifying and quantitating the common MPN-associated mutations are considered herein with particular regard to their clinical utility. The evolution of molecular diagnostic applications and platforms has occurred in parallel with the discovery of MPN-associated mutations, and it therefore appears likely that emerging technologies such as next-generation sequencing and digital PCR will in the future play an increasing role in the molecular diagnosis of MPN.
Asunto(s)
Trastornos Mieloproliferativos/diagnóstico , Trastornos Mieloproliferativos/genética , Calreticulina/genética , Exones , Humanos , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Mutación , Trastornos Mieloproliferativos/metabolismo , Garantía de la Calidad de Atención de Salud , Receptores de Trombopoyetina/genéticaAsunto(s)
Alelos , Calreticulina/genética , Transformación Celular Neoplásica/genética , Leucemia Mieloide Aguda/genética , Mutación , Trombocitemia Esencial/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Aberraciones Cromosómicas , Progresión de la Enfermedad , Expresión Génica , Humanos , Cariotipo , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/etiología , Masculino , Persona de Mediana Edad , Polimorfismo de Longitud del Fragmento de Restricción , Trombocitemia Esencial/complicaciones , Trombocitemia Esencial/diagnóstico , Trombocitemia Esencial/tratamiento farmacológicoAsunto(s)
Trastornos de las Plaquetas Sanguíneas/genética , Predisposición Genética a la Enfermedad , Leucemia Mieloide Aguda/genética , Aneuploidia , Análisis Mutacional de ADN , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/patología , Linaje , TrombocitopeniaAsunto(s)
Células de la Médula Ósea/metabolismo , Calreticulina/genética , Mielofibrosis Primaria/diagnóstico , Mielofibrosis Primaria/genética , Trombocitemia Esencial/diagnóstico , Trombocitemia Esencial/genética , Alelos , Células de la Médula Ósea/patología , Células Clonales , Expresión Génica , Heterocigoto , Humanos , Janus Quinasa 2/genética , Mutación , Mielofibrosis Primaria/metabolismo , Mielofibrosis Primaria/patología , Estudios Retrospectivos , Trombocitemia Esencial/metabolismo , Trombocitemia Esencial/patologíaAsunto(s)
Proteínas de Fusión bcr-abl/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Mutación , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Estudios RetrospectivosAsunto(s)
Calreticulina/genética , Mutación , Circulación Esplácnica , Trombosis de la Vena/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Estudios de Cohortes , Femenino , Humanos , Lactante , Masculino , Persona de Mediana EdadAsunto(s)
Calreticulina/genética , Mutación INDEL , Trombocitemia Esencial/genética , Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , MasculinoAsunto(s)
Trasplante de Células Madre Hematopoyéticas , Janus Quinasa 2/genética , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/cirugía , Adolescente , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Trastornos Mieloproliferativos/diagnóstico , Trastornos Mieloproliferativos/patología , Neoplasia Residual/diagnóstico , Neoplasia Residual/genética , Reacción en Cadena de la Polimerasa , Trasplante Homólogo , Adulto JovenAsunto(s)
Fusión Génica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Receptores de Citocinas/genética , Receptores Purinérgicos P2Y/genética , Niño , Estudios de Cohortes , Humanos , Pronóstico , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Citocinas/metabolismo , Receptores Purinérgicos P2Y/metabolismo , Factores de RiesgoRESUMEN
Although transformation of the myeloproliferative neoplasms (MPNs) to acute myeloid leukemia (AML) is well documented, development of an MPN in patients previously treated for, and in remission from, AML is exceedingly rare. A case is described in which a patient was successfully treated for AML and in whom a JAK2 V617F-positive MPN was diagnosed after seven years in remission. Retrospective evaluation of the JAK2 V617F detected a low allele burden at AML diagnosis and following one course of induction chemotherapy. This putative chemoresistant clone subsequently expanded over the intervening seven years, resulting in a hematologically overt MPN. As AML relapse has not occurred, the MPN may have arose in a separate initiating cell from that of the AML. Alternatively, both malignancies possibly evolved from a common precursor defined by a predisposition mutation with divergent evolution into MPN through acquisition of the JAK2 V617F and AML through acquisition of different mutations. This case emphasizes the protracted time frame from acquisition of a disease-driving mutation to overt MPN and further underscores the clonal complexity in MPN evolution.
RESUMEN
BACKGROUND: The classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), consisting of polycythemia vera, essential thrombocythemia, and primary myelofibrosis, are a heterogeneous group of neoplasms that harbor driver mutations in the JAK2, CALR, and MPL genes. The detection of mutations in these genes has been incorporated into the recent World Health Organization (WHO) diagnostic criteria for MPN. Given a pressing clinical need to screen for mutations in these genes in a routine diagnostic setting, a targeted next-generation sequencing (NGS) assay for the detection of MPN-associated mutations located in JAK2 exon 14, JAK2 exon 12, CALR exon 9, and MPL exon 10 was developed to provide a single platform alternative to reflexive, stepwise diagnostic algorithms. METHODS: Polymerase chain reaction (PCR) primers were designed to target mutation hotspots in JAK2 exon 14, JAK2 exon 12, MPL exon 10, and CALR exon 9. Multiplexed PCR conditions were optimized by using qualitative PCR followed by NGS. Diagnostic genomic DNA from 35 MPN patients, known to harbor driver mutations in one of the target genes, was used to validate the assay. RESULTS: One hundred percent concordance was observed between the previously-identified mutations and those detected by NGS, with no false positives, nor any known mutations missed (specificity = 100%, CI = 0.96, sensitivity = 100%, CI = 0.89). Improved resolution of mutation sequences was also revealed by NGS analysis. CONCLUSION: Detection of diagnostically relevant driver mutations of MPN is enhanced by employing a targeted multiplex NGS approach. This assay presents a robust solution to classical MPN mutation screening, providing an alternative to time-consuming sequential analyses.