RESUMEN
We developed genetic-epigenetic tissue mapping (GETMap) to determine the tissue composition of plasma DNA carrying genetic variants not present in the constitutional genome through comparing their methylation profiles with relevant tissues. We validated this approach by showing that, in pregnant women, circulating DNA carrying fetal-specific alleles was entirely placenta-derived. In lung transplant recipients, we showed that, at 72 hr after transplantation, the lung contributed only a median of 17% to the plasma DNA carrying donor-specific alleles, and hematopoietic cells contributed a median of 78%. In hepatocellular cancer patients, the liver was identified as the predominant source of plasma DNA carrying tumor-specific mutations. In a pregnant woman with lymphoma, plasma DNA molecules carrying cancer mutations and fetal-specific alleles were accurately shown to be derived from the lymphocytes and placenta, respectively. Analysis of tissue origin for plasma DNA carrying genetic variants is potentially useful for noninvasive prenatal testing, transplantation monitoring, and cancer screening.
Asunto(s)
ADN/sangre , Epigenómica/métodos , Neoplasias/genética , Trasplante de Órganos/métodos , Diagnóstico Prenatal/métodos , Adulto , Anciano , Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/genética , ADN/genética , Metilación de ADN , ADN de Neoplasias/sangre , ADN de Neoplasias/genética , Epigénesis Genética , Femenino , Feto/metabolismo , Variación Genética , Humanos , Neoplasias Hepáticas/genética , Linfoma/genética , Masculino , Persona de Mediana Edad , Neoplasias/sangre , Placenta/metabolismo , Embarazo , Análisis de Secuencia de ADN/métodosRESUMEN
PURPOSE: Persistently elevated posttreatment plasma EBV DNA is a robust predictor of relapse in nasopharyngeal carcinoma (NPC). However, assay standardization is necessary for use in biomarker-driven trials. We conducted a study to harmonize the method between four centers with expertise in EBV DNA quantitation. EXPERIMENTAL DESIGN: Plasma samples of 40 patients with NPC were distributed to four centers. DNA was extracted and EBV DNA copy number was determined by real-time quantitative PCR (BamHI-W primer/probe). Centers used the same protocol but generated their own calibrators. A harmonization study was then conducted using the same calibrators and PCR master mix and validated with ten pooled samples. RESULTS: The initial intraclass correlations (ICC) for the first 40 samples between each center and the index center were 0.62 [95% confidence interval (CI): 0.39-0.78], 0.70 (0.50-0.83), and 0.59 (0.35-0.76). The largest variability was the use of different PCR master mixes and calibrators. Standardization improved ICC to 0.83 (0.5-0.95), 0.95 (0.83-0.99) and 0.96 (0.86-0.99), respectively, for ten archival frozen samples. For fresh plasma with spiked-in EBV DNA, correlations were more than 0.99 between the centers. At 5 EBV DNA copies per reaction or above, the coefficient of variance (CV) was less than 10% for the cycle threshold (Ct) among all centers, suggesting this concentration can be reliably used as a cutoff for defining the presence of detectable EBV DNA. CONCLUSIONS: Quantitative PCR assays, even when conducted in experienced clinical labs, can yield large variability in plasma EBV DNA copy numbers without harmonization. The use of common calibrators and PCR master mix can help to reduce variability.
Asunto(s)
ADN Viral/sangre , Infecciones por Virus de Epstein-Barr/virología , Herpesvirus Humano 4/genética , Neoplasias Nasofaríngeas/virología , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/normas , Calibración/normas , Carcinoma , ADN Viral/genética , ADN Viral/normas , Infecciones por Virus de Epstein-Barr/sangre , Infecciones por Virus de Epstein-Barr/diagnóstico , Humanos , Cooperación Internacional , Laboratorios/normas , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/sangre , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Estándares de Referencia , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
The presence of fetal DNA in the plasma of pregnant women has opened up new possibilities for noninvasive prenatal diagnosis. Over the past decades, different types of fetal markers have been developed, initially based on discriminative genetic markers such as male-specific signals or paternally-inherited polymorphisms, and gradually evolved to the detection of fetal-specific transcripts or epigenetic signatures. This development has extended the coverage of the application of cell-free fetal DNA to essentially all pregnancies, regardless of the gender of the fetus or its polymorphic status. In this review, we present an overview of the development of noninvasive prenatal diagnosis through epigenetics. We introduce the basis of how fetal DNA could be detected from a large background of maternal DNA in maternal plasma based on fetal-specific DNA methylation patterns. We evaluate the methodologies involved and discuss the factors that affect the robustness of the detection. We review the progress in adopting fetal epigenetic markers for noninvasive prenatal assessment of fetal chromosomal aneuploidies and pregnancy-associated disorders. We conclude with comments on the future directions regarding the search for new fetal epigenetic markers and the clinical implementation of epigenetic approaches for noninvasive prenatal diagnosis.