Lisa Y.S. Chan

Noninvasive prenatal diagnosis of fetal trisomy 18 and trisomy 13 by maternal plasma DNA sequencing.

Massively parallel sequencing of DNA molecules in the plasma of pregnant women has been shown to allow accurate and noninvasive prenatal detection of fetal trisomy 21. However, whether the sequencing approach is as accurate for the noninvasive prenatal diagnosis of trisomy 13 and 18 is unclear due to the lack of data from a large sample set. We studied 392 pregnancies, among which 25 involved a trisomy 13 fetus and 37 involved a trisomy 18 fetus, by massively parallel sequencing. By using our previously reported standard z-score approach, we demonstrated that this approach could identify 36.0% and 73.0% of trisomy 13 and 18 at specificities of 92.4% and 97.2%, respectively. We aimed to improve the detection of trisomy 13 and 18 by using a non-repeat-masked reference human genome instead of a repeat-masked one to increase the number of aligned sequence reads for each sample. We then applied a bioinformatics approach to correct GC content bias in the sequencing data. With these measures, we detected all (25 out of 25) trisomy 13 fetuses at a specificity of 98.9% (261 out of 264 non-trisomy 13 cases), and 91.9% (34 out of 37) of the trisomy 18 fetuses at 98.0% specificity (247 out of 252 non-trisomy 18 cases). These data indicate that with appropriate bioinformatics analysis, noninvasive prenatal diagnosis of trisomy 13 and trisomy 18 by maternal plasma DNA sequencing is achievable.

Plasma Epstein-Barr Viral Deoxyribonucleic Acid Quantitation Complements Tumor-Node-Metastasis Staging Prognostication in Nasopharyngeal Carcinoma

PURPOSE To evaluate the effect of combining circulating Epstein-Barr viral (EBV) DNA load data with TNM staging data in pretherapy prognostication of nasopharyngeal carcinoma (NPC). PATIENTS AND METHODS Three hundred seventy-six patients with all stages of NPC were studied. Pretreatment plasma/serum EBV DNA concentrations were quantified by a polymerase chain reaction assay. Determinants of overall survival were assessed by multivariate analysis. Survival probabilities of patient groups, segregated by clinical stage (I, II, III, or IV) alone and also according to EBV DNA load (low or high), were compared. RESULTS Pretherapy circulating EBV DNA load is an independent prognostic factor for overall survival in NPC. Patients with early-stage disease were segregated by EBV DNA levels into a poor-risk subgroup with survival similar to that of stage III disease and a good-risk subgroup with survival similar to stage I disease. CONCLUSION Pretherapy circulating EBV DNA load is an independent prognostic factor to International Union Against Cancer (UICC) staging in NPC. Combined interpretation of EBV DNA data with UICC staging data leads to alteration of risk definition of patient subsets, with improved risk discrimination in early-stage disease. Validation studies are awaited.

Time Course of Early and Late Changes in Plasma DNA in Trauma Patients

BACKGROUND Cell-free DNA concentrations increase in the circulation of patients after trauma and may have prognostic potential, but little is know concerning the temporal changes or clearance of the DNA or its relationships with posttraumatic complications. We investigated temporal changes in plasma DNA concentrations in patients after trauma with use of real-time quantitative PCR. METHODS Serial plasma samples were taken from two trauma populations. In the first study, samples were collected every 20 min from 25 patients within the first 3 h of trauma. In the second study, samples were collected every day from 36 other trauma patients admitted to the intensive care unit (ICU). RESULTS In the first study, plasma DNA was increased within 20 min of injury and was significantly higher in patients with severe injury and in patients who went on to develop organ failure. In patients with less severe injuries, plasma DNA concentrations decreased toward reference values within 3 h. In the second study, plasma DNA concentrations were higher in patients who developed multiple organ dysfunction syndrome between the second and fourth days of admission than in patients who did not develop the syndrome. In patients who remained in the ICU with continuing organ dysfunction, plasma DNA remained higher than in healthy controls even at 28 days after injury. Most survivors with multiple organ dysfunction syndrome showed an initial very high peak followed by a prolonged smaller increase. CONCLUSIONS Plasma DNA concentrations increase early after injury and are higher in patients with severe injuries and in those who develop organ failure. Increased plasma DNA persists for days after injuries, especially in patients with multiple organ dysfunction syndrome.

Systematic search for placental DNA-methylation markers on chromosome 21: Toward a maternal plasma-based epigenetic test for fetal trisomy 21

BACKGROUND The presence of fetal DNA in maternal plasma represents a source of fetal genetic material for noninvasive prenatal diagnosis; however, the coexisting background maternal DNA complicates the analysis of aneuploidy in such fetal DNA. Recently, the SERPINB5 gene on chromosome 18 was shown to exhibit different DNA-methylation patterns in the placenta and maternal blood cells, and the allelic ratio for placenta-derived hypomethylated SERPINB5 in maternal plasma was further shown to be useful for noninvasive detection of fetal trisomy 18. METHODS To develop a similar method for the noninvasive detection of trisomy 21, we used methylation-sensitive single nucleotide primer extension and/or bisulfite sequencing to systematically search 114 CpG islands (CGIs)-76% of the 149 CGIs on chromosome 21 identified by bioinformatic criteria-for differentially methylated DNA patterns. The methylation index (MI) of a CpG site was estimated as the proportion of molecules methylated at that site. RESULTS We identified 22 CGIs which were shown to contain CpG sites that were either completely unmethylated (MI = 0.00) in maternal blood cells and methylated in the placenta (MI range, 0.22-0.65), or completely methylated (MI = 1.00) in maternal blood cells and hypomethylated in the placenta (MI range, 0.00-0.75). We detected, for the first time, placental DNA-methylation patterns on chromosome 21 in maternal plasma during pregnancy and observed their postpartum clearance. CONCLUSION Twenty-two (19%) of the 114 studied CGIs on chromosome 21 showed epigenetic differences between samples of placenta and maternal blood cells; these CGIs may provide a rich source of markers for noninvasive prenatal diagnosis.

Quantitative Analysis of Circulating Mitochondrial DNA in Plasma

BACKGROUND Recent studies have demonstrated the existence of circulating mitochondrial DNA in plasma and serum, but the concentrations and physical characteristics of circulating mitochondrial DNA are unknown. The aim of this study was to develop an assay to quantify mitochondrial DNA in the plasma of healthy individuals. METHODS We adopted a real-time quantitative PCR approach and evaluated the specificity of the assay for detecting mitochondrial DNA with a cell line (rho(0)) devoid of mitochondria. The concentrations and physical characteristics of circulating mitochondrial DNA were investigated by experiments conducted in three modules. In module 1, we evaluated the concentrations of mitochondrial DNA in plasma aliquots derived from four blood-processing protocols. In module 2, we investigated the existence of both particle-associated and free forms of mitochondrial DNA in plasma by subjecting plasma to filtration and ultracentrifugation. In module 3, we used filters with different pore sizes to investigate the size characteristics of the particle-associated fraction of circulating mitochondrial DNA. RESULTS The mitochondrial DNA-specific, real-time quantitative PCR had a dynamic range of five orders of magnitude and a sensitivity that enabled detection of one copy of mitochondrial DNA in plasma. In module 1, we found significant differences in the amounts of circulating mitochondrial DNA among plasma aliquots processed by different methods. Data from module 2 revealed that a significant fraction of mitochondrial DNA in plasma was filterable or pelletable by ultracentrifugation. Module 3 demonstrated that filters with different pore sizes removed mitochondrial DNA from plasma to different degrees. CONCLUSIONS Both particle-associated and free mitochondrial DNA are present in plasma, and their respective concentrations are affected by the process used to harvest plasma from whole blood. These results may have implications in the design of future studies on circulating mitochondrial DNA measured in different disease conditions.

Fetal Cell-free Plasma DNA Concentrations in Maternal Blood Are Stable 24 Hours after Collection: Analysis of First- and Third-Trimester Samples

The discovery of intact fetal cells in maternal blood has led to the possibility of noninvasive prenatal genetic testing. Although extensive work has been performed on maternal blood in the hope of developing a reliable method for the recovery of intact fetal cells, the total number of recoverable cells is quite small, averaging 19 nucleated fetal cells in 16 mL of blood (1)(2). This is contrasted by a relatively large amount of cell-free fetal DNA detectable in maternal plasma (3). Recent studies suggest that cell-free fetal DNA in maternal plasma can be used as a diagnostic tool for diseases of pregnancy, such as preeclampsia or preterm labor, or for fetal anomalies, such as Down syndrome (4)(5)(6)(7). Because of these potential applications, it is of great importance to determine whether cell-free fetal DNA concentrations remain stable in a test tube after phlebotomy. In this report we evaluate the effects of time after phlebotomy on the quantity of cell-free fetal DNA in blood samples from a pregnant woman and a woman who has just undergone termination of pregnancy. The post-termination samples were used because in the first trimester, there are relatively few fetal cells present in maternal blood. The termination procedure increases fetomaternal transfusion (8). We hypothesized that after phlebotomy, apoptotic fetal cells that die in the venipuncture tube would release their DNA. We also asked whether more fetal cells undergo apoptosis as a result of exposure to the maternal bloodstream and cytokines. In both cases, we hypothesized that cell-free fetal DNA concentrations would increase in the blood samples over time. This study was performed with Institutional Review Board approval and with informed consent from participants at Tufts-New England Medical Center. For early-gestation samples, 29 women who were 6–17 weeks pregnant were recruited from …

Plasma Cell-free Epstein-Barr Virus DNA Quantitation in Patients with Nasopharyngeal Carcinoma: Correlation with Clinical Staging

Nasopharyngeal carcinoma (NPC) is an important cancer in southern China and Southeast Asia.1 In Hong Kong, nearly all NPC cases are undifferentiated or poorly differentiated and harbor Epstein-Barr virus (EBV) in tumoral tissues.1 The recent interest in the presence of tumor-derived DNA in the plasma and serum of cancer patients has prompted our group2 and Mutirangura et al.3 to look for EBV DNA in the plasma and serum of NPC patients. Our preliminary data indicate that this approach is sensitive and specific for NPC.2 Our core technology for EBV DNA detection is real-time quantitative PCR,4 which is able to provide a quantitative measurement of the concentrations of EBV DNA in the plasma of NPC patients. In this communication, we report our latest results based on an expanded sample size, thus allowing a better understanding of the relationship between circulating EBV DNA and NPC staging.

Serial Analysis of Fetal DNA Concentrations in Maternal Plasma in Late Pregnancy

The discovery of fetal DNA in maternal plasma/serum has suggested new possibilities for noninvasive prenatal diagnosis (1) with applications in investigations of sex-linked diseases (2), rhesus D status (3), and β-thalassemia (4). Biologically, fetal DNA concentrations have been found to increase with gestational age, with a sharp increase toward the end of pregnancy (5). This latter phenomenon has been postulated to be related to the imminence of delivery (6). Consistent with the latter hypothesis, women with preterm labor have been found to have a higher concentration of fetal DNA in their plasma (7)(8). These results suggest that circulating fetal DNA concentrations are undergoing a time of flux during the late third trimester, but detailed information on the dynamics of maternal plasma fetal DNA is not available for this stage of gestation. In this study, we performed a serial analysis of fetal DNA concentrations in maternal plasma during the late third trimester. We recruited 33 healthy pregnant women from the Department of Obstetrics and Gynaecology, Prince of Wales Hospital, Hong Kong. All had ultrasonic evidence of male fetuses. Fetal sex was confirmed at delivery. Ethics approval was obtained from the Clinical Research Ethical Committee of the Chinese University of Hong Kong. Informed consent was obtained in all …

Derivation of a prediction rule for posttraumatic organ failure using plasma DNA and other variables.

Abstract: The early identification of patients at high risk of developing posttraumatic organ failure would allow preventive therapies to be studied. In this study, highly sensitive and specific guidelines for the early prediction of post‐traumatic organ failure (OF) and multiple organ dysfunction syndrome (MODS) using cell‐free (plasma) DNA and other predictors of posttraumatic complications were derived. As plasma DNA increases after injury and may be used to predict acute lung injury (ALI), we hypothesized that in combination with other predictors it would predict the later development of OF and MODS. Eighty‐three patients (69 males; median age, 36 years) were studied as a consequence of major trauma within 3.5 hours of injury (median time to sampling and assessment, 60 min). Plasma DNA was measured using a real‐time, quantitative, polymerase chain reaction assay for the β‐globin gene. OF and MODS occurred in 20/83 (24%) and 9/79 (11%) cases, respectively. At selected cutoff points, the sensitivity of plasma DNA for predicting OF and MODS ranged from 50% to 100%, specificity ranged from 74% to 95%, and the likelihood ratio ranged from 3.89 to 10.50. Other variables studied included serum albumin, creatine kinase, aspartate transaminase, lactate dehydrogenase, leukocyte count, hematocrit, injury severity score, maximal abbreviated injury score, and shock index. Using a classification and regression tree, plasma DNA and aspartate transaminase at optimal cutoffs predicted OF and MODS with an overall correct classification of 93% and 87%, respectively.

Nasopharyngeal carcinoma in situ (NPCIS)--pathologic and clinical perspectives.

Dysplasia or carcinoma in situ lesions (NPCIS) of the nasopharynx have rarely been reported. The prevalence, biologic behavior, and the transformation period of the pure preinvasive lesions have not been fully explained.