Publications by authors named "Lisa Y S Chan"

28 Publications

  • Page 1 of 1

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

PLoS One 2011 6;6(7):e21791. Epub 2011 Jul 6.

Centre for Research into Circulating Fetal Nucleic Acids, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong SAR, China.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0021791PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3130771PMC
November 2011

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

Clin Chem 2008 Mar 17;54(3):500-11. Epub 2008 Jan 17.

Centre for Research into Circulating Fetal Nucleic Acids, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1373/clinchem.2007.098731DOI Listing
March 2008

Hypermethylation of RASSF1A in human and rhesus placentas.

Am J Pathol 2007 Mar;170(3):941-50

Department of Chemical Pathology, The Chinese University of Hong Kong, Shatin, China.

The pseudomalignant nature of the placenta prompted us to search for tumor suppressor gene hypermethylation, a phenomenon widely reported in cancer, in the human placenta. Nine tumor suppressor genes were studied. Hypermethylation of the Ras association domain family 1 A (RASSF1A) gene was found in human placentas from all three trimesters of pregnancy but was absent in other fetal tissues. Hypermethylation of Rassf1 was similarly observed in placentas from the rhesus monkey but not the mouse. An inverse relationship between RASSF1A promoter methylation and gene expression was demonstrated by bisulfite sequencing of microdissected placental cells and immunohistochemical staining of placental tissue sections using an anti-RASSF1A antibody. Treatment of choriocarcinoma cell lines, JAR and JEG3, by 5-aza-2'-deoxycytidine and trichostatin A led to reduction in RASSF1A methylation but increased expression. These observations extend the analogy between the primate placenta and malignant tumors to the epigenetic level.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2353/ajpath.2007.060641DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1864885PMC
March 2007

Plasma Epstein-Barr viral deoxyribonucleic acid quantitation complements tumor-node-metastasis staging prognostication in nasopharyngeal carcinoma.

J Clin Oncol 2006 Dec;24(34):5414-8

Department of Clinical Oncology, Sir YK Pao Centre for Cancer, Centre for Clinical Trials, Hong Kong, People's Republic of China.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1200/JCO.2006.07.7982DOI Listing
December 2006

Reduced plasma RNA integrity in nasopharyngeal carcinoma patients.

Clin Cancer Res 2006 Apr;12(8):2512-6

Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong SAR, China.

Purpose: Recent research has shown the feasibility of detecting cell-free RNA markers in human subjects. As elevated RNase activity has previously been described in the circulation of cancer patients, we hypothesized that cancer patients may have reduced plasma RNA integrity. In this study, we used nasopharyngeal carcinoma (NPC) as a model system to test this hypothesis.

Experimental Design: Plasma RNA integrity was determined using the ratio of the concentrations of transcript sequences corresponding to the 3' to those from the 5' end of a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Transcript concentrations were measured using real-time quantitative reverse transcription-PCR assays targeting the 5' and 3' regions. We analyzed the plasma RNA integrity in 49 untreated NPC patients and 53 healthy controls. We also assessed the plasma samples from 19 NPC patients before and after radiotherapy to further show the clinical potential of this marker.

Results: The 3' to 5' GAPDH ratio was significantly lower in the plasma of untreated NPC patients when compared with healthy controls (0.0252 versus 0.0485, P = 0.024). Statistical analysis showed that plasma GAPDH ratio was correlated with tumor stage but not with sex and age. Moreover, 14 of 19 NPC patients (74%) showed significant increase in the plasma GAPDH ratio following radiotherapy (P = 0.003). All of these patients were in clinical remission after treatment.

Conclusions: Our findings suggest that NPC is associated with disturbances in the integrity of cell-free circulating RNA, raising the possibility that measurement of plasma RNA integrity may serve as a useful marker for the diagnosis and monitoring of malignant diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1078-0432.CCR-05-2572DOI Listing
April 2006

Plasma cell-free DNA as an indicator of severity of injury in burn patients.

Clin Chem Lab Med 2006 ;44(1):13-7

Plastic and Reconstructive Surgery, Department of Surgery, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, SAR.

Background: Raised levels of plasma cell-free DNA have been detected in various patient groups, including trauma patients. We hypothesized that plasma DNA is increased in burn patients and may represent an objective indicator of burn severity and have predictive as well as prognostic significance.

Methods: This was a prospective clinical study with full ethical approval. With informed consent, blood samples were collected from 28 burn patients within 24 h of injury and from 12 control subjects. Plasma cell-free DNA was measured by real-time quantitative polymerase chain reaction (PCR) assay for the beta-globin gene. Descriptive analysis, non-parametric data comparison tests (Mann-Whitney) and correlation tests (Spearman rank) were performed on the data.

Results: Samples were taken at a mean time of 5.7 h after injury from 13 patients with flame/flash burns and 15 patients with scalds. Median plasma DNA levels in the control, scald and flame/flash burn patient groups were 287, 648 and 2685 kilogenome-equivalents/L, respectively. Plasma DNA levels correlated with the length of hospital stay, but not with admission to the intensive care unit (ICU) nor the length of ICU stay. DNA levels correlated with the burn surface area (Spearman rank r = 0.54, p = 0.04) and the number of operations needed (Spearman rank r = 0.55, p = 0.03) for scalds, but not for flame/flash burns.

Conclusions: Plasma DNA is increased after burn injury and is significantly correlated with some outcome measures, including the length of hospital stay. DNA levels are higher in flame/flash patients than in scald patients; the difference may provide an objective indication of burn depth and inhalation injury.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1515/CCLM.2006.003DOI Listing
March 2006

Detection of the placental epigenetic signature of the maspin gene in maternal plasma.

Proc Natl Acad Sci U S A 2005 Oct 3;102(41):14753-8. Epub 2005 Oct 3.

Department of Chemical Pathology, Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong Special Administrative Region, China.

The discovery of fetal DNA in the plasma of pregnant women has opened up new approaches for noninvasive prenatal diagnosis and monitoring. Up to now, the lack of a fetal DNA marker that can be universally detected in maternal plasma has limited the clinical application of this technology. We hypothesized that epigenetic differences between the placenta and maternal blood cells could be used for developing such a marker. By using bisulfite DNA sequencing, the methylation status of the maspin gene promoter in placental tissues and paired maternal blood cells from pregnant women was analyzed. The maspin gene promoter was found to be hypomethylated in placental tissues and densely methylated in maternal blood cells. Genotyping of a single nucleotide polymorphism within the unmethylated maspin sequences in maternal plasma demonstrated that these sequences were derived from the fetus. By using real-time quantitative methylation-specific PCR, unmethylated maspin sequences were detected in maternal plasma in all three trimesters of pregnancy and were cleared within 24 h after delivery. The maternal plasma concentration of unmethylated maspin sequences was elevated by a median of 5.7 times in preeclamptic pregnancies compared with nonpreeclamptic pregnancies. Hypomethylated maspin DNA is the first universal marker for fetal DNA in maternal plasma, thus allowing the measurement of fetal DNA concentrations in pregnancy-associated disorders, irrespective of fetal gender and genetic polymorphisms. Differential DNA methylation between the placenta and maternal blood cells may be exploited to develop further markers for noninvasive prenatal assessment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.0503335102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1253547PMC
October 2005

Cell-free DNA in serum and plasma: comparison of ELISA and quantitative PCR.

Clin Chem 2005 Aug;51(8):1544-6

Institute of Clinical Chemistry, University of Munich, Munich, Germany.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1373/clinchem.2005.049320DOI Listing
August 2005

Distribution of cell-free and cell-associated Epstein-Barr virus (EBV) DNA in the blood of patients with nasopharyngeal carcinoma and EBV-associated lymphoma.

Clin Chem 2004 Oct 19;50(10):1842-5. Epub 2004 Aug 19.

Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1373/clinchem.2004.036640DOI Listing
October 2004

Improved accuracy of detection of nasopharyngeal carcinoma by combined application of circulating Epstein-Barr virus DNA and anti-Epstein-Barr viral capsid antigen IgA antibody.

Clin Chem 2004 Feb 18;50(2):339-45. Epub 2003 Dec 18.

Department of Clinical Oncology, The Chinese University of Hong Kong, Hong Kong SAR, Peoples Republic of China.

Background: Circulating Epstein-Barr viral (EBV) DNA and anti-EBV capsid antigen IgA (IgA VCA) represent two of the most sensitive peripheral blood markers of nasopharyngeal carcinoma (NPC), but direct comparative studies of these two markers are lacking.

Methods: The sensitivities and specificities of IgA-VCA and EBV DNA for diagnosis of NPC were determined in 139 new cases of NPC and 178 healthy individuals, respectively. EBV DNA was also assessed in 36 healthy family members identified as having false-positive IgA-VCA results at a screening clinic. EBV DNA was measured by a real-time quantitative PCR assay with a detection limit of 60 copies/mL. IgA-VCA was measured by semiquantitative indirect immunofluorescent method; a titer > or =1/10 was taken as positive.

Results: The sensitivities of EBV DNA and IgA-VCA for diagnosis of NPC were 95% (95% confidence interval, 91-98%) and 81% (73-87%), respectively. The combined marker panel had an overall sensitivity (positive result by either marker) of 99%. The concentrations of both markers showed dependence on cancer stage. The specificities of EBV DNA and IgA-VCA were 98% (96-99%) and 96% (91-98%), respectively. Among 36 healthy family members with false-positive IgA-VCA results, three-fourths had undetectable EBV DNA, whereas the others had increased EBV DNA concentrations that were significantly lower than in NPC patients.

Conclusions: For diagnosis of NPC, EBV DNA identifies almost all false-negative IgA-VCA cases and gives a 99% diagnostic sensitivity when combined with IgA-VCA. In the screening setting, EBV DNA identifies three-fourths of false-positive IgA-VCA cases. The selective application of EBV DNA in an IgA-VCA-based screening protocol could improve screening accuracy with only moderate increases in cost.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1373/clinchem.2003.022426DOI Listing
February 2004

Disparity of sensitivities in detection of radiation-naïve and postirradiation recurrent nasopharyngeal carcinoma of the undifferentiated type by quantitative analysis of circulating Epstein-Barr virus DNA1,2.

Clin Cancer Res 2003 Aug;9(9):3431-4

Department of Clinical Oncology, The Chinese University of Hong Kong, Hong Kong SAR, China.

Purpose: The purpose of this research was to compare the sensitivities of plasma EBV DNA in detection of postirradiation locally recurrent nasopharyngeal carcinoma (NPC), postirradiation distant metastatic NPC, and radiation-naïve NPC.

Experimental Design: Twenty-four patients with postirradiation local recurrence of NPC were assessed for plasma EBV DNA levels by a real-time quantitative PCR system. The results were compared with those of a cohort of 140 patients with newly diagnosed NPC and with those of 25 patients with distant metastatic relapse. EBV-encoded RNA positivity was also assessed in locally recurrent tumors and newly diagnosed tumors with undetectable plasma EBV DNA levels.

Results: Postirradiation locally recurrent tumors were associated with a significantly lower rate of detectable plasma EBV DNA compared with radiation-naïve tumors of comparable stage [stage I-II tumors: 5 of 12 (42%) versus 47 of 51 (92%), P = 0.0002; stage III-IV tumors: 10 of 12 (83%) versus 88 of 89 (99%), P = 0.01; Fisher's exact test], and compared with distant metastatic recurrences [15 of 24 (63%) versus 24 of 25 (96%), P < 0.02; Fisher's exact test]. The median EBV DNA level in patients with detectable EBV DNA was also significantly lower in locally recurrent tumors than in radiation-naïve tumors. All of the tissue samples of tumors associated with undetectable EBV DNA levels, where available, were EBV-encoded RNA positive.

Conclusions: The sensitivity of EBV DNA in the detection of tumors regrowing from an irradiated site is much lower than that from a radiation-naïve site. Although plasma EBV DNA is very effective in detecting distant metastatic relapse of NPC, it cannot be relied on as the sole surveillance tool for detection of local relapse.
View Article and Find Full Text PDF

Download full-text PDF

Source
August 2003

Rapid clearance of plasma Epstein-Barr virus DNA after surgical treatment of nasopharyngeal carcinoma.

Clin Cancer Res 2003 Aug;9(9):3254-9

Department of Surgery, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong Special Administrative Region, China.

Purpose: Circulating EBV DNA analysis has been shown to be valuable in the detection, prognostication, and monitoring of nasopharyngeal carcinoma (NPC) patients. A previous study has shown that, after radiotherapy, plasma EBV DNA levels of NPC patients would decline exponentially with a median half-life of 3.8 days. We postulate that this decline in plasma EBV DNA reflects the decrease in cancer cell population and, therefore, the rate of decline reflects the radiosensitivity of the tumor. However, this postulation would hold true only if EBV DNA is rapidly eliminated from the circulation. In this study, we determined the in vivo elimination rate of plasma EBV DNA in NPC patients.

Experimental Design: We monitored the level of plasma EBV DNA in NPC patients during and after surgical resection of NPC. The half-life of plasma EBV DNA was then calculated by plotting the natural logarithm of EBV DNA concentrations against time.

Results: The median half-life of plasma EBV DNA after surgical resection of NPC was 139 min. After a median follow-up of 6.7 days, EBV DNA was undetectable in 8 of 11 patients. One of 8 patients with undectable EBV DNA and all of the patients with detectable EBV DNA developed clinical relapse.

Conclusions: The in vivo elimination of EBV DNA is very rapid after surgical resection of NPC. The failure of complete and rapid elimination of EBV DNA from the circulation predicts disease recurrence.
View Article and Find Full Text PDF

Download full-text PDF

Source
August 2003

[Mutation-function analysis in the lipoprotein lipase gene of Chinese patients with hypertriglyceridemic type 2 diabetes].

Zhongguo Yi Xue Ke Xue Yuan Xue Bao 2003 Apr;25(2):134-41

Department of Chemical Pathology, Chinese University of Hong Kong, Hong Kong, China.

Objective: To investigate the role of lipoprotein lipase (LPL) gene on Chinese patients with hypertriglyceridemic type 2 diabetes.

Methods: Three subject groups, including hypertriglyceridemic group, normalipidemic type 2 diabetes group and healthy controls, were recruited and screened for sequence changes in LPL gene with PCR, SSCP, restriction analysis and direct DNA sequencing. LPL mass and activity in post-heparin plasma and in in vitro expression were investigated. Comparative modeling was performed via Swiss-PDB Viewer to provide the potential 2-D structures of wildtype and mutant proteins.

Results: Four missense mutations, Ala71Thr, Val18Ile, Gly188Glu and Glu242Lys, were identified in patients with hypertriglyceridemic type 2 diabetes, and not in both normalipidemic diabetes and the control subjects. The four missense mutations were located in the highly conserved amino acid sites, which are involved in highly conserved exon 3, 5, or 6 regions. They led to reduced LPL mass and enzyme activities in both post-heparin plasma and in vitro expression. The modeled structures displayed the differences to a great extent between the mutant and wide-type molecules.

Conclusion: These results indicated that the 4 missense mutations lead to LPL deficiency and subsequent hypertriglyceridemia. The LPL deficiency predispose a progressive diabetic pathway to those affected individuals. LPL gene is one of susceptibility gene for hypertriglyceridemic type 2 diabetes.
View Article and Find Full Text PDF

Download full-text PDF

Source
April 2003

Time course of early and late changes in plasma DNA in trauma patients.

Clin Chem 2003 Aug;49(8):1286-91

Accident & Emergency Medicine Academic Unit, Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1373/49.8.1286DOI Listing
August 2003

Pretherapy quantitative measurement of circulating Epstein-Barr virus DNA is predictive of posttherapy distant failure in patients with early-stage nasopharyngeal carcinoma of undifferentiated type.

Cancer 2003 Jul;98(2):288-91

Department of Clinical Oncology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong.

Background: Patients with International Union Against Cancer (UICC) Stage I-II nasopharyngeal carcinoma (NPC) appear to have a relatively favorable prognosis and generally are excluded from trials of combined modality treatment. More recently, plasma/serum cell-free Epstein-Barr virus (EBV) DNA has been shown to be measurable in the majority of NPC patients at the time of diagnosis, and appears to have prognostic significance. However, within Stage I-II disease, in which failure events are infrequent, the prognostic impact of the pretreatment EBV DNA level has not been addressed to our knowledge. This issue has management implications because different therapeutic strategies currently are employed for patients with good-risk and those with poor-risk NPC.

Methods: A cohort of 90 patients with UICC Stage I-II NPC (World Health Organization Grade 2/3 histology) had their pretherapy plasma/serum EBV DNA levels determined by a quantitative polymerase chain reaction assay and correlated with the probability of posttherapy failure. All patients received radiation therapy only, except for three patients who also received concurrent chemotherapy. Kaplan-Meier plots of the probability of locoregional failure, distant failure, and cancer-specific survival were compared with reference to clinical stage and EBV DNA levels.

Results: With a median follow-up time of 45 months, 12 patients and 7 patients, respectively, had developed locoregional and distant failures, including 2 patients with both local and distant failures. Patients with distant failure had significantly higher pretherapy EBV DNA levels than those without failure (a median of 13,219 copies/mL [interquatile-range, 274,635 copies/mL] vs. a median of 423 copies/mL [interquatile-range, 2753 copies/mL]). The probability of distant failure was significantly higher in patients with high (>4000 copies/mL plasma) compared with low EBV DNA levels (P=0.0001, log-rank test) and for Stage IIB disease compared with Stage I and Stage IIA disease combined (P=0.0149, log-rank test), but was not significantly different between patients with Stage II and those with Stage I disease. The risks of locoregional failure were not significantly different between patients with high and those with low EBV DNA levels, and also was not significantly different between clinical substages. Approximately 35% of patients with Stage IIB disease were in the at-risk group for distant failure, as identified by high EBV DNA levels.

Conclusions: Within a group of patients with UICC Stage I-II NPC, the pretherapy plasma EBV DNA level was found to identify a poor-risk group with a probability of distant failure similar to that of patients with advanced stage disease. This group of patients may warrant management considerations currently applicable only to cases of Stage III-IV disease. The prognostic significance of designating Stage IIB disease as per the 1997 UICC staging was confirmed, although the pretherapy EBV DNA level appears to be a more powerful prognostic discriminator in patients with early-stage NPC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cncr.11496DOI Listing
July 2003

Molecular characterization of circulating EBV DNA in the plasma of nasopharyngeal carcinoma and lymphoma patients.

Cancer Res 2003 May;63(9):2028-32

Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong Special Administrative Region, China.

Despite the increasing clinical applications of circulating EBV DNA analysis as a tumor marker, the molecular nature of these EBV DNA molecules remains unclear. We subjected plasma/serum samples of nasopharyngeal carcinoma and lymphoma patients to DNase digestion and ultracentrifugation and showed that circulating EBV DNA molecules are "naked" DNA fragments instead of being contained inside virions. We further showed that these EBV DNA fragments were relatively short, and 87% of them were shorter than 181 bp. These results provide fundamental information that may improve our understanding of the release of tumor-derived nucleic acids into the blood of cancer patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
May 2003

Quantitative analysis of pleural fluid cell-free DNA as a tool for the classification of pleural effusions.

Clin Chem 2003 May;49(5):740-5

Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR.

Background: Recently, much interest has been focused on the quantification of DNA in miscellaneous body fluids. In this study, the application is extended to classifying pleural effusions by measuring cell-free DNA in pleural fluid.

Methods: We recruited 50 consecutive patients with pleural effusions with informed consent. Pleural fluids were centrifuged at 13000 g, with supernatants aliquoted for extraction and analysis of beta-globin DNA sequence by quantitative real-time PCR. Serum and pleural fluid biochemistries were performed to classify pleural effusions using the modified criteria of Light et al. (Ann Intern Med 1972;77:507-13). The ROC curve was plotted to determine the cutoff DNA concentration for classifying pleural fluids as transudates or exudates. Indicators of diagnostic accuracy were calculated for both pleural fluid DNA and modified criteria of Light et al., using the discharge, microbiologic, and histologic diagnoses as the reference standard.

Results: The area under the ROC curve was 0.95 [95% confidence interval (CI), 0.84-0.99]. At 509 genome-equivalents/mL, pleural fluid DNA alone correctly classified 46 of 50 pleural effusions with 91% sensitivity (95% CI, 76-98%), 88% specificity (95% CI, 64-98%), and positive and negative likelihood ratios of 7.7 (95% CI, 3.1-19.5) and 0.10 (95% CI, 0.04-0.27), respectively. With the modified criteria of Light et al., 43 of 50 pleural effusions were correctly classified with 97% sensitivity (95% CI, 91-100%) and 67% specificity (95% CI, 45-89%). There were significant correlations between cell-free DNA and both lactate dehydrogenase and total protein in pleural fluid, suggesting their common origin.

Conclusions: Pleural fluid DNA concentrations are markedly increased in exudative effusions, making it a potential new tool to evaluate the etiologic causes of pleural effusions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1373/49.5.740DOI Listing
May 2003

Quantitative analysis of circulating mitochondrial DNA in plasma.

Clin Chem 2003 May;49(5):719-26

Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1373/49.5.719DOI Listing
May 2003

Pathogenic mutations of the lipoprotein lipase gene in Chinese patients with hypertriglyceridemic type 2 diabetes.

Hum Mutat 2003 Apr;21(4):453

Department of Chemical Pathology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.

Elevated plasma triglyceride and nonesterified fatty acid concentrations may cause insulin resistance. Lipoprotein lipase (LPL) is a rate-determining enzyme in lipid metabolism. To investigate the role of the LPL gene in Chinese patients with hypertriglyceridemic type 2 diabetes, 277 patients with type 2 diabetes and 241 healthy control subjects were recruited and screened for sequence changes in the LPL gene by PCR, SSCP, restriction analysis and direct DNA sequencing. Ten mutations were identified: four missense mutations, Ala71Thr, Val181Ile, Gly188Glu and Glu242Lys; one nonsense mutation Ser447Ter; and five silent mutations. Ser447Ter was found in both patients and controls with no significant difference in frequency. The four missense mutations were located in the highly conserved exon 3, 5, and 6 regions and in highly conserved amino acid sites. They led to reduced LPL mass and enzyme activities in both post-heparin plasma and in vitro expression. The modeled structures displayed major differences between the mutant and wildtype molecules. These results indicated that the four missense mutations lead to LPL deficiency and subsequent hypertriglyceridemia. Based on our study and published data, a putative pathogenic pathway was suggested: LPL enzyme deficiency causes elevated plasma triglyceride level and subsequent insulin resistance; both increased free fatty acids and insulin resistance promote gluconeogenesis and hyperglycaemia, a vicious circle leading to type 2 diabetes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/humu.9134DOI Listing
April 2003

Serial analysis of fetal DNA concentrations in maternal plasma in late pregnancy.

Clin Chem 2003 Apr;49(4):678-80

Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1373/49.4.678DOI Listing
April 2003

Fetal cell-free plasma DNA concentrations in maternal blood are stable 24 hours after collection: analysis of first- and third-trimester samples.

Clin Chem 2003 Jan;49(1):195-8

Division of Genetics, Department of Pediatrics, Tufts-New England Medical Center and Tufts University School of Medicine, Boston, MA 02111, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1373/49.1.195DOI Listing
January 2003

Fetal DNA clearance from maternal plasma is impaired in preeclampsia.

Clin Chem 2002 Dec;48(12):2141-6

Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR.

Background: Increased fetal DNA in maternal plasma/serum has been reported in pregnancies complicated by preeclampsia. We hypothesized that impaired clearance of fetal DNA might contribute, at least in part, to the above-mentioned phenomenon.

Methods: We studied 7 preeclamptic and 10 control pregnant women. All had male fetuses. Serial blood samples were obtained from before delivery to 6 h postpartum. Male fetal DNA in maternal plasma was measured by real-time quantitative PCR for the SRY gene on the Y chromosome.

Results: The median fetal DNA concentrations before delivery were significantly higher in the preeclamptic women than in the controls (521 vs 227 genome-equivalents/mL for preeclamptic and control women, respectively; Mann-Whitney rank-sum test, P = 0.017). The median fetal DNA concentrations at 6 h after delivery were also significantly different between the two groups (208 vs 0 genome-equivalents/mL for preeclamptic and control women, respectively; Mann-Whitney rank-sum test, P = 0.002). A first-order clearance model was found to best describe the kinetics of maternal plasma fetal DNA clearance. Moreover, we observed a significant difference in the median apparent clearance half-lives of fetal DNA between the preeclamptic women (114 min) and controls (28 min; Mann-Whitney rank-sum test, P = 0.007).

Conclusions: This study represents the first documentation of impaired fetal DNA clearance from maternal plasma in preeclampsia. Such an abnormality in circulating DNA clearance may also be present in other medical conditions associated with quantitative aberrations in circulating DNA concentrations.
View Article and Find Full Text PDF

Download full-text PDF

Source
December 2002

Plasma Epstein-Barr virus DNA and residual disease after radiotherapy for undifferentiated nasopharyngeal carcinoma.

J Natl Cancer Inst 2002 Nov;94(21):1614-9

Department of Clinical Oncology, Sir Y. K. Pao Centre for Cancer, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong Special Administrative Region.

Background: Epstein-Barr virus (EBV) DNA can be detected and quantified in the plasma of patients with EBV-related tumors, such as nasopharyngeal carcinoma (NPC). Although NPC at early stages can be cured by radical radiotherapy, there is a high recurrence rate in patients with advanced NPC. The pretreatment level of circulating EBV DNA is a prognostic factor for NPC, but the prognostic value of post-treatment EBV DNA has not been studied. We designed a prospective study in Hong Kong, China, to investigate the value of plasma EBV DNA as a prognostic factor for NPC.

Methods: One hundred seventy NPC patients, without metastatic disease at presentation, were treated with a uniform radiotherapy protocol. Circulating EBV DNA was measured by real-time quantitative polymerase chain reaction before treatment and 6-8 weeks after radiotherapy was completed. Risk ratios (RRs) were determined with a Cox regression model, and associations of various factors with progression-free and overall survival and recurrence rates were determined with a stepwise Cox proportional hazards model. All statistical tests were two-sided.

Results: Ninety-nine percent of patients achieved complete clinical remission. Levels of post-treatment EBV DNA dominated the effect of levels of pretreatment EBV DNA for progression-free survival. The RR for NPC recurrence was 11.9 (95% confidence interval [CI] = 5.53 to 25.43) for patients with higher post-treatment EBV DNA and 2.5 (95% CI = 1.14 to 5.70) for patients with higher pretreatment EBV DNA. Higher levels of post-treatment EBV DNA were statistically significantly associated with overall survival (P<.001; RR for NPC recurrence = 8.6, 95% CI = 3.69 to 19.97). The positive and negative predictive values for NPC recurrence for a higher level of post-treatment EBV DNA were 87% (95% CI = 58% to 98%) and 83% (95% CI = 76% to 89%), respectively.

Conclusion: Levels of post-treatment plasma EBV DNA in patients with NPC appear to strongly predict progression-free and overall survival and to accurately reflect the post-treatment residual tumor load.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/jnci/94.21.1614DOI Listing
November 2002

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

Head Neck 2002 Nov;24(11):989-95

Division of Otorhinolaryngology, Department of Surgery, The Chinese University of Hong Kong, Prince of Wales Hospital, 30-32 Ngan Shing Street, Shatin, New Territories, Hong Kong, SAR, China.

Background: 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.

Methods: All cases of NPCIS were retrospectively reviewed during the period between 1990 and 2000. The clinical features of all cases were studied. The biopsy samples were examined using light microscopy and in situ hybridization (ISH) for EBV-encoded RNA (EBER). The sera taken before and after the transformation were analyzed for anti-viral capsid antigen (VCA) EBV titers and circulating cell-free EBV DNA concentration.

Results: Three cases of NPCIS were identified. Two of the three cases subsequently developed into invasive NPC after initial presentation. The interval of transformation varied from 40 to 48 months. In all three cases, the specimens showed abnormal findings on light microscopy and positive staining for EBER. Elevated anti-VCA titers were present in two of the preinvasive lesions. No cell-free EBV DNA was detected in the sera of these patients during the preinvasive phase of the disease.

Conclusions: Preinvasive NPC is a rare but distinct entity. Its transformation period can be as long as 4 to 5 years. Elevated anti-VCA titers, in the presence of abnormal cells on light microscopy, should alert the pathologist to perform ISH EBER studies to diagnose this rare condition.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/hed.10161DOI Listing
November 2002

Genotype-phenotype studies of six novel LPL mutations in Chinese patients with hypertriglyceridemia.

Hum Mutat 2002 Sep;20(3):232-3

Department of Chemical Pathology, The Chinese University of Hong Kong, Hong Kong, China.

We screened 160 unrelated Chinese hypertriglyceridemic subjects for sequence alterations in the promoter and the 10 exons of the lipoprotein lipase (LPL) gene. We identified one reported mutation (L252R), one common polymorphism (S447X), and six novel mutations: V181I, C283Y, S298R and S338F (found in single individuals), L252V (in two individuals), and A71T (in three individuals). Screening of family members of the above probands revealed a total of 19 mutation carriers, most of whom, though not all, displayed reduced LPL activity and mass when compared to normolipidemic control subjects. In in vitro expression studies, A71T, V181I, L252R, L252V and C283Y decreased the specific activity of the gene product. Interestingly, S298R had no effect on the catalytic activity while S338F increased it. A71T and C283Y reduced the secretion of the mutant proteins significantly while V181I, S298R and S338F had mild effects only. The total LPL mass of all the mutant constructs was reduced compared to that of the wild type construct, probably due to the instabilities of the mutant mRNA or the mutant protein. The heterogeneity in phenotypic effects of these mutations is a likely consequence of their variable effects on proteoglycan binding, conformation and interactions with other secondary genetic or environmental factors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/humu.9054DOI Listing
September 2002

Novel mitochondrial 16S rRNA mutation, 3200T-->C, associated with adult-onset type 2 diabetes.

Chin Med J (Engl) 2002 May;115(5):753-8

Department of Chemical Pathology, Chinese University of Hong Kong, China.

Objective: To investigate the role of a potential diabetes-related mitochondrial region, which includes two previously reported mutations, 3243A-->G and 3316G-->A, in Chinese patients with adult-onset type 2 diabetes.

Methods: A total of 277 patients and 241 normal subjects were recruited for the study. Mitochondrial nt 3116 - 3353, which spans the 16S rRNA, tRNA(leu(UUR)) and the NADH dehydrogenase 1 gene, were detected using polymerase chain reaction (PCR), direct DNA sequencing, PCR-restriction fragment length polymorphism and allele-specific PCR. Variants were analyzed by two-tailed Fisher exact test. The function of the variants in 16S rRNA were predicted for minimal free energy secondary structures by RNA folding software mfold version 3.

Results: Four homoplasmic nucleotide substitutions were observed, 3200T-->C, 3206C-->T, 3290T-->C and 3316G-->A. Only the 3200T-->C mutation is present in the diabetic population and absent in the control population. No statistically significant associations were found between the other three variants and type 2 diabetes. The 3200T-->C and 3206C-->T nucleotide substitutions located in 16S rRNA are novel variants. The 3200T-->C caused a great alteration in the minimal free energy secondary structure model while the 3206C-->T altered normal 16S rRNA structure little.

Conclusions: The results suggest that the 3200T-->C mutation is linked to the development of type 2 diabetes, but that the other observed mutations are neutral. In contrast to the Japanese studies, the 3316G-->A does not appear to be related to type 2 diabetes.
View Article and Find Full Text PDF

Download full-text PDF

Source
May 2002

Diagnostic and prognostic implications of circulating cell-free Epstein-Barr virus DNA in natural killer/T-cell lymphoma.

Clin Cancer Res 2002 Jan;8(1):29-34

Department of Clinical Oncology and The Sir Y. K. Pao Cancer Centre, The Chinese University of Hong Kong, Prince of Wales Hospital, The New Territories, Hong Kong Special Administrative Region, China.

Purpose: Natural killer/T-cell (NK/T-cell) lymphoma is a highly aggressive tumor for which no serological tumor marker has yet been established to be useful clinically. We investigated the potential of circulating EBV DNA as a tumor marker for this malignancy.

Experimental Design: A real-time quantitative PCR assay was used to measure circulating EBV DNA.

Results: Plasma EBV DNA levels were measured in 18 patients with NK/T-cell lymphoma at presentation and during therapy. Plasma EBV DNA was detected in 17 of the 18 patients (median, 659 copies/ml; interquartile range, 181-17,042 copies/ml) but in none of 35 control subjects (p < 0.0001). Serial measurements of plasma EBV DNA levels during therapy showed a close correlation between clinical response and changes in plasma EBV DNA levels. Clinically responding patients showed a fall of plasma EBV DNA levels to low or undetectable levels, whereas those who failed therapy showed a rapid increase in plasma EBV DNA levels. Most importantly, patients with high baseline plasma EBV DNA levels (> or = 600 copies/ml) demonstrated a significantly inferior survival than those with low baseline plasma EBV DNA (< 600 copies/ml; 21% versus 78%; P = 0.024).

Conclusions: Plasma EBV DNA, as measured by real-time quantitative PCR, is a useful tumor marker for diagnosis, disease monitoring, and prediction of outcome in patients with NK/T-cell lymphoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
January 2002