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BACKGROUND: Circulating levels of cell-free DNA increase in many pathologic conditions. However, notable discrepancies in the quantitative analysis of cell-free DNA from a large number of laboratories have become a considerable pitfall, hampering its clinical application. METHODS: We designed a novel recombinant DNA fragment that could be applied as an internal standard in a newly developed and validated duplex real-time PCR assay for the quantitative analysis of total cell-free plasma DNA, which was tested in 5,442 healthy adults and 200 trauma patients. RESULTS: Compared with two traditional methods, this novel assay showed a lower detection limit of 0.1 ng/mL, lower intra- and inter-assay CVs, and higher accuracy in the recovery test. The median plasma DNA concentration of healthy males (20.3 ng/mL, n=3,092) was significantly higher than that of healthy females (16.1 ng/mL, n=2,350) (Mann-Whitney two-sample rank sum test, P<0.0001). The reference intervals of plasma DNA concentration were 0-45.8 ng/mL and 0-52.5 ng/mL for healthy females and males, respectively. The plasma DNA concentrations of the majority of trauma patients (96%) were higher than the upper normal cutoff values and were closely related to the corresponding injury severity scores (R²=0.916, P<0.0001). CONCLUSIONS: This duplex real-time PCR assay with a new internal standard could eliminate variation and allow for more sensitive, repeatable, accurate, and stable quantitative measurements of plasma DNA, showing promising application in clinical diagnosis.
Subject(s)
Adult , Female , Humans , Male , Middle Aged , DNA/blood , Healthy Volunteers , Real-Time Polymerase Chain Reaction/methods , Reference Values , Wounds and Injuries/bloodABSTRACT
The cell-free plasma DNA of the 132 critically ill patients admitted to the emergency intensive care unit ( EICU) was measured by real-time quantitative polymerase chain reaction assay for the humanβ-globin gene. Ac-cording to the prognosis, the patients were divided into survival group (95 cases) and death group (37 cases). Blood samples were collected after EICU admission, 24 hours after admission, 48 hours after admission, and 1 week after admission. 30 age-mached controls were collected from healthy volunteers healthy care centre. Com-pared with control group, at each time point, cell-free plasma DNA concentration was significantly higher in EICU patients (P<0. 05). Cell-free plasma DNA concentrations were higher in hospital non-survivors than in survivors, of which plasma DNA concentration of 24 hours after admission and 48 hours after admission was statistically signifi-cant ( P<0 . 05 ) . The area under the ROC curves ( AUC ) for plasma DNA was bigger than the lactate and A-PACHE IIscore, the maximum of AUC was 0. 847(95% CI 0.648~1.000) at the point of 48 hours after admis-sion.
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The cell-free plasma DNA (cfpDNA) has been suggested as a useful tumor marker for its quantitative and qualitative tumor-specific alterations that reflect the biological characteristics and the progression and outcomes of tumors.Therefore,it has been used as liquid biopsy to detect cfpDNA in peripheral blood for the diagnosis,monitoring of clinical effects,and prognosis of malignancies
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Objective: To improve the sensitivity of Human Papillomavirus (HPV) detection in plasma from high-grade cervical neoplasia patients (CIN III) and cervical cancer (CC) evaluating any likely correlation with disease stage.Method: We subjected plasma DNA isolates from 112 patients (CIN and ICC) to a pre-PCR restoration treatment to improve detection sensitivity. HPV-specific sequences were detected by conventional PCR both in cervical scrapes and plasma DNA obtained from each patient. For every single DNA sample, both non-restored and restored isolates were PCR analyzed.Results: We detected HPV in plasma DNA isolates with significantly higher efficiency on restored plasma-DNA as compared to each non-restored equivalent, still maintaining close correlation with the clinical stage of the cases. By analyzing plasma-DNA isolates we could classify as HPV positive >50.0% of the cases that were previously known to be positive from the cervical scrape based assay. Interestingly, 100% of the cases in which subtype HPV18 was detected in cervical scrapes were also positive in plasma DNA.Conclusions: Restoration of plasma DNA from cervical cancer patients allows a more sensitive PCR-based HPV detection, maintaining the correlation to disease stage traditionally observed.
Objetivo: Mejorar la sensibilidad y la detección del virus del papiloma humano (VPH) en el plasma de pacientes con neoplasias intraepiteliales de alto grado (NIEAG) y cáncer de cuello uterino (CCU) para evaluar si existe una relación con el estadío de la enfermedad.Método: Los ADN de plasma aislados de 112 pacientes (NIC y CCU) se sometieron a restauración mediante reacción de la polimerasa en cadena (siglas en inglés, PCR), para mejorar su calidad como sustrato para PCR. En cada paciente se detectaron secuencias específicas del VPH por PCR convencional, tanto en exudados cérvico-vaginales como en ADN del plasma. Por cada muestra se analizaron por PCR, cantidades equivalentes del ADN aislado, tanto no restaurado como restaurado.Resultados: Para las muestras pareadas se pudo detectar VPH en ADN de plasma de forma más eficiente en los materiales restaurados, pues se mantuvo una estrecha correlación con el estadío clínico de los casos. Mediante el análisis de ADN de plasma es posible detectar como VPH positivas más de 50% de los casos que se identificaron previamente como positivos en citología de cuello uterino. Se enfatiza el hecho que 100% de los casos en los que el subtipo VPH18 fue descubierto en exudado cérvico-vaginal también fueron positivos en el ADN de plasma.Conclusiones: El proceso de restauración de ADN de plasma de pacientes con cáncer de cuello uterino permite mejorar la detección de VPH, por PCR, y mantener la correlación con el estadío de la enfermedad.
Subject(s)
Humans , Female , DNA , DNA Probes, HPV , Plasma , Uterine Cervical Neoplasms , Cell BiologyABSTRACT
Objectives To evaluate accurately hepatocyte injury degree of severe hepatitis patients by quantifying plasma DNA of severe hepatitis patients and study its clinical application in diagnosis of severe hepatitis comparing with ALT.Methods Six milliliters of peripheral blood samples were collected from 185 patients with hepatitis B which are divided into four groups, severe hepatitis with 30 cases, acute hepatitis with 20 cases, chronic B hepatitis with 90 cases, and liver cirrhosis with 45 cases. 100 healthy controls were enrolled. Circulating DNA was extracted from plasma by the BILATEST virus DNA/RNA extraction kit and quantified with a novel duplex real-time PCR assay, respectively.Results Plasma DNA levels of hepatitis B patients were significantly higher than those of healthy controls (104.2 ng/ml vs. 23.4 ng/ml (median),P=0.0000).A significant difference of plasma DNA concentration was found between severe hepatitis and acute hepatitis (P=0.0018), and chronic B hepatitis (P=0.0000), and liver cirrhosis (P=0.0000).The median value of serum ALT of hepatitis B patients was 107.5 U/L, much higher than that of the healthy controls (24.1 U/L,P=0.0000).The levels of serum ALT were significantly different between severe hepatitis and acute hepatitis (P=0.0024), while there was no remarkable difference between severe hepatitis and chronic B hepatitis (P=0.0600), liver cirrhosis (P=1.0000). Moreover, for distinguishing severe hepatitis from liver cirrhosis and chronic B hepatitis, the plasma DNA assay was notably superior to ALT by the analysis of receive operating characteristic (ROC) curves (AUC, 0.95 vs. 0.51,P=0.0000; 0.86 vs. 0.34,P=0.0000).Conclusion The results by measuring plasma DNA of hepatitis B patients with the novel duplex real-time quantitative PCR showed that plasma DNA may be considered as a robust predictive marker for accurately evaluating hepatocyte injury degree of severe hepatitis patients.
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Objective To investigate the loss of heterozygosity (LOH) of 3p D3S1300 and D3S1289 loci of plasma DNA of lung cancer (LC) patients and evaluate its value for a tumor marker of lung cancer.Methods Two microsatellite markers,D3S1300 and D3S1289,were analyzed by PCR and silver staining to investigate LOH of the plasma DNA and cancer tissue DNA from 69 cases of primary lung cancer,and the plasma DNA from 40 control subjects.Results The percentages of D3S1300 LOH detected in the tumor tissue and plasma DNA of LC patients were 40.6% and 29.0% respectively,while the percentages of D3S1289 LOH were 31.9% and 24.6% respectively.In contrast,only 2 cases of D3S1300 LOH and 3 cases of D3S1289 LOH were found in plasma DNA of control group (P
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BACKGROUND/AIMS: The plasma DNA concentration of patients with cancer is known to be higher than normal controls. Increased DNA concentration and tumor-specific genes in plasma can be used as tumor markers in some cancers. This study was designed to evaluate whether quantification of plasma DNA concentration by using real-time PCR is useful as a tumor marker in the diagnosis of pancreatic cancer. METHODS: Twenty-four patients (M:F=16:8, mean age; 60.5+/-11.5 years) with pancreatic cancer were recruited for this study. Fifteen patients with chronic pancreatitis and fifteen healthy persons were selected as controls (M:F=26:4, 53.5+/-11.2 years). The concentration of plasma DNA was determined by real-time PCR for telomerase reverse transcriptase gene. RESULTS: Plasma DNA concentration in patients with pancreatic cancer (46.4+/-63.2 ng/mL) was higher than that of chronic pancreatitis (p=0.041) and normal controls (p=0.030). The sensitivity and specificity in detecting pancreatic cancer were 75% and 70% respectively when the cut-off value of plasma DNA concentration was set at 46.9 ng/mL. CONCLUSIONS: Plasma DNA concentration in patients with pancreatic cancer was higher than that of controls. However, its sensitivity and specificity is not high enough to be used as a tumor marker for pancreatic cancer.
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , DNA, Neoplasm/blood , Diagnosis, Differential , Pancreatic Neoplasms/diagnosis , Pancreatitis, Chronic/diagnosis , Polymerase Chain Reaction , Biomarkers, Tumor/bloodABSTRACT
Microsatellites are short tandem repeated nucleotide sequences that are present throughout the human genome. Variations in the repeat number or a loss of heterozygosity around the microsatellites have been termed a microsatellite alteration (MA). A MA reflects the genetic instability caused by an impairment in the DNA mismatch repair system and is suggested to be a novel tumorigenic mechanism. A number of studies have reported that MA in the DNA extracted from the plasma occurs at varying frequencies among patients with a non-small cell lung carcinoma (NSCLC). The genomic DNA from 9 subjects with a non-small cell lung cancer (squamous cell cancer 6, adenocarcinoma 2, non-small cell lung cancer1) and 9 age matched non-cancer control subjects (AMC: tuberculosis 3, other inflammatory lung disease 6) and 12 normal control subjects (NC) were extracted from the peripheral blood leukocytes and plasma. Three microsatellite loci were amplified with the primers targeting the Gene Bank sequence D21S1245, D3S1300, and D3S1234. MA in the form of an allelic loss or a band shift was examined with 6% polyacrylamide gel electrophoresis and silver staining. None (0/12) of the NC subjects less than 40 years of age showed a MA in any of the three markers, while 88.9%(8/9) of the AMC above 40 showed a MA in at least one of the three markers (p0.05). In conclusion, a MA in the D21S1245, D3S1300, and D3S1234 loci using DNA extracted from the plasma was detected in 66.7% of lung cancer while no MA was found in the young non-smoking control subjects. However, many of the non-cancer control subjects (aged smokers) also showed a MA, which compromised the specificity of the MA analysis as a screening test. Therefore, a further study with a larger sample size will be needed.
Subject(s)
Humans , Adenocarcinoma , Base Sequence , Carcinoma, Non-Small-Cell Lung , DNA Mismatch Repair , DNA , Electrophoresis, Polyacrylamide Gel , Genome, Human , Leukocytes , Loss of Heterozygosity , Lung Diseases , Lung Neoplasms , Lung , Mass Screening , Microsatellite Repeats , Plasma , Sample Size , Silver Staining , Smoke , Smoking , TuberculosisABSTRACT
OBJECTIVE: Recent studies demonstrated that soluble tumor DNA is found in the plasma of cancer patients, and some microsatellite alteration have been identified in ovarian carcinoma. The aim of study was to detect microsatellite abnormalities in the plasma of patients with ovarian carcinoma and to evaluate their efficacy as molecular screening or diagnostic tool for ovarian cancer. METHODS: In fifteen ovarian carcinoma patients, DNA was extracted from the plasma samples and microsatellite analysis was done with 11 microsatellite markers. RESULTS: All fifteen cases showed at least one tumor specific alteration in microsatellite analysis. The frequency of genetic alteration varies from 14.2% to 85.7%. Highly frequent tumor specfic alteration markers are D18S69 (85.7%), D10S215 (69.2%), D16S504 (66.7%), D8SNEFL (62.5%) and D11S1340 (60.0%). CONCLUSION: These results suggest that the mutation of tumor DNA can be detected in plasma of patients with ovarian carcinoma. LOH is more frequent event and the frequency of genetic alteration is relatively higher than that of previous reports.
Subject(s)
Humans , DNA , Loss of Heterozygosity , Mass Screening , Microsatellite Instability , Microsatellite Repeats , Ovarian Neoplasms , PlasmaABSTRACT
Objective To study the diagnostic value of LOH and MSI of free DNA 3p microsatellite point in plasma of lung cancer patients. Methods Using PCR and sliver dye techniques, LOH and MSI of three microsatellite sites of plasma DNA on chromosome 3p in resected fresh tissue from 37 patients and whole blood sample from 94 patients were detected. Results The plasma free DNA concentration of most lung cancer patients was above microgramme level. Above 70% of tissues and plasma were positive. No significant difference was found at different stages and in different pathologic types. The result of patients with lymph node metastasis was significantly different from that of the patients without lymph node metastasis. Conclusion Plasma DNA of lung cancer patients is a good medium for gene diagnosis and may be used widely in the future.
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Objective To study the diagnostic and monitoring values of the loss of heteroxygosity(LOH) of 3p loci in free plasma DNA of small cell lung cancer. Methods PCR silver staining was used for the detection of LOH of three microsatellite sites in the 3p loci of plasma free DNA and tissue of SCLC. Results In 33 cases of tissue, the incidence rate of LOH was 54.5%(18/33), but the positive rate of LOH in plasma free DNA was 42.4%(14/33). The correlation between them was 78.8%(14/18). Conclusion The plasma free DNA in patients with lung cancer is primarily originated from the tumor tissue. LOH of plasma free DNA may be valuable molecular markers in the diagnosis of SCLC.