Total Plasma DNA Research Articles

Quantification of circulating cell‐free DNA in the plasma of cancer patients during radiation therapy

Cell-free plasma DNA is elevated in cancer patients and decreases in response to effective treatments. Consequently, these nucleic acids have potential as new tumor markers. In our current study, we investigated whether the plasma DNA concentrations in patients with cancer are altered during the course of radiation therapy. To first determine the origin of cell-free plasma DNA, plasma samples from mice bearing transplanted human tumors were analyzed for human-specific and mouse-specific cell-free DNA. Human-specific DNA was detectable only in plasma from tumor-bearing mice. However, mouse-specific plasma DNA was significantly higher in tumor-bearing mice than in normal mice, suggesting that cell-free plasma DNA originated from both tumor and normal cells. We measured the total cell-free plasma DNA levels by quantitative polymerase chain reaction in 15 cancer patients undergoing radiation therapy and compared these values with healthy control subjects. The cancer patients showed higher pretreatment plasma DNA concentrations than the healthy controls. Eleven of these patients showed a transient increase of up to eightfold in their cell-free plasma DNA concentrations during the first or second week of radiation therapy, followed by decreasing concentrations toward the end of treatment. In two other cancer patients, the cell-free plasma DNA concentrations only decreased over the course of the treatment. The total cell-free plasma DNA levels in cancer patients thus show dynamic changes associated with the progression of radiation therapy. Additional prospective studies will be required to elucidate the potential clinical utility and biological implications of dynamic changes in cell-free plasma DNA during radiation therapy.

Phase II study of docetaxel, carboplatin and trastuzumab (THC) in patients with locally advanced breast cancer

1062 Background: Preclinical and clinical data suggest that docetaxel, trastuzumab and platinum compounds act synergistically. Studies in the metastatic disease are conflicting. In this phase II study the activity of this regimen was assessed and correlated with changes in circulating biomarkers. Methods: Patients with locally advanced, FISH+ HER2 amplified, breast cancer were treated in this protocol with TCH as initial neo-adjuvant treatment. Patients received Docetaxel 75 mg/sqm, carboplatinum AUC 6mg/mL.min and trastuzumab 6 mg/kg (with a loading dose in cycle 1). Six cycles were intended provided no progression occured. No prophylactic G-CSF or antibiotics were administered. Measurement of circulating ECD HER2, CTC (RT-PCR mammaglobin and CK-19), and total plasma DNA was performed prior to the start of therapy, after cycle 1 and after 3 and 6 cycles. A cardiac MUGA exam was performed every three cycles. All patients were intented to undego mastectomy and axillary clearance after the end of chemotherapy. Results: 40 patients have been treated with a median age of 56 year. No patient suffered from progression of disease while on THC. All patients received 6 cycles, 4 patients received respectively 7, 7, 8 and 9 cycles. In total 247 cycles were administered. No cases of CHF were observed. 5 patients suffered from a asymptomatic decline > 10% in LVEF. A clinical CR and PR was obtained in 32 (80%). A pathological CR both in breast axilla was obtained in 16/39 (41%). Tumor response was predicted by changes in both CTC and ECD HER-2. Conclusions: THC was confirmed to be a very active and safe regimen in patients with HER-2 amplified breast cancer. Changes in biomarkers rapidly predicted response. No significant financial relationships to disclose.

Phase II study of Docetaxel, Carboplatin, and Trastuzumab (THC) as first-line treatment in patients with HER-2 amplified advanced breast cancer. Changes in circulating tumor cells (CTC), total plasma DNA and circulating HER-2 ECD

Biodegradable cationic polymers have become promising alternatives to traditional polycationic gene delivery systems in which the high charge densities of high molecular weight polymers contribute significantly to cellular toxicities. Previous research has shown that biodegradable, multiblock copolymers (MBC), PEG-PLL-g-16% His, are efficient gene carriers with negligible cellular toxicities. The present research was designed to characterize the polymer degradation as well as to determine the biodistribution of the MBC after systemic administration. Polymer degradation was performed in buffer as a function of pH, in serum and within polymer/pDNA complexes. The MBC exhibited exponential decay with a half-life (t1/2) of ∼14 min at pH 9.0, ∼5 h at pH 7.4 and ∼2 h in serum. However, there was little or no degradation observed at pH 4.0 and the MBC within the complexes degraded between 4 and 8 h in serum. Biodistribution data performed with fluorescently labeled polymer and pDNA revealed that intact complexes remained in the blood up to 3 days, which was also reflected in the organs as a function of time. Therefore, the cumulative data suggest that PEG may be sterically stabilizing complexes in vivo via dysopsonization in which serum proteins mask the complexes from elements of the reticuloendothelial system (RES).

Methylated Tumor-Specific DNA as a Plasma Biomarker in Patients with Glioma

Objective: Patients with systemic malignancies have substantial quantities of tumor-specific DNA in their plasma which may serve as a potential biomarker for tumor burden. This approach has not been studied in gliomas. Methods: Methylation specific polymerase chain reaction (MSP) was used to determine the methylation status the promoters for p16/INK4a, MGMT, p73, and RARβ within glioma tissue and plasma. Blood was collected prior to craniotomy in 10 patients with glioma. DNA was extracted from tumor and plasma samples and assayed with MSP. Total plasma DNA also was quantified. Tumor-specific plasma DNA was defined as identification of the same methylated promoter (MP) in both tumor and plasma. Results: Total plasma DNA concentration was markedly elevated (mean 6,503 ng/ml, SEM 1,400 ng/ml). Glioma tissue contained methylation of at least one promoter in 9 out of 10 (90 percent) of patients studied. Of these patients, 6 out of 9 (67 percent) demonstrated methylation of at least one of the same promoters in plasma. Five of these had one MP identified in the plasma and one had 2 MP. Overall, glioma-specific plasma DNA was present in plasma of 6 out of 10 (60 percent) of patients. Each MP DNA marker found in the plasma also was present in the intracranial tumor. Conclusions: Patients with high grade gliomas have large amounts of DNA in the plasma. Of these primary brain tumors, 90 percent contained methylated gene promoters, and in over 60 percent of these patients the same methylated promoters present in the tumor also were found in the plasma. This represents the first step to developing a quantitative plasma biomarker that could be used to monitor glioma status.

Non-invasive fetal sex determination using real-time PCR

Objective. The aim of this study was to evaluate the specificity and sensitivity of the real-time quantitative PCR method for fetal gender determination in early pregnancy.Methods. Blood samples were collected from 46 pregnant women prior to amniocentesis. DNA was extracted from maternal plasma using a QIAmp DNA Blood Mini Kit. DNA samples were subjected to real-time quantitative PCR amplification of SRY (as a fetus-specific marker) and β-globin (as a marker for total plasma DNA) genes.Results. The β-globin gene sequence was detected in all samples. The SRY gene was detected in 25 of 28 plasma samples from women with male fetuses and in none of the 18 samples from women with female fetuses (sensitivity 89.2% and specificity 100%). The fetal gender was correctly determined in 43 (93.5%) of 46 maternal plasma samples. The concentration of the β-globin gene ranged from 161 to 25 568 genome-equivalents (GE)/mL (median 1051.1), while the concentration of the SRY gene ranged from 5 to 166 GE/mL (median 27.4). The percentage of free fetal DNA ranged from 0.1% to 46.1% (median 2.0%).Conclusion. Amplification of fetal DNA from maternal plasma by real-time quantitative PCR is a promising method for fetal sex determination in early pregnancy. However, further studies are necessary before this procedure can be included into a clinical routine.

Investigation of the Genomic Representation of Plasma DNA in Pregnant Women by Comparative Genomic Hybridization Analysis: A Feasibility Study

Prenatal diagnosis is part of the established obstetric care in most developed countries. However, current methods for obtaining fetal tissues for prenatal diagnosis, including chorionic villus sampling and amniocentesis, are invasive and carry a risk of fetal loss. Analysis of circulating cell-free DNA from pregnant women provides new possibilities for noninvasive prenatal diagnosis. Clinical applications of plasma fetal DNA analysis in maternal plasma include fetal RhD genotyping (1)(2), fetal aneuploidy detection (3)(4), and the prenatal diagnosis of several genetic diseases, including myotonic dystrophy (5), congenital adrenal hyperplasia (6), and β-thalassemia (7)(8). In addition to exploring the clinical applications of circulating DNA analysis, our group has investigated the molecular characteristics of cell-free DNA in maternal plasma and has shown that plasma DNA fragments in pregnant women are longer than those in nonpregnant women (9). More importantly, we have shown that the fragment size of fetal DNA in maternal circulation is shorter than that of the maternally derived DNA (9). This observation has allowed the enrichment of fetal DNA from maternal plasma (10). In this study, we used comparative genomic hybridization (CGH), a technique that can provide gene dosage information for the entire genome, to investigate the genomic representation of plasma DNA in pregnant women. The generation of this information is important because any potential unequal representation of any particular region would raise fundamental questions regarding the underlying mechanisms of DNA release into and degradation from the plasma. Diagnostically, a variety of genes [e.g., β-globin (11)(12) and glyceraldehyde-3-phosphate dehydrogenase (13)] have been chosen for the quantitative analysis of total plasma DNA. If unequal representation is observed, additional care must be exercised in the design and performance of DNA analyses and interpretation of quantitative data obtained from plasma. Healthy pregnant women without pregnancy-associated complications, …

Detection for fetal DNA in maternal plasma by fluorescence quantitative polymerase chain reaction

目的 探讨荧光定量PCR(fluorescence quantitative PCR,FQ-PCR)方法检测孕妇血浆中胎儿DNA的可行性,探讨胎儿DNA在孕妇血浆中的含量及其在孕期的变化规律.方法以胎儿SRY基因序列作为胎儿DNA在孕妇血浆中的标志,应用荧光MGB(Minor Groove Binder)探针实时定量PCR方法连续测定30例孕妇在不同孕期和产后共237份血浆标本中胎儿DNA的含量.结果使用该方法最低可检测到20 000个女性DNA中的一个男性DNA.孕妇血浆中最早检出SRY序列的时间为孕6+6周.孕8周起,所有怀男胎孕妇的标本中均可检出SRY序列,其含量随着妊娠的进展而增高,在晚期妊娠达高峰,产后24～48 h血浆中SRY序列检测均为阴性.胎儿DNA在孕妇血浆总DNA含量中的相对浓度分别为孕早期4.88%、孕中期6.10%、孕晚期4.77%.全部实验无假阳性和假阴性出现.结论FQ-PCR方法是一种灵敏度和准确性高,特异性强的定量检测孕妇血浆中胎儿游离DNA的方法,孕妇血浆中存在高浓度的胎儿DNA,可用于无创伤性产前基因诊断。

Geranylgeraniol and β-ionone inhibit hepatic preneoplastic lesions, cell proliferation, total plasma cholesterol and DNA damage during the initial phases of hepatocarcinogenesis, but only the former inhibits NF-κB activation

Chemopreventive activities of the isoprenoids geranylgeraniol (GGO) and beta-ionone (BI) were evaluated during initial phases of hepatocarcinogenesis. Rats received 8 or 16 mg/100 g body wt GGO (GGO8 and GGO16 groups) or BI (BI8 and BI16 groups), or only corn oil (CO group, controls) daily for 7 weeks. Incidence (%) and the mean number of visible hepatocyte nodules/animal were inhibited in the GGO8 (64% and 21 +/- 40), GGO16 (33% and 3 +/- 5), BI8 (50% and 13 +/- 34) and BI16 (42% and 9 +/- 19) groups compared with the CO group (100% and 34 +/- 51) (P < 0.05, except for the GGO8 group). Number/cm(2) liver section, mean area (mm(2)) and % liver section area occupied by persistent hepatic placental glutathione S-transferase positive preneoplastic lesions (PNL) were reduced in the GGO8 (11 +/- 9; 0.26 +/- 0.35; 2.7 +/- 3.0), GGO16 (6 +/- 6; 0.18 +/- 0.16; 0.9 +/- 0.9), BI8 (9 +/- 5; 0.13 +/- 0.20; 1.1 +/- 1.2) and BI16 (8 +/- 6; 0.08 +/- 0.09; 0.6 +/- 0.4) groups compared with the CO group (26 +/- 18; 0.29 +/- 0.34; 7.0 +/- 5.5) (P < 0.05). GGO16 and BI16 groups showed smaller visible hepatocyte nodules, reduced PNL cell proliferation and total plasma cholesterol levels compared with the CO group (P < 0.05), but did not show any differences (P > 0.05) in PNL apoptosis. DNA damage expressed as comet length (microm) was reduced in the GGO8 (96.7 +/- 1.5), GGO16 (94.2 +/- 1.5), BI8 (97.1 +/- 1.1) and BI16 (95.1 +/- 1.5) groups compared with the CO group (102.1 +/- 1.7) (P < 0.05). In comparison with normal animals, the CO group animals showed increased (P < 0.05) nuclear levels of nuclear factor kappa B (NF-kappaB) p65 subunit in hepatic cells, which were decreased (P < 0.05) in the GGO16 group animals. Anticarcinogenic actions of these isoprenoids seem to follow a dose-response relationship. Results indicate that GGO and BI could be represented as promising chemopreventive agents against hepatocarcinogenesis. Inhibition of cell proliferation and DNA damage seems to be important for the anticarcinogenic actions of isoprenoids, while the inhibition of NF-kappaB activation seems to be specifically related to GGO actions.

Apoptosis as an early event in the development of multiple organ failure?

We have recently developed a simple method of plasma free DNA detection, which enables us to distinguish between apoptotic and genomic (necrotic) DNA. After applying this method to the critically ill, we revealed apoptotic DNA on the day of admission to be higher than later when multiple-organ failure developed. Moreover, apoptotic DNA contributed to total plasma DNA much more than DNA from necrotic cells and its increase predicted future development of multiple-organ failure and death.

Improved prenatal detection of a fetal point mutation for achondroplasia by the use of size‐fractionated circulatory DNA in maternal plasma—case report

The efficacious analysis of fetal loci involving point mutations from circulatory fetal DNA in maternal plasma is hindered by the preponderance of maternal DNA. It has recently been shown that the size difference between fetal and maternal DNA species can be used for the selective enrichment of circulatory fetal DNA in maternal plasma. We have now tested this approach for the detection of a fetal point mutation in the fibroblast growth factor receptor 3 (FGFR3) gene that causes achondroplasia. Circulatory DNA was extracted from maternal plasma and size-fractionated by agarose gel electrophoresis. The fraction with a size less than 300 bp was examined by a touchdown PCR assay specific for the FGFR3 gene, and the mutation was identified by SfcI restriction analysis. Our analysis indicated that although the fetal mutation was discernible in the analysis of total plasma DNA, the result using size-fractionated DNA was much more evident. The enrichment of circulatory fetal DNA in maternal plasma by size-fractionation facilitates the detection of subtle feto-maternal genetic differences, such as those involving point mutations. This approach can easily be extended for the non-invasive prenatal determination of other fetal loci.

Quantitative Analysis of Fetal DNA in Maternal Plasma and Serum: Implications for Noninvasive Prenatal Diagnosis

We have developed a real-time quantitative PCR assay to measure the concentration of fetal DNA in maternal plasma and serum. Our results show that fetal DNA is present in high concentrations in maternal plasma, reaching a mean of 25.4 genome equivalents/ml (range 3.3-69. 4) in early pregnancy and 292.2 genome equivalents/ml (range 76. 9-769) in late pregnancy. These concentrations correspond to 3.4% (range 0.39%-11.9%) and 6.2% (range 2.33%-11.4%) of the total plasma DNA in early and late pregnancy, respectively. Sequential follow-up study of women who conceived by in vitro fertilization shows that fetal DNA can be detected in maternal serum as early as the 7th wk of gestation and that it then increases in concentration as pregnancy progresses. These data suggest that fetal DNA can be readily detected in maternal plasma and serum and may be a valuable source of material for noninvasive prenatal diagnosis.

Delayed clearance of HBV-DNA detected by PCR in the absence of viral replication.

The polymerase chain reaction (PCR) was used to study the in vivo persistence and clearance of HBV-DNA in the serum of a monkey (Macaca mulatta) known to be naturally resistant to the HBV infection. Total infectious plasma, virus pellet, and viral DNA were inoculated into 3 different monkeys. Degradation of infectious particles and clearance of HBV-DNA were analyzed by detecting HBV-DNA sequences in serial dilutions of serum by dot blot hybridization and PCR. Semi-quantitation was carried out by comparison with minimal positive dilutions of known HBV-DNA. The presence of hepatitis B surface antigen (HB-sAg) and hepatitis B e antigen (HBeAg) in serum was also investigated. PCR assays were found positive in serial samples of the 3 monkeys, and passively transmitted viral DNA remained detectable for 3 months after inoculation. HBsAg and HBeAg were detectable for 3 weeks and 2 weeks, respectively, in a monkey inoculated with total infectious plasma. Semi-quantitation of HBV viremia showed that the amount of virus detected 1 day after injection was markedly decreased and persisted at a low level, showing a rapid and important sequestration of viral particles. Since replication can be excluded in these monkeys, the data show that HBV-DNA sequences can remain detectable at a low level in the serum for long periods. It is thus conceivable that PCR may also detect the HBV genome, eventually degraded in vivo, and a weakly positive result does not always mean ongoing viral replication.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlates of plasma cortisol and DNA repair in human peripheral lymphocytes: suppression of repair in women taking estrogen.

We have examined the relationship between total plasma cortisol concentration and DNA repair capacity in human peripheral lymphocytes cultured in vitro; the data indicate that high concentrations of cortisol (> 20 micrograms/dl) inhibit DNA repair. The inhibitory effect can be abrogated by the addition of RU38486, a cortisol antagonist. In addition, we compared plasma cortisol concentration and in vitro DNA repair capacity in 52 healthy individuals. Females on therapeutic estrogen (oral contraceptive or estrogen replacement therapy) had significantly elevated plasma cortisol and suppression of DNA repair capacity.