Plasma DNA Analysis Research Articles

Quantitative and qualitative characterization of plasma DNA identifies primary and recurrent colorectal cancer

Because plasma DNA may be a useful tool for cancer detection, we screened primary tumors and related multiple plasma samples at the time of surgery and during the follow-up period for plasma DNA level as well as for K-Ras mutations and p16INK4a promoter hypermethylation in colorectal cancer patients. At the time of surgery, DNA levels were higher in tumor patients than in healthy donors, and K-Ras and p16INK4a alterations were detected in 7 and 11 cancers respectively, and in all related plasma samples. During the follow-up, plasma DNA levels decrease progressively but rapidly increased when a relapse occurred, whereas K-Ras and p16INK4a alterations were detected only in relapsed patients. Therefore, combined quantitative and qualitative analyses of plasma DNA confirm the presence of colorectal cancer, define disease-free status and indicate the presence of relapse.

Application of maternal plasma DNA analysis for noninvasive prenatal diagnosis of Hb E-β-thalassemia

To establish simple noninvasive prenatal diagnosis of common beta-thalassemia in Southeast Asia, we have evaluated the possibility of identifying the 3 most common beta-thalassemia genes [beta(E), beta(17A-T), and beta(41/42(-CTCC))] by analysis of fetal DNA in maternal plasma using combined conventional polymerase chain reaction (PCR) and real-time quantitative PCR. Maternal plasma was obtained from peripheral blood of Thai pregnant women collected during the first and second trimesters of gestation. DNA was prepared from 200 microL plasma using a QIAmp Blood Mini Kit. Identifications of the beta(E), beta(41/42(-CTTT)), and beta(17A-T) in plasma DNA were carried out using semi-nested (for beta(E)) and nested (for beta(41/42) and beta(17)) real-time allele-specific PCR methodologies, and the results were compared with those obtained on fetal tissue analysis with routine invasive procedure. Twenty-six fetal beta(E) genes were correctly identified by maternal plasma DNA analysis of 39 pregnant women investigated. The fetal beta(41/42) and beta(17) mutations were detectable in 6 of 12 and 4 of 9 maternal plasma specimens, respectively, which were in concordance with the results obtained by routine invasive procedure. The noninvasive prenatal diagnostic methods developed should potentially prove useful for detection of paternally inherited mutation and for providing the exclusion of pregnancies at risk for this common genetic disorder in the region.

Mass Spectrometric Detection of an SNP Panel as an Internal Positive Control for Fetal DNA Analysis in Maternal Plasma

Applications of fetal DNA detection in maternal plasma have been reported for the prenatal assessment of fetal RhD status, sex-linked disorders, and β-thalassemia. Because fetal DNA constitutes only 3% to 6% of the total DNA in maternal plasma (1), fetal sequences may occasionally go undetected because of low fetal DNA concentrations or fetal DNA loss during sample processing. Such false-negative results may lead to misinterpretation of the fetal genotype and consequently, false diagnoses. Thus the incorporation of analytical controls to confirm the presence of fetal DNA is recommended. Other investigators have developed a panel of insertion-deletion polymorphisms to serve this purpose(2). Single nucleotide polymorphisms (SNPs), however, are the most abundant class of polymorphisms in the human genome. We have recently developed a mass spectrometry based protocol, the s ingle a llele b ase e xtension r eaction (SABER), that allows the sensitive and specific detection of fetal SNPs in maternal plasma(3). We applied SABER to develop an SNP panel to serve as an …

Noninvasive Prenatal Detection of Fetal Trisomy 18 by Epigenetic Allelic Ratio Analysis in Maternal Plasma: Theoretical and Empirical Considerations

The discovery of cell-free fetal DNA in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis. However, the use of maternal plasma fetal DNA for the direct detection of fetal chromosomal aneuploidies has not been reported. We postulate that the aneuploidy status of a fetus could be revealed by an epigenetic allelic ratio approach, i.e., by analyzing the allelic ratio of a single-base variation present within DNA molecules exhibiting a placental-specific epigenetic signature in maternal plasma. Placental-derived fetal-specific unmethylated maspin (SERPINB5) promoter sequences on human chromosome 18 were detectable in placental-maternal DNA mixtures and in maternal plasma by bisulfite modification followed by methylation-specific PCR (MSP) and primer extension. The ratios between the extension products of the 2 alleles were calculated for heterozygous placentas, placental-maternal blood cell DNA mixtures, and maternal plasma samples. The allelic ratios were compared between pregnancies carrying trisomy 18 and euploid fetuses. The epigenetic allelic ratios of all tested trisomy 18 samples deviated from the reference range obtained from euploid samples (placental DNA, 1.135 to 2.052; placental-maternal DNA mixtures, 1.170 to 1.985; maternal plasma, 0.330 to 3.044; without skew correction on the raw mass spectrometric data). A theoretical model was established and validated that predicted that a minimum of 200 copies of genomic DNA after bisulfite conversion were required for distinguishing euploid and aneuploid fetuses with confidence. Epigenetic allelic ratio analysis of maternal plasma DNA represents a promising approach for noninvasive prenatal diagnosis of fetal chromosomal aneuploidies.

Quantitative Analysis of Plasma DNA in Colorectal Cancer Patients

Extracellular DNA in the plasma or serum of cancer patients has been recently proposed as a source of analyzable cancer-related gene sequences (qualitative approach). Furthermore, patients with different tumor types show high levels of cell-free circulating DNA both in plasma and serum (quantitative approach) at the time of surgery. Our aim was to verify whether the level of cell-free DNA in plasma might help in detecting recurrences during follow-up of colorectal cancer (CRC) patients. We studied 70 patients undergoing surgery for primary CRC. Plasma samples were obtained at the time of surgery and during follow-up. The cell-free circulating DNA in plasma was quantified by the Dipstick Kit method. At the time of surgery, in all patients, cell-free DNA levels in plasma were about 25 times higher in comparison with 20 healthy donors. In contrast, the carcinoembryonic antigen (CEA) value of this cohort of patients was altered in only about 37% of cases. During follow-up, cell-free DNA levels decreased progressively in tumor-free patients, while it increased in those developing recurrences or metastases. The results were further supported by qualitative analysis of circulating tumor-specific DNA, such as K-Ras mutations and p16(INK4a) promoter hypermethylation. These preliminary data confirm that plasma tumor DNA levels (i) are significantly higher in patients with CRC, (ii) decrease progressively in the follow-up period in tumor-free patients, and (iii) increase in patients with recurrence or metastasis. We suggest, therefore, that the quantification of plasma cell-free DNA might represent a useful tool for monitoring of CRC and, prospectively, for identifying high-risk individuals.

P-921: Prevention of rhesus alloimmunization: Determination of paternal rhesus D zygosity by plasma DNA analysis prior to preimplantation genetic diagnosis (PGD)

P-921: Prevention of rhesus alloimmunization: Determination of paternal rhesus D zygosity by plasma DNA analysis prior to preimplantation genetic diagnosis (PGD)

Recent developments in fetal nucleic acids in maternal plasma: implications to noninvasive prenatal fetal blood group genotyping

The discovery of circulating cell-free fetal DNA in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis. Fetal DNA in maternal plasma has been used for the noninvasive prenatal determination of the RhD status of fetuses carried by RhD-negative pregnant women. In such analysis, the possible need of an internal control for the presence of detectable amounts of fetal DNA in a particular maternal plasma sample has been actively discussed. Recently, the development of a robust method for discriminating single nucleotide differences in plasma DNA using single allele base extension reaction (SABER) followed by matrix-assisted laser-desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) has opened up the possibilities of using a panel of single nucleotide polymorphisms as such a positive control. A second approach is the recent successful development of fetal epigenetic markers which can be developed into universal fetal DNA markers. These developments hold promise to allow the eventual widespread utilization of maternal plasma DNA analysis for the noninvasive prenatal diagnosis of blood group mismatches between the mother and fetus.

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, …

Plasma DNA as a prognostic marker for stroke patients with negative neuroimaging within the first 24 h of symptom onset

Modern neuroimaging safely and reliably diagnoses stroke and provides information for outcome prediction. However, some patients with clinical stroke have no detectable abnormality on neuroimaging and other patients are not fit for such investigations. Therefore, we evaluated the potential of plasma DNA and serum S100 protein concentrations to predict post-stroke mortality and morbidity in patients with negative neuroimaging results. Patients with stroke-like symptoms but negative neuroimaging results were recruited. Both plasma and serum were collected from each patient for plasma DNA and serum S100 analysis. The primary outcome measures were 6-month mortality and morbidity using the post-stroke modified Rankin score (mRS). Forty-four patients were recruited to the study. Seventeen (39%) patients were classified as post-stroke mRS grades 3-6. The median plasma DNA concentration of this group of patients was significantly higher than that of patients with post-stroke mRS grades 0-2. Median serum S100 protein concentrations did not show significant differences between the two groups. Plasma DNA concentrations > 800 kilogenome-equivalent/l have a sensitivity of 42% and a specificity of 100% for predicting 6-month post-stroke mRS (grades 0-2), with an area under the receiver operator characteristic (ROC) curve of 0.742. By comparison, serum S100 protein concentrations > 0.09 microg/l have a sensitivity of 48% and specificity of 75% for predicting 6-month post-stroke mRS (grades 0-2), and the area under the curve is 0.542. Plasma DNA concentration predicts post-stroke morbidity and mortality in patients with negative neuroimaging, and may be more effective than S100 protein measurement.

231 POSTER Quantitative and qualitative analyses of plasma DNA in colorectal cancer patients as prognostic tools

231 POSTER Quantitative and qualitative analyses of plasma DNA in colorectal cancer patients as prognostic tools

Effects of Preanalytical Factors on the Molecular Size of Cell-Free DNA in Blood

Recently, plasma DNA analysis has been increasingly explored for different clinical diagnostic purposes. Previous studies have shown that the concentration of plasma DNA is increased in patients with neoplastic diseases (1), pregnancy-related complications (2)(3), trauma(4), and certain autoimmune diseases (5). In addition to quantitative analysis, qualitative changes in plasma DNA have also been investigated in different physiologic and pathologic conditions. In this regard, our group has shown that fetal DNA is shorter than the maternal counterpart in the plasma of pregnant women (6), and this has allowed the enrichment of fetal DNA from maternal plasma by size fractionation (7). It has also been shown that plasma DNA fragments are longer in patients suffering from a variety of neoplastic diseases (8). In addition, circulating Epstein–Barr virus DNA molecules in patients with nasopharyngeal carcinoma have been found to consist mainly of short DNA fragments <180 bp in size (9). Because the samples collected from patients and healthy controls in these studies were usually collected at different time points and under different conditions, it is important to ensure that the observed variations in the sizes of plasma DNA are the result of biological changes rather than artifacts attributable to differences in sample handling. Therefore, in this study, we investigated the effects of several preanalytical factors, including clotting, delayed separation of blood cells from plasma, freezing–thawing, and storage, on the integrity of circulating cell-free DNA. We recruited 27 healthy volunteers for modules 1 to 4 of the study. Venous blood (30 mL) was collected from each volunteer into eight tubes containing EDTA and one plain tube. The samples serving as references were processed immediately after collection. Plasma was separated from the blood cells by centrifugation at 1600 g for 10 min, and the plasma was then microcentrifuged at 16 000 g …

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.

Recent developments in fetal DNA in maternal plasma.

It has been several years since the presence of fetal DNA in maternal plasma was first discovered. Numerous potential applications have been reported to date. However, the study of maternal plasma for the prenatal diagnosis of fetal genetic traits has largely centered on paternally inherited autosomal dominant traits. Recently, a number of approaches have been developed that allow the prenatal assessment of autosomal recessive conditions through fetal DNA analysis in maternal plasma. On the other hand, aberrations in fetal DNA concentrations in maternal plasma have been reported in a number of pregnancy-associated conditions. Recent studies have begun to address the possible underlying mechanisms causing such quantitative aberrations. Current studies on circulating fetal DNA have focused on the detection of the paternally inherited fetal-specific alleles in maternal plasma. However, it has been demonstrated that the development of fetal epigenetic markers has great potential to expand the field.

Detection of p53 mutations in the plasma DNA of patients with ovarian cancer

Mutation of p53 is one of the most common genetic abnormalities detected in up to 81% of cases of ovarian cancer. To evaluate the use of plasma DNA analysis as a method for somatic mutation screening, we measured the presence of p53 mutations in DNA isolated from plasma and cancer tissue from patients with ovarian cancer. We analyzed the plasma DNA for the presence of p53 mutations (exons 5–8). Of 27 cases of ovarian cancer, 12 cases (44%) had mutations of p53 in cancer tissue. In two of the 12 cases (16.7%), identical mutations were detected in DNA of their preoperative plasma. In our follow-up of the two patients with p53 mutations in their plasma, mutant DNA was undetectable in their plasma after surgery. In one case, the p53 mutation re-surfaced in their plasma 16 months after surgery, and the patient died 2 months later. We have shown that tumor-derived DNA can be detected in the plasma of some patients with ovarian cancer, particularly in those with more advanced stage.

Use of magnetic beads for plasma cell-free DNA extraction: toward automation of plasma DNA analysis for molecular diagnostics.

Urine, breast milk, plasma, and serum have been shown to contain cell-free DNA (1)(2)(3)(4)(5)(6)(7). For plasma DNA detection, several recent studies addressed the need for careful evaluation and standardization of preanalytical processes (8)(9)(10)(11)(12). Key problems appear, such as possible contamination of plasma by white blood cells; the generally low and variable amount of circulating DNA, making extraction/quantification difficult and time-consuming; poor DNA quality; and the presence of PCR inhibitors. In any case, automation of DNA extraction, which is a prerequisite for introduction of these diagnostic approaches in clinical laboratories, is difficult to achieve because of the volumes of plasma necessary to get sufficient DNA. In this study (summarized in Fig. 1⇓ ), we propose a new semiautomated, time-saving process for extraction of plasma cell-free DNA that provides high yields and is suitable for PCR amplification. Two 5-mL blood samples from each of 23 patients being monitored for lung (n = 19) or colon cancer (n = 4) and 20 healthy controls were collected in EDTA-containing tubes. Informed consent was obtained for each. Blood was centrifuged at 800 g for 10 min, and the collected plasma was transferred to a 15-mL BD FalconTM polypropylene tube for an additional centrifugation step of 10 min at 1500 g to remove any remaining leukocytes and platelets. An equal volume of 2× concentrated proteolytic buffer (2×: 20 mmol/L Tris-HCl, 50 mmol/L EDTA, 200 mmol/L NaCl, 10 g/L sodium dodecyl sulfate, and 400 mg/L proteinase K) was added to the plasma, mixed, and incubated 1 h at 37 °C. After digestion, plasma samples were concentrated by centrifugation at room temperature (to avoid sodium dodecyl sulfate precipitation) for 20 min at 2600 g in Amicon …

Fetal expressed gene analysis in maternal blood: a new tool for noninvasive study of the fetus.

Noninvasive approaches to prenatal diagnosis can avoid the risk of fetal loss associated with invasive procedures such as chorionic villus sampling, amniocentesis, and cordocentesis. Isolation of fetal cells from maternal blood requires further improvements before it can be applied in a clinical setting (1), but the reliability of cell-free fetal DNA analysis in maternal plasma or serum is now well established (2)(3)(4). As a result, it is currently used in specialized centers for the determination of fetal sex and fetal RhD status for the management of pregnant women at risk for X-linked disorders (5) or RhD alloimmunization (6). Because fetal DNA in maternal serum is circulating in an excess background of maternal DNA, clinical applications are restricted mainly to the detection of fetal sequences distinct from the mother’s DNA sequences. Fetal RNA in maternal blood may be an alternative source of fetal nucleic acids. Al-Mufti et al. (7) detected specific RhD mRNA in mononuclear fetal cells isolated from blood of RhD-negative pregnant women, and Lo’s group demonstrated the presence of Y-chromosome-specific ( ZFY ) mRNA in maternal plasma of women carrying a male fetus (8). These two applications, however, again require fetal sequences that differ from the mother’s. We have investigated the presence in maternal blood of fetal transcripts that may have the same sequence as that of the mother. Human chorionic gonadotropin (hCG) mRNA is a good candidate because it is a pregnancy-specific polypeptide hormone produced by the placenta and is specifically expressed in the fetal syncytiotrophoblast. We studied 43 pregnant women and 20 nonpregnant women who had previously given birth to at least one neonate. After receiving informed consent, we collected blood (2.5 mL) into PAXgeneTM tubes to reduce RNA degradation (9). All pregnancy samples were obtained before …

Plasma DNA microsatellite panel as sensitive and tumor-specific marker in lung cancer patients.

The majority of lung cancer patients have tumor-derived genetic alterations in circulating plasma DNA that could be exploited as a diagnostic tool. We used fluorescent microsatellite analysis to detect alterations in plasma and tumor DNA in 34 patients who underwent bronchoscopy for lung cancer, including 11 small cell lung cancer (SCLC) and 23 nonsmall cell lung cancer (NSCLC) (12 adenocarcinomas, 11 squamous cell carcinomas) and 20 controls. Allelotyping was performed with a selected panel of 12 microsatellites from 9 chromosomal regions 3p21, 3p24, 5q, 9p, 9q, 13q, 17p, 17q and 20q. Plasma DNA allelic imbalance (AI) was found in 88% (30 of 34 patients), with a similar sensitivity in SCLC and NSCLC. In the 24 paired available tumor tissues, 83% (20 of 24) presented at least 1 AI. Among these patients, 85% (17 of 20) presented also at least 1 AI in paired plasma DNA, but the location of the allelic alterations in paired plasma and tumor DNA could differ, suggesting the presence of heterogeneous tumor clones. None of the 20 controls displayed plasma or bronchial DNA alteration. A reduced panel of six markers (at 3p, 5q, 9p, 9q) showed a sensitivity of 85%. Moreover, a different panel of microsatellites at 3p and 17p13 in SCLC and at 5q, 9p, 9q and 20q in NSCLC patients could be specifically used. Analysis of plasma DNA using this targeted panel could be a valuable noninvasive test and a useful tool to monitor disease progression without assessing the tumor.

Non-invasive prenatal diagnosis: on the horizon?

The launch of the genomics and postgenomics era has greatly expanded our understanding of the genetic basis of many diseases. In conjunction with the sociocultural trend to delay childbirth and to maintain smaller family units, extra demand may be placed on the existing prenatal diagnostic services. The inherent risk of fetal loss associated with current prenatal diagnostic procedures, such as amniocentesis and chorionic villus sampling, has spurred research into non-invasive prenatal diagnosis. Much research has been conducted on the exploitation of fetal genetic material present in the maternal circulation. The initial focus was on the isolation of intact fetal cells and subsequently, the existence of extracellular fetal DNA in maternal plasma was realized. Exciting developments have been achieved in recent years. A large-scale trial to evaluate the clinical utility of fetal cell isolation from maternal blood for fetal aneuploidy diagnosis was launched and data were recently published. Much has taken place in the research of fetal DNA analysis in maternal plasma and in one example, namely prenatal RhD determination, this type of analysis has been used in the clinical setting. This paper reviews the technological developments in non-invasive prenatal diagnosis.

Allelic imbalance and microsatellite instability in plasma DNA released from polyclonal pancreatic adenocarcinoma.

Evidence of circulating soluble DNA in blood-stream of cancer patients has emerged. Because the plasma DNA is largely derived from cancer cells, genetic analysis of plasma DNA is important to understand the molecular events occurred in cancer patient. Seven microsatellite markers in the soluble plasma DNA from patients with pancreatic adenocarcinoma and other pancreato-biliary malignant tumors were examined for microsatellite instability (MSI) and allelic imbalance (AI). A variety of genetic alterations including MSI and AI were detected in the plasma DNA. Some alterations were detected before recurrence of the tumor was verified. Analysis of five primary pancreatic adenocarcinomas by microdissection revealed that the heterogeneous nature of pancreatic tumors is associated with both MSI and AI in the same tumor. The presence of altered plasma DNA including MSI and/or AI from the same pancreatic cancer patient may be important evidence for the presence of these alterations in heterogeneous primary tumors. Analysis of plasma DNA could become one of the diagnostic or therapeutic measures for this type of pancreatic adenocarcinoma.

Effects of Blood-Processing Protocols on Fetal and Total DNA Quantification in Maternal Plasma

Recently, apoptotic cells have been found in plasma obtained by centrifugation of blood from pregnant women, raising the question of what constitutes plasma and whether plasma is truly cell free. We compared the effects of different blood-processing protocols on the quantification, DNA composition, and day-to-day fluctuation of fetal and total DNA in maternal plasma. Blood samples were collected from healthy pregnant women. The blood sample from each individual was simultaneously processed by different means, including the following: Percoll separation, centrifugation, microcentrifugation, and filtration. The resulting plasma aliquots were subjected to real-time quantitative amplification of the beta-globin (for total DNA) and SRY (for fetal DNA) genes. The differences in the beta-globin and SRY DNA concentrations and the degree of variation between the various plasma aliquots were assessed statistically. Different protocols of blood processing significantly affected the quantification and the day-to-day fluctuation of total (P <0.001), but not fetal (quantification, P = 0.336; fluctuation, P = 0.206), DNA in maternal plasma. The quantitative difference could be attributed to the fact that efficacies of different protocols for generating cell-free plasma vary. Processing blood samples by centrifugation followed by filtration or microcentrifugation is effective in producing cell-free plasma. Standardization in plasma-processing protocols is needed for maternal plasma DNA analysis, especially for quantification of total DNA in maternal plasma. Such preanalytic factors may also affect other applications of plasma DNA analysis.