Methylation of the imprinted GNAS1 gene in cell-free plasma DNA : equal steady-state quantities of methylated and unmethylated DNA in plasma

Background Genomic DNA sequences in cell-free plasma are biomarkers of cancer prognosis, where characteristic changes in methylation of tumour suppressor or oncogene DNA regions are indicative of changes in gene activity. Also, cell-free fetal DNA can be distinguished, by its methylation status, from the maternal DNA in the plasma of pregnant women, hence providing DNA biomarkers for the proposed minimally-invasive diagnosis of fetal aneuploidies, including Down's syndrome. However, the production and clearance of cell-free DNA from plasma in relation to its methylation status, are poorly understood processes. Methods We studied the methylation status of DNA derived from the imprinted GNAS1 locus, in cell-free plasma DNA of healthy adults. Heterozygotes were identified that carried the SNP rs1800905 in the imprinted region. The parent-of-origin-dependent DNA methylation was analysed by bisulfite conversion, followed by cloning and sequencing. Results Genomic DNA molecules derived from both the methylated, maternal, allele and the unmethylated, paternal, allele were found in plasma. Methylated and unmethylated DNA molecules were present in equal numbers. Conclusions Our data indicate that the methylation status of a DNA sequence has no effect on its steady state concentration in the cell-free DNA component of plasma, in healthy adults

Nucleosomes in serum as a marker for cell death

The concentration of nucleosomes is elevated in blood of patients with diseases which are associated with enhanced cell death. In order to detect these circulating nucleosomes, we used the Cell Death Detection-ELISA(Plus) (CDDE) from Roche Diagnostics (Mannheim, Germany) (details at http:\textbackslash{}\textbackslash{}biochem.roche.com). For its application in liquid materials we performed various modifications: we introduced a standard curve with nucleosome-rich material, which enabled direct quantification and improved comparability of the values within (CVinterassay:3.0-4.1%) and between several runs (CVinterassay:8.6-13.5%), and tested the analytical specificity of the ELISA. Because of the fast elimination of nucleosomes from circulation and their limited stability, we compared plasma and serum matrix and investigated in detail the pre-analytical handling of serum samples which can considerably influence the test results. Careless venipuncture producing hemolysis, delayed centrifugation and bacterial contamination of the blood samples led to false-positive results; delayed stabilization with EDTA and insufficient storage conditions resulted in false-negative values. At temperatures of -20 degreesC, serum samples which were treated with 10 mM EDTA were stable for at least 6 months. In order to avoid possible interfering factors, we recommend a schedule for the pre-analytical handling of the samples. As the first stage, the possible clinical application was investigated in the sera of 310 persons. Patients with solid tumors (n = 220; mean = 361 Arbitrary Units (AU)) had considerably higher values than healthy persons (n = 50; mean = 30 AU; P = 0.0001) and patients with inflammatory diseases (n = 40; mean = 296 AU; p = 0.096). Within the group of patients with tumors, those in advanced stages (UICC 4) showed significantly higher values than those in early stages (UICC 1-3) (P = 0.0004)

Microsatellite alterations and TP53 mutations in plasma DNA of small-cell lung cancer patients: Follow-up study and prognostic significance

Background: Small-cell lung cancer (SCLC), one of the major types of lung cancer, is associated with many different somatic molecular genetic changes. These alterations, observed in tumor DNA, have also been identified in the plasma DNA of patients. We undertook the present study to make a prospective investigation into the correlation between abnormal plasma DNA and patient survival. Patients and methods: Thirty-five patients with SCLC were selected after histological diagnosis. Polymorphic markers (ACTBP2, UT762 and AR) were chosen for their reported high rate of alterations in SCLC and analyzed in tumor tissue, normal blood cells and plasma DNA. Furthermore, we looked for mutations of the TP53 gene in tumor and plasma DNA. Results: In 25 patients (71%) at least one molecular change precisely matching that of the primary tumor was detected in the plasma DNA. No difference in survival was observed between patients with aberrant plasma DNA and patients without plasma DNA alterations. However, patients with microsatellite modifications and TP53 mutations concomitantly, showed a significant difference (P = 0.02) in survival compared with patients bearing only one of these molecular changes. In 15 cases it was possible to find a correlation either between tumor response and disappearance of abnormal plasma DNA, or tumor progression and persistence of plasma DNA alterations. Conclusions: Free plasma DNA with molecular alterations is present to a high degree in plasma DNA of SCLC patients and may have a role as a prognostic facto

Circulating Cell-Free DNA in Dogs with Mammary Tumors: Short and Long Fragments and Integrity Index

Circulating cell-free DNA (cfDNA) has been considered an interesting diagnostic/prognostic plasma biomarker in tumor-bearing subjects. In cancer patients, cfDNA can hypothetically derive from tumor necrosis/apoptosis, lysed circulating cells, and some yet unrevealed mechanisms of active release. This study aimed to preliminarily analyze cfDNA in dogs with canine mammary tumors (CMTs). Forty-four neoplastic, 17 non-neoplastic disease-bearing, and 15 healthy dogs were recruited. Necrosis and apoptosis were also assessed as potential source of cfDNA on 78 CMTs diagnosed from the 44 dogs. The cfDNA fragments and integrity index significantly differentiated neoplastic versus non-neoplastic dogs (P<0.05), and allowed the distinction between benign and malignant lesions (P<0.05). Even if without statistical significance, the amount of cfDNA was also affected by tumor necrosis and correlated with tumor size and apoptotic markers expression. A significant (P<0.01) increase of Bcl-2 in malignant tumors was observed, and in metastatic CMTs the evasion of apoptosis was also suggested. This study, therefore, provides evidence that cfDNA could be a diagnostic marker in dogs carrying mammary nodules suggesting that its potential application in early diagnostic procedures should be further investigated

Origin and quantification of circulating DNA in mice with human colorectal cancer xenografts

Although circulating DNA (ctDNA) could be an attractive tool for early cancer detection, diagnosis, prognosis, monitoring or prediction of response to therapies, knowledge on its origin, form and rate of release is poor and often contradictory. Here, we describe an experimental system to systematically examine these aspects. Nude mice were xenografted with human HT29 or SW620 colorectal carcinoma (CRC) cells and ctDNA was analyzed by Q–PCR with highly specific and sensitive primer sets at different times post-graft. We could discriminate ctDNA from normal (murine) cells and from mutated and non-mutated tumor (human) cells by using species-specific KRAS or PSAT1 primers and by assessing the presence of the BRAF V600E mutation. The concentration of human (mutated and non-mutated) ctDNA increased significantly with tumor growth. Conversely, and differently from previous studies, low, constant level of mouse ctDNA was observed, thus facilitating the study of mutated and non-mutated tumor derived ctDNA. Finally, analysis of ctDNA fragmentation confirmed the predominance of low-size fragments among tumor ctDNA from mice with bigger tumors. Higher ctDNA fragmentation was also observed in plasma samples from three metastatic CRC patients in comparison to healthy individuals. Our data confirm the predominance of mononucleosome-derived fragments in plasma from xenografted animals and, as a consequence, of apoptosis as a source of ctDNA, in particular for tumor-derived ctDNA. Altogether, our results suggest that ctDNA features vary during CRC tumor development and our experimental system might be a useful tool to follow such variations

Liquid biopsy by NGS: differential presence of exons (DPE) in cell-free DNA reveals different patterns in metastatic and nonmetastatic colorectal cancer

Next-generation sequencing (NGS) has been proposed as a suitable tool for liquid biopsy in colorectal cancer (CRC), although most studies to date have focused almost exclusively on sequencing of panels of potential clinically actionable genes. We evaluated the clinical value of whole-exome sequencing (WES) of cell-free DNA (cfDNA) circulating in plasma, with the goal of identifying differential clinical profiles in patients with CRC. To this end, we applied an original concept, "differential presence of exons" (DPE). We determined differences in levels of 379 exons in plasma cfDNA and used DPE analysis to cluster and classify patients with disseminated and localized disease. The resultant bioinformatics analysis pipeline allowed us to design a predictive DPE algorithm in a small subset of patients that could not be initially classified based on the selection criteria. This DPE suggests that these nucleic acids could be actively released by both tumor and nontumor cells as a means of intercellular communication and might thus play a role in the process of malignant transformation. DPE is a new technique for the study of plasma cfDNA by WES that might have predictive and prognostic value in patients with CRC.This study was funded by a grant from “Fondo de Investigaciones Sanitarias-FEDER,” Ministry of Health, Spain (FIS; PI13/01924). Work in the laboratory of Susana OlmedillasLópez is further supported by the Spanish Ministry of Health and Consumer Affairs (via a cooperative network-FEDER [TerCel RD12-0019-0035]). The CBMSO receives an institutional grant from the Fundación Ramón Arece

Hypermethylation of p16INK4a in Korean Non-small Cell Lung Cancer Patients

Promoter hypermethylation of the p16INK4a gene was investigated in 81 sets of samples of tumor tissue and adjacent normal tissue from Korean patients with primary lung cancer, using the modified real-time polymerase chain reaction (PCR)/ SYBR Green detection method. The results showed hypermethylation of p16INK4a in 27.2% of tumor tissues, and in 11.1% of adjacent normal tissue. No significant association was found between the overall aberrant methylation in tumor and corresponding normal specimens (r=0.137, p=0.219). In 22 cases with p16INK4a hypermethylation in tumor tissues, only 4 (18.1%) cases were found to have a hypermethylated normal tissue specimen. The findings of this study show that smoking can influence the methylation level of the promoter region of p16INK4a, and that this occurs in tumor tissues more frequently than in normal tissues. Other clinicopathological characteristics, including age, sex, tumor stage, and histologic type were not found to be correlated with p16INK4a methylation