Plasma ccfDNA Research Articles

Correlation between plasma ccfDNA, mtDNA changes, CTCs, and epithelial-mesenchymal transition in breast cancer patients undergoing NACT.

Breast cancer is the most prevalent cancer in women, emphasizing need for noninvasive blood biomarkers to aid in treatment selection. Previous studies have demonstrated elevated levels of plasma circulating cell-free DNA (ccfDNA) in breast cancer patients. Both ccfDNA and mitochondrial DNA (mtDNA) are fragments released into the bloodstream. In this study, we investigated effectiveness of ccfDNA and mtDNA as indicators of treatment response and explored their potential as monitoring biomarkers. Additionally, we compared these markers with circulating tumor cell (CTC) data and assessed their relationship with epithelial-mesenchymal transition (EMT). Thirty-six female breast cancer patients and 21 healthy females were included in the study. Quantitative polymerase chain reaction (qPCR) was performed on plasma samples to measure levels of ND1, ND4, ALU115, ALU247, and GAPDH, and DNA integrity was determined by calculating ratios of ALU247/ALU115 and ND4/ND1. After treatment, patients had a significant decrease in ccfDNA levels and a significant increase in mtDNA copy number (mtDNAcn). However, there was no significant change in ccfDNA and mtDNA integrity. When comparing all groups, patients exhibited higher levels of ALU115 and ALU247 compared to controls. Moreover, patients demonstrated significantly lower ccfDNA integrity than controls. This study represents the first comprehensive investigation of plasma ccfDNA levels, mtDNAcn, and integrities collectively. Furthermore, it is the first study to explore the relationship between these markers and CTCs, cancer stem cell markers, treatment response, and metastatic status. Our findings suggest that plasma ccfDNA and mtDNA may serve as potential biomarkers for assessing chemotherapy response and can be employed alone or in combination with other biomarkers to monitor treatment efficacy in breast cancer patients.

Mitochondrial Fraction of Circulating Cell-Free DNA as an Indicator of Human Pathology.

Circulating cell-free DNA (ccfDNA) of mitochondrial origin (ccf-mtDNA) consists of a minor fraction of total ccfDNA in blood or in other biological fluids. Aberrant levels of ccf-mtDNA have been observed in many pathologies. Here, we introduce a simple and effective standardized Taqman probe-based dual-qPCR assay for the simultaneous detection and relative quantification of nuclear and mitochondrial fragments of ccfDNA. Three pathologies of major burden, one malignancy (Breast Cancer, BrCa), one inflammatory (Osteoarthritis, OA) and one metabolic (Type 2 Diabetes, T2D), were studied. Higher levels of ccf-mtDNA were detected both in BrCa and T2D in relation to health, but not in OA. In BrCa, hormonal receptor status was associated with ccf-mtDNA levels. Machine learning analysis of ccf-mtDNA datasets was used to build biosignatures of clinical relevance. (A) a three-feature biosignature discriminating between health and BrCa (AUC: 0.887) and a five-feature biosignature for predicting the overall survival of BrCa patients (Concordance Index: 0.756). (B) a five-feature biosignature stratifying among T2D, prediabetes and health (AUC: 0.772); a five-feature biosignature discriminating between T2D and health (AUC: 0.797); and a four-feature biosignature identifying prediabetes from health (AUC: 0.795). (C) a biosignature including total plasma ccfDNA with very high performance in discriminating OA from health (AUC: 0.934). Aberrant ccf-mtDNA levels could have diagnostic/prognostic potential in BrCa and Diabetes, while the developed multiparameter biosignatures can add value to their clinical management.

Interlaboratory evaluation of quality control methods for circulating cell-free DNA extraction

Analysis of circulating cell-free DNA (ccfDNA) isolated from liquid biopsies is rapidly being implemented into clinical practice. However, diagnostic accuracy is significantly impacted by sample quality and standardised approaches for assessing the quality of ccfDNA are not yet established. In this study we evaluated the application of nucleic acid “spike-in” control materials to aid quality control (QC) and standardisation of cfDNA isolation for use in in vitro diagnostic assays. We describe an approach for the design and characterisation of in-process QC materials, illustrating it with a spike-in material containing an exogenous Arabidopsis sequence and DNA fragments approximating to ccfDNA and genomic DNA lengths. Protocols for inclusion of the spike-in material in plasma ccfDNA extraction and quantification of its recovery by digital PCR (dPCR) were assessed for their suitability for process QC in an inter-laboratory study between five expert laboratories, using a range of blood collection devices and ccfDNA extraction methods. The results successfully demonstrated that spiking plasmid-derived material into plasma did not deleteriously interfere with endogenous ccfDNA recovery. The approach performed consistently across a range of commonly-used extraction protocols and was able to highlight differences in efficiency and variability between the methods, with the dPCR quantification assay performing with good repeatability (generally CV <5%). We conclude that initial findings demonstrate that this approach appears “fit for purpose” and spike-in recovery can be combined with other extraction QC metrics for monitoring the performance of a process over time, or in the context of external quality assessment.

Circulating Cell Free DNA as Predictive Biomarker in Cervical Cancer Patients Treated with Radical Chemo-Radiation

<h3>Purpose/Objective(s)</h3> Liquid biopsy is a new technique for cancer detection. Evaluation of circulating cell-free DNA (ccfDNA) through liquid biopsy is a minimally invasive and cost-effective method that may serve as a potential biomarker for early detection, treatment monitoring, status of residual disease, and distant tumor metastasis in cervical cancer patients. We evaluated plasma ccfDNA levels in patients of cervical cancer undergoing chemo-radiation and correlated their levels with treatment response and short-term clinical outcome. <h3>Materials/Methods</h3> This prospective, single arm, open–label, observational pilot study included patients of cervical cancer, being radically treated with three-dimensional conformal radiotherapy or intensity modulated radiotherapy to a dose of 46Gy/23 fractions over 4.5weeks followed by high dose rate (HDR) brachytherapy to deliver EQD2 of >80Gy. Concurrent weekly cisplatin 40mg/m² was administered weekly throughout the course of external radiotherapy. Peripheral blood samples were collected in EDTA tubes before starting treatment and at 6months after completion of treatment. On the same day, tubes were centrifuged at 800g for 10 minutes. The supernatant was transferred to 10ml tubes and centrifuged at 1600g for 10 minutes to remove debris. Plasma supernatants were transferred in 1mL aliquots and stored at -80°C. Total genomic DNA of plasma aliquots were extracted using the commercially available Circulating Nucleic Acid Kits. Categorical variables were tested using the chi-square test. Univariate analysis was done to analyze the factors affecting ccfDNA levels and if found significant, multivariate analysis was used. <h3>Results</h3> Of 22 patients of histologically confirmed cervical cancer, 11 (50%) were of stage II disease followed by 7 (31.8%) with stage III disease. The median age was 53.5 years (range 39-70 years). The baseline mean ccfDNA levels was 69.68±58.335 ng/μL which was significantly reduced post-treatment to 0.452±0.199 ng/μL (p<0.001). The stage of the disease significantly affected the baseline ccfDNA levels (p=0.029). Positive pelvic and para-aortic lymph nodes showed a trend towards higher baseline ccfDNA levels (p=0.068 and 0.38 respectively). Patients with progressive disease had a trend towards higher post-treatment ccfDNA levels (p=0.19). <h3>Conclusion</h3> Our study revealed the role of ccfDNA as a potential biomarker in cervical cancer, which, however requires further longitudinal studies for confirmation.

BIOM-18. METHYLATION ANALYSIS TO REVEAL THE CELLULAR ORIGIN OF PROGNOSTIC CIRCULATING CELL-FREE DNA IN GLIOBLASTOMA

Abstract We have previously demonstrated an independent association between high levels of plasma circulating cell-free DNA (ccfDNA) concentration and poor survival outcomes in patients with newly diagnosed glioblastoma. Given that somatic mutations are rarely detected in glioblastoma patient plasma, we reasoned that DNA shed by tumor (circulating tumor DNA, ctDNA) was not likely to be a driver of prognostic increases in ccfDNA. To investigate the tissue of origin for this prognostic ccfDNA, we analyzed the plasma ccfDNA methylation signatures of 23 patients with newly diagnosed glioblastoma using the Illumina Infinium EPIC array, a genome-wide DNA methylation technique covering over 850,000 CpG sites. Deconvolution of the ccfDNA methylation signatures based on published reference methylomes revealed an increased proportion of ccfDNA derived from granulocytes in glioblastoma specimens compared to healthy controls (p &amp;lt; 0.0001). Further, this granulocyte proportion was increased in patients with high ccfDNA levels (above median value) compared to patients with low ccfDNA (p = 0.0001). Granulocyte proportion correlated with ccfDNA concentration (Spearman r = 0.64, p = 0.001). The top two gene sets identified by differential methylation analysis followed by gene set enrichment analysis (methylGSA) between high- and low-ccfDNA specimens were “neutrophil activation involved in immune response” (GO:0002283, p = 3.4E-23) and “neutrophil degranulation” (GO:0043312, p = 3.4E-23). Additional analysis of ccfDNA fragment size identified larger fragments ( &amp;gt; 700 bp) as the major source of the prognostic signal (log-rank p = 0.002 for progression-free survival) compared to traditionally sized ccfDNA fragments (50-700 bp, log-rank p = 0.12). Taken together, these data suggest that granulocytes are the primary contributing source of prognostic ccfDNA in glioblastoma. Future studies are needed to determine the mechanism through which granulocyte-derived ccfDNA is associated with clinical outcomes in glioblastoma and to explore related therapeutic opportunities.

Optimization of Sources of Circulating Cell-Free DNA Variability for Downstream Molecular Analysis

Circulating cell-free DNA (ccfDNA) is used increasingly as a cancer biomarker for prognostication, as a correlate for tumor volume, or as input for downstream molecular analysis. Determining optimal blood processing and ccfDNA quantification are crucial for ccfDNA to serve as an accurate biomarker as it moves into the clinical realm. Whole blood was collected from 50 subjects, processed to plasma, and used immediately or frozen at -80°C. Plasma ccfDNA was extracted and concentration was assessed by real-time quantitative PCR (qPCR), fluorimetry, and droplet digital PCR (ddPCR). For the 24 plasma samples from metastatic pancreatic cancer patients, the variant allele fractions (VAF) of KRAS G12/13 pathogenic variants in circulating tumor DNA (ctDNA) were measured by ddPCR. Using a high-speed (16,000 × g) or slower-speed (4100 × g) second centrifugation step showed no difference in ccfDNA yield or ctDNA VAF. A two- versus three-spin centrifugation protocol also showed no difference in ccfDNA yield or ctDNA VAF. A higher yield was observed from fresh versus frozen plasma by qPCR and fluorimetry, whereas a higher yield was observed for frozen versus fresh plasma by ddPCR, however, no difference was observed in ctDNA VAF. Overall, our findings suggest factors to consider when implementing a ccfDNA extraction and quantification workflow in a research or clinical setting.

Whole genome sequencing of low input circulating cell-free DNA obtained from normal human subjects.

Cell‐free DNA circulates in plasma at low levels as a normal by‐product of cellular apoptosis. Multiple clinical pathologies, as well as environmental stressors can lead to increased circulating cell‐free DNA (ccfDNA) levels. Plasma DNA studies frequently employ targeted amplicon deep sequencing platforms due to limited concentrations (ng/ml) of ccfDNA in the blood. Here, we report whole genome sequencing (WGS) and read distribution across chromosomes of ccfDNA extracted from two human plasma samples from normal, healthy subjects, representative of limited clinical samples at <1 ml. Amplification was sufficiently robust with ~90% of the reference genome (GRCh38.p2) exhibiting 10X coverage. Chromosome read coverage was uniform and directly proportional to the number of reads for each chromosome across both samples. Almost 99% of the identified genomic sequence variants were known annotated dbSNP variants in the hg38 reference genome. A high prevalence of C>T and T>C mutations was present along with a strong concordance of variants shared between the germline genome databases; gnomAD (81.1%) and the 1000 Genome Project (93.6%). This study demonstrates isolation and amplification procedures from low input ccfDNA samples that can detect sequence variants across the whole genome from amplified human plasma ccfDNA that can translate to multiple clinical research disciplines.

Identification of Somatic Mutations in Papanicolaou Smear DNA and Plasma Circulating Cell-Free DNA for Detection of Endometrial and Epithelial Ovarian Cancers: A Pilot Study.

ObjectivesThe aim of this study was to identify tumor-derived DNA from Papanicolaou (Pap) smear and plasma specimens collected from patients with endometrial cancer or atypical hyperplasia (EC/AH) or epithelial ovarian cancer (OC).MethodsTumor tissues, peripheral blood, and Pap smear samples were collected from patients with EC/AH and patients with epithelial OC. Somatic mutations of tumor specimens in EC/AH and OC were examined by whole-exome sequencing using a 127-driver gene panel from The Cancer Genome Atlas (TCGA). A nine-gene EC/AH panel and an eight-gene OC panel were established based on the identified significantly mutated genes in the EC/AH and OC tumor specimens. Circulating single-molecule amplification and resequencing technology (cSMART) was applied to evaluate somatic mutations in Pap smear DNA and plasma circulating cell-free DNA (ccfDNA) using the EC/AH and OC gene panels.ResultsIn EC/AH group, there existed 22 tumors and 14 of the 22 tumors contributed hot spot mutations for the EC/AH nine-gene panel. In the Pap smear subgroup, all 21 Pap smears tested positive. Nine out of 11 (81.8%) identified the same gene mutations with their matched tumors and the remaining 10 Pap smears all tested positive. In the plasma subgroup, 10 out of 26 (38.5%) plasmas tested positive. One out of 13 (7.7%) identified the same gene mutation with its matched tumor and 5 out of the remaining 13 plasmas (38.5%) tested positive. In OC group, there existed 17 tumors and 16 of the 17 tumors contributed hot spot mutations for the OC eight-gene panel. In the Pap smear subgroup, all 11 Pap smears tested positive. Five out of 10 (50.0%) identified the same gene mutations with their matched tumors and the remaining one Pap smear also tested positive. In the plasma subgroup, all 22 plasmas tested positive. Ten out of 14 (71.4%) identified the same gene mutation with their matched tumors and the remaining 4 plasmas all tested positive.ConclusionsTumor-derived DNA can be detected in Pap smears and plasmas from patients with EC/AH or epithelial OC. Using a small gene-panel, early detection of EC/AH and OC might be promising. However, the value of plasma ccfDNA for EC/AH requires further investigation.

Sequencing of Circulating Cell-Free DNA in Patients with AML Detects Clinically Significant Mutations Not Detected in Bone Marrow: The Role for Complementary Peripheral Blood and Bone Marrow Genomic Analysis

Sequencing of Circulating Cell-Free DNA in Patients with AML Detects Clinically Significant Mutations Not Detected in Bone Marrow: The Role for Complementary Peripheral Blood and Bone Marrow Genomic Analysis

Changes in circulating cell-free nuclear DNA and mitochondrial DNA of patients with adolescent idiopathic scoliosis

BackgroundAdolescent idiopathic scoliosis (AIS) which characterized by complex three-dimensional deformity of spine has been difficult to cure because of the unknown etiopathology and uncertainty of progression. Nowadays, circulating cell-free (ccf) DNA was found to be a potential biomarker for several benign and malignant diseases. However, whether ccf DNA can be a biomarker for AIS has not been reported yet. In this study, we investigate the circulating cell-free nuclear DNA (ccf n-DNA) and mitochondrial DNA (ccf mt-DNA) concentrations in the plasma of patients with AIS and controls (CT), and the changed plasma ccf n-DNA and ccf mt-DNA levels and their association with clinical parameters were assessed.MethodsThe plasma of peripheral blood from 69 AIS patients and 21 age-matched CT was collected for ccf DNA analysis. Quantitative PCR was used to detect ccf n-DNA and ccf mt-DNA levels, and correlation analyses between the ccf n-DNA and ccf mt-DNA levels and clinical characteristics were conducted. Receiver operator curves (ROC) were used to analyze the sensitivity and specificity of ccf n-DNA and ccf mt-DNA levels to different characteristics.ResultsThe plasma ccf n-DNA levels of both GAPDH and ACTB were significantly decreased in AIS patients compared with those in controls, while the plasma ccf mt-DNA levels did not changed. According to sex-related analyses, the ccf n-DNA levels in male CT-M was higher than that in female CT and male AIS, but the ccf n-DNA levels in female AIS was not significantly changed when compared with male AIS or female CT. However, the concentration of ccf mt-DNA in female AIS increased significantly when compared with male AIS. Surprisingly, Lenke type-related analyses suggested that Lenke type 1 patients had lower ccf n-DNA levels, whereas Lenke type 5 patients had higher ccf mt-DNA levels compared with those of controls. However, a lower sensitivity and specificity of AIS predicted by ccf n-DNA or ccf mt-DNA levels was observed, whether in total, by sex, or by Lenke type.ConclusionAlthough with no/little predictive accuracy of AIS/progressed AIS by ccf DNA levels, significantly changed plasma ccf DNA levels were observed in AIS patients compared with those in controls.

PF212 NEXT‐GENERATION SEQUENCING OF CIRCULATING CELL‐FREE DNA PROVIDES COMPLEMENTARY INFORMATION TO GENOMIC PROFILING OF BONE MARROW IN PATIENTS WITH ACUTE LEUKEMIAS

Background:Circulating cell‐free DNA (ccfDNA) allows for non‐invasive peripheral blood sampling of cancer‐associated mutations and has established clinical utility in several solid tumors. The ability of ccfDNA to detect and track leukemia‐associated mutations may also be useful in leukemias, both by identifying prognostic or targetable mutations at diagnosis or as a non‐invasive marker of measurable residual disease (MRD).Aims:To evaluate the feasibility of genomic analysis of ccfDNA to detect somatic mutations in patients (pts) with acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL).Methods:Plasma ccfDNA was collected and analyzed using a capture‐based next‐generation sequencing (NGS) assay (Qia275) targeting 275 genes. NGS was performed on DNA extracted from the bone marrow (BM) in a CLIA‐certified molecular diagnostics laboratory for the detection of mutations in the coding sequence of 28 genes, all of which were also assessed in the ccfDNA panel. An established bioinformatics pipeline was used to identify somatic variants.Results:A total of 24 pts (AML, n = 22; ALL, n = 2) underwent paired ccfDNA and BM sequencing at diagnosis. ccfDNA was also collected in 11 pts at ≥1 time point during remission. Among baseline samples, ccfDNA was collected a median of 6 days after BM collection (range, 0 to 27 days) and 0.5 days after the start of induction (range, −7 to 7 days). Using the 28 overlapping genes between the two assays, 55 somatic mutations were detected. 12 mutations (22%) were detected in ccfDNA but not BM. The median number of mutations detected in the BM and in ccfDNA per pt were 1.5 (range, 0–5) and 1 (range, 0–4), respectively (P = 0.31). Time from the start of chemotherapy until ccfDNA collection did not impact the concordance with BM mutation analysis. Among mutations detected in baseline ccfDNA samples, the median variant allelic frequency (VAF) was 33.7% (range, 2.7% to 90.8%). ccfDNA VAF was higher in mutations also detected in BM compared to those detected only in ccfDNA (median 38.3% vs 25.4%; P = 0.01), suggesting that these may be small subclonal populations more likely to be missed with BM sampling. Conversely, BM VAF was higher in those also detected in ccfDNA compared to those only found in BM (39.7% vs 21.0%; P &lt; 0.05). Mutations detected with ccfDNA but not BM included FLT3‐ITD in 4 pts, ASXL1 in 2 pts, WT1 in 1 pt, IDH1 in 1 pt, and BRAF and two EGFR mutations in 1 pt. In the 4 pts with FLT3‐ITD, the mutation was detected by PCR of the BM but not by BM sequencing. Among the other 5 pts with mutations detected in ccfDNA but not BM, 2 pts eventually relapsed; in both pts, the discordant mutation (IDH1 and ASXL1) was detected in the relapse BM. Using ccfDNA, the persistence of leukemia‐associated mutations during remission or the emergence of new mutations appeared to herald relapse. Two pts with t(8;21) AML developed new RUNX1 mutations detected by ccfDNA while in remission and relapsed 3 months and 14 months later; in both pts the emergent RUNX1 mutation was also detected in the relapse BM. Another pt with AML in morphological remission on day +30 after stem cell transplant had persistent TP53 and TET2 mutations detected by ccfDNA and relapsed 1 month later.Summary/Conclusion:This study demonstrates the feasibility of ccfDNA monitoring in pts with acute leukemias. ccfDNA can identify clinically significant prognostic or targetable mutations not detected by BM sequencing. The potential use of ccfDNA as a non‐invasive method of MRD assessment needs to be evaluated in larger, prospective cohorts.

Plasma free DNA: Evaluation of temperature-associated storage effects observed for Roche Cell-Free DNA collection tubes.

IntroductionStandardized pre-analytical blood sample procedures for the analysis of circulating cell-free DNA (ccfDNA) are still not available. Therefore, the present study aimed at evaluating the impact of storage conditions related to different times (24 and 48 h) and temperatures (room temperature (RT) and 4 - 8 °C) on the plasma ccfDNA concentration of blood samples drawn into Cell-Free DNA collection tubes (Roche Diagnostics GmbH, Mannheim, Germany).Materials and methodsVenous blood from 30 healthy individuals was collected into five 8.5 mL Cell-Free DNA Collection Tubes (Roche Diagnostics GmbH) each. Plasma samples were processed at time point of blood collection (tube 1), and after storage under the following conditions: 24 h at RT (tube 2) or 4-8 °C (tube 3), and 48 h at RT (tube 4) or 4 - 8 °C (tube 5). Circulating cell-free DNA concentrations were determined by EvaGreen chemistry-based droplet digital PCR (ddPCR).ResultsNo statistically significant differences between median (interquartile range) plasma ccfDNA concentrations (ng/mL) at time point of blood collection (3.17 (2.13 – 3.76)) and after storage for 24 h (RT: 3.02 (2.41 – 3.68); 4-8 °C: 3.21 (2.19 – 3.46)) and 48 h (RT: 3.13 (2.10 – 3.76); 4-8 °C: 3.09 (2.19 – 3.50)) were observed (P values from 0.102 – 0.975).ConclusionsNo unwanted release of genomic DNA from white blood cells could be detected in plasma samples after tube storage for 24 and 48 h regardless of storage temperature.

DNA damage and circulating cell free DNA in greenhouse workers exposed to pesticides.

Pesticides widely used in agriculture and other applications have been linked to cancer and other diseases through several potential mechanisms. The goals of this study were to assess DNA damage in lymphocytes by the cytokinesis-block micronucleus assay (CBMN), and to measure circulating cell free DNA (ccf-DNA) in the blood of pesticide-exposed greenhouse workers and matched controls living in the same area. CBMN was applied to peripheral blood lymphocyte samples taken at different times (spring and autumn) for each individual. We measured plasma ccf-DNA levels using a Qubit® fluorometer. The results indicated that the MNL, BNMN, and NBUDs frequencies of pesticide-exposed individuals were significantly higher than non-exposed individuals. Apart from MNL, BNMN and CBPI, a season-related effect was found for the NPB and NBUD frequencies. With MNL and BNMN as the dependent variables, multiple regression analysis showed that age and gender affected MN formation. The ccf-DNA level in the pesticide-exposed group was significantly higher than the control group. There was no seasonal variation regarding the free DNA amount. Ccf-DNA in males was found to be higher than females. The MNL, BNMN, NPB, and CBPI did not correlate with the ccf-DNA amount. It can be concluded that pesticide exposure can modulate DNA integrity via different mechanisms. Also, elevated levels of ccf-DNA could be recommended as a biomarker of pesticide exposure. Environ. Mol. Mutagen. 59:161-169, 2018. © 2017 Wiley Periodicals, Inc.

Aberrant single-minded homolog 1 methylation as a potential biomarker for cervical cancer.

The aim of this study is to evaluate the possibility of using the methylation status of single-minded homolog 1 (SIM1) as a diagnostic biomarker for cervical cancer. All the patient and normal specimens including the normal cervix (n = 10), cervical cancer tissues (n = 45), blood (n = 45), and cervical brush specimens (n = 110) were retrospectively obtained. Quantitative methylation-specific PCR was performed to detect SIM1 methylation in primary tumors, cervical brush specimens, and plasma circulating cell-free DNA (ccfDNA). SIM1 expression was detected by western blot analysis. We found that SIM1 was highly methylated in the majority of the cervical cancer tissues that we tested, but not in any of the normal tissues. Hypermethylation of SIM1 led to a pronounced reduction in SIM1 expression in cervical cancer tissues compared with normal cervix. SIM1 methylation status on cervical brush specimens also distinguished cervical cancer from normal, cervical intraepithelial neoplasia (CIN) 1 and 2. The degree of SIM1 methylation was significantly associated with the severity of the disease (Ptrend < .0001). We also investigated the possibility of detecting methylated SIM1 in plasma ccfDNA from cervical cancer patients. Methylated SIM1 was detected in 36.6% (15/41) of ccfDNA samples, and concordance rate with the matched cancer tissues was 41.5% (17/41) with sensitivity 38.5% and specificity 100%. This study has shown that SIM1 is frequently hypermethylated in cervical cancer, compared with normal cervix tissue, CIN1 and 2 samples, suggesting that the methylation status of SIM1 could be a potential diagnostic biomarker for cervical cancer.

Detection of copy number alterations in cell-free tumor DNA from plasma

BackgroundSomatic copy number alterations (SCNAs) occurring in tumors can provide information about tumor classification, patient's outcome or treatment targets. Liquid biopsies, incl. plasma samples containing circulating cell-free tumor DNA (ccfDNA) can be used to assess SCNAs for clinical purposes, however specify and reliability of methods have to be tested. MethodsSNP microarrays (Affymetrix) were used to generate whole-genome copy number profiles from plasma ccfDNA (OncoScan) and paired tumor biopsies (CytoScan) from ten patients with metastatic cancers. Numerical, segmental and focal SCNAs were assessed using ASCAT/TuScan and SNP-FASST2. ResultsAberrations in ccfDNA in 4 patients resembled numerical (76%) and segmental (80%) aberrations in tDNA. Three patients represented low correlation due to postponed sampling time, ccfDNA quality and possible treatment interference. Breakpoints of high-amplitude amplification were assessed with high accuracy and relative breakpoints difference of only 7% (0.02–37%). Similarly, biallelic losses were reliably detected. Array was 100% successful in detection of SCNAs on clinically relevant genes compared to SCNAs in tumor biopsies. Tracking of SCNAs changes during the treatment course of one patient also indicated that apoptosis/necrosis of non-cancerous cells presumably induced by treatment can influence ccfDNA composition and introduce false-negative findings into the analysis of liquid biopsies. ConclusionsGenomic alterations detected in ccfDNA from liquid biopsies by comprehensive SNP array are reliable source for information for stratification of patients for targeted treatment. General significanceClinically relevant SCNAs can be detected in ccfDNA with high resolution and can therefore serve as an alternative to tumor biopsy in defining treatment targets.

Diagnostic Validity of Serum and Peritoneal TNF-alpha , High Sensitivity CRP and Plasma Cell-Free Nuclear DNA ( ccf nDNA ) as Biomarkers of Pelvic Endometriosis : A Case Control Study

Introduction:Endometriosis is a disease defined by the presence of endometrial glands and stroma located outside the uterine cavity. These ectopic implants can be found throughout the pelvis, on and within the ovaries, abutting the uterine ligaments, occupying the rectovaginal septum, invading the intestinal serosa, and along the parietal peritoneum. Endometrial implantation at distant sites such as the pleura, lung, within surgical scars, and along the diaphragm also has been reported. (1). It results often in subfertility and pain, occurs mainly in women of reproductive age (16–50 years) and has a progressive character in at least 50%, but the rate and risk factors for progression are unknown. Endometriosis can be classified into four stages: minimal, mild, moderate and severe. The gold standard for the diagnosis of endometriosis is laparoscopic inspection, ideally with histological confirmation. (2), however, is an invasive technique and should be performed only after imaging techniques prove insufficient for confident diagnosis. (3) Lack of a non-invasive diagnostic test contributes to the long delay between onset of symptoms and diagnosis of endometriosis. (2) Additional tools are needed for non-invasive classifications in order to reduce the number of unnecessary laparoscopies without adversely affecting outcomes. Finding specific and more sensitive biomarkers in endometriosis is critical, because endometriosis is usually diagnosed only in advanced stages, and there is a high rate of morbidity for this disease. (4) Aim of the work: The aim of the current study is to assess the validity of serum and peritoneal high sensitivity CRP and TNF-alpha and plasma cell-free nuclear DNA (ccf nDNA) as biomarkers in early diagnosis of pelvic endometriosis. Methods: This study was conducted at the Obstetrics & Gynecology department, Maternity Hospital, Ain Shams University. This is a case control study of 120 women scheduled for diagnostic laparoscopy. Laparoscopy was indicated in these women whether for various causes of subfertility or for chronic pelvic pain between January 2011 and January 2012. The patients were divided into the following groups: Group I (endometriosis group/ study group) consisted of 80 patients diagnosed to have endometriosis during laparoscopy. Group I cases were subdivided into two subgroups, Group IA: consisted of 34 cases with stage 1or minimal endometriosis and Group IB: consisted of 46 cases with stage 2 or mild endometriosis. Group II (non-endometriosis group/Control group): consisted of 40 cases with no detected pelvic pathology. During the laparoscopy procedure, both peripheral venous blood and peritoneal samples were withdrawn. Serum and peritoneal levels of high sensitivity CRP and TNF-alpha as well as plasma levels of ccf nDNA were compared in both groups and in early stages (minimal and mild) of endometriosis within the study group. Results: Serum TNF-a, serum hs-CRP and plasma ccf DNA were significantly elevated in cases compared with the control group. They were also elevated in patients with group IA and group IB as compared to control group. However, there was no statistically significant difference between cases and control group as regards peritoneal TNF-a and peritoneal hs-CRP. There was no significant difference between the group IA and group IB as regards all biomarkers. Conclusion: Our results showed that serum TNF-alpha , serum hs-CRP and plasma ccf DNA are highly reliable biomarkers for screening and early diagnosis of endometriosis, but they can not be used to discriminate between stage I and stage II. On the other hand, peritoneal TNF-a and peritoneal hs-CRP are non reliable for early diagnosis of endometriosis and can not be used to discriminate between stage I and stage II of endometriosis.