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Elevated level of cell-free plasma DNA is associated with advanced-stage breast cancer and metastasis
Clinical Chemistry and Laboratory Medicine
... The techniques mostly used so far for ccfDNA quantification is quantitative PCR (qPCR) in BC for the short and long sequences ALU115/247 [80,81] and LINE1 sequences [82] or using the reference gene GAPDH [83,84]. Both methods have repetitively confirmed higher ccfDNA levels in BC in relation to healthy individuals [79,81,82,[84][85][86][87][88][89][90][91][92][93]. Increased levels of ccfDNA in BC have also been correlated to metastasis [55,81,86], tumor size [79,[82][83][84], other histopathological parameters [79,89] and BC outcome [55,90]. ...
... Both methods have repetitively confirmed higher ccfDNA levels in BC in relation to healthy individuals [79,81,82,[84][85][86][87][88][89][90][91][92][93]. Increased levels of ccfDNA in BC have also been correlated to metastasis [55,81,86], tumor size [79,[82][83][84], other histopathological parameters [79,89] and BC outcome [55,90]. In our recent study, elevated levels of ccfDNA were correlated to the incidence of death, shorter PFS and non-response to pharmacotherapy in metastatic patients [55]. ...
... Most interestingly from a clinical aspect is the construction of a single-parametric linear model using ccfDNA plasma concentration values with great discriminating power to predict response to chemotherapy [55]. However, in our patient group we did not detect correlations of quantity to clinicopathological parameters, possibly due to the different quantification methods and patient classification criteria, in concordance with some researchers [86,88]. Other studies have assessed the ccfDNA quantity in relation to diagnosis. ...
Simple Summary
Our research focuses in the elucidation of the nature of circulating cell-free DNA (ccfDNA) as a biological entity and its exploitation as a liquid biopsy biomaterial. Working on breast cancer, it became clear that although a promising biosource, its clinical exploitation is burdened mainly by gaps in knowledge about its biology and specific characteristics. The current review covers multiple aspects of ccfDNA in breast cancer. We cover key issues such as quantity, integrity, releasing structures, methylation specific changes, release mechanisms, biological role. Machine learning approaches for analyzing ccfDNA-generated data to produce classifiers for clinical use are also discussed.
Abstract
Breast cancer (BC) is a leading cause of death between women. Mortality is significantly raised due to drug resistance and metastasis, while personalized treatment options are obstructed by the limitations of conventional biopsy follow-up. Lately, research is focusing on circulating biomarkers as minimally invasive choices for diagnosis, prognosis and treatment monitoring. Circulating cell-free DNA (ccfDNA) is a promising liquid biopsy biomaterial of great potential as it is thought to mirror the tumor’s lifespan; however, its clinical exploitation is burdened mainly by gaps in knowledge of its biology and specific characteristics. The current review aims to gather latest findings about the nature of ccfDNA and its multiple molecular and biological characteristics in breast cancer, covering basic and translational research and giving insights about its validity in a clinical setting.
... Circulating tumor DNA (ctDNA) in the bloodstream has emerged as a promising biomarker of disease status for breast cancer [20,[87][88][89][90]]. An elevated level of ctDNA has been found to be associated with advanced-stage breast cancer and metastasis [91,92]. Breast cancer patients had significantly higher levels of ctDNA than healthy controls [91]. ...
... An elevated level of ctDNA has been found to be associated with advanced-stage breast cancer and metastasis [91,92]. Breast cancer patients had significantly higher levels of ctDNA than healthy controls [91]. The ctDNA concentrations at levels ≥0.75% could be detected in breast cancer patients with a sensitivity of >90% and a specificity of >99% [19]. ...
Simple Summary
Early diagnosis of breast cancer greatly increases the chance of cure and survival from the disease. The mammogram is widely used for early detection of breast cancer, but its effectiveness and accuracy have been a concern for a long time as well as its inability in detecting small cancers, especially in women with dense breast tissues. Therefore, it is an unmet clinical need to develop a simple, convenient test to overcome the shortcomings of mammography. Liquid biopsy, which is based on the analysis of body fluids, has attracted much attention in the search for cancer biomarkers. Recent advances in analytical techniques have gradually made it possible to detect breast cancer early through a biomarker analysis of blood, nipple aspirate fluid, sweat, urine, tears, or the breath. We envision that a simple blood or breath test holds great promise as a biomarker for early detection of breast cancer in the near future.
Abstract
Breast cancer is the most common cancer in women worldwide. Accurate early diagnosis of breast cancer is critical in the management of the disease. Although mammogram screening has been widely used for breast cancer screening, high false-positive and false-negative rates and radiation from mammography have always been a concern. Over the last 20 years, the emergence of “omics” strategies has resulted in significant advances in the search for non-invasive biomarkers for breast cancer diagnosis at an early stage. Circulating carcinoma antigens, circulating tumor cells, circulating cell-free tumor nucleic acids (DNA or RNA), circulating microRNAs, and circulating extracellular vesicles in the peripheral blood, nipple aspirate fluid, sweat, urine, and tears, as well as volatile organic compounds in the breath, have emerged as potential non-invasive diagnostic biomarkers to supplement current clinical approaches to earlier detection of breast cancer. In this review, we summarize the current progress of research in these areas.
... With regard to lymph node involvement, the level of nDNA was significantly elevated in breast cancer patients with lymph node metastasis than in those without lymph node metastasis. This agreed with other studies (Roth et al., 2011 andNicolini et al., 2013) which explained that the more lymph node involvement, the more necrosis and apoptosis and the more DNA released into the circulation (Thyagarajan et al., 2013). Plasma mtDNA was significantly higher in breast cancer patients with node positive involvement and distant metastasis than in patients without node involvement or distant metastasis, and to our knowledge no studies have proved this. ...
... In the present study, there was a significant increase in the level of plasma nuclear DNA in advanced tumour stages when comparing stage III to stages I and II. This finding was in agreement with other studies (Nicolini et al., 2013). ...
Background:
In Egypt, breast cancer is estimated to be the most common cancer among females. It is also a leading cause of cancer-related mortality. Use of circulating cell-free DNA (ccf-DNA) as non-invasive biomarkers is a promising tool for diagnosis and follow-up of breast cancer (BC) patients.
Objective:
To assess the role of circulating cell free DNA (nuclear and mitochondrial) in diagnosing BC.
Materials and methods:
Multiplex real time PCR was used to detect the level of ccf nuclear and mitochondrial DNA in the peripheral blood of 50 breast cancer patients together with 30 patients with benign lesions and 20 healthy controls. Laboratory investigations, histopathological staging and receptor studies were carried out for the cancer group. Receiver operating characteristic curves were used to evaluate the performance of ccf-nDNA and mtDNA.
Results:
The levels of both nDNA and mtDNA in the cancer group were significantly higher in comparison to the benign and the healthy control group. There was a statistically significant association between nDNA and mtDNA levels and well established prognostic parameters; namely, histological grade, tumour stage, lymph node status andhormonal receptor status.
Conclusions:
Our data suggests that nuclear and mitochondrial ccf-DNA may be used as non-invasive biomarkers in BC.
... On the basis of that Plasma free DNA concentration is related to progression and metastasis and it can be used as a remarkable biomarker for treatment effect and prognosis assessment [7,8,9,10,11,12] Our study suggests that the measurement of plasma free DNA may be used for the assessment of breast cancer development and distant metastasis and so on. ...
... Similar studies have been performed in breast cancer, some of which found higher ctDNA levels in breast cancer patients than in control individuals, whereas other studies reported that there was no difference in ctDNA levels between the two groups. 67 One factor that contributes to this current limitation in ctDNA detection is that copy numbers of ctDNA are generally very low compared with those of wild-type cfDNA. Another factor is the limited accuracy of the current sequencing methods in cancer. ...
Circulating cell-free DNA (cfDNA) released by tumor cells, termed ctDNA, closely reflects the heterogeneity of primary cancers and their metastases. As a noninvasive, real-time monitoring biomarker, ctDNA is a promising tool for detecting driver gene mutations, assessing tumor burden and acquired resistance, and early diagnosis. However, isolation and enrichment of cfDNA is a big challenge due to the high degree of DNA fragmentation and its relatively low abundance in the bloodstream. This review aims to provide insights into the recent technological advances in acquisition of optimal quality cfDNA, the use of preservatives, isolation methods, processing timelines, and detection techniques. It also describes clinical applications of ctDNA in cancer patient management.
... In primary review, a total of 45 publications dealing with abnormal concentration [8,[17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36], methylation alterations [37][38][39][40][41][42][43][44], microsatellite instability [45][46][47][48][49][50] and other characteristics [10,14,[51][52][53][54][55][56][57][58] of plasma or serum DNA for the diagnosis of breast cancer were retrieved. After full-text review, 20 studies [8, 10, 14, 29-35, 42-44, 48-50, 56-59] were excluded because they did not allow the calculation of sensitivity or specificity, included very rare indicators, or consisted of less than 10 breast cancer patients ( Figure 1). ...
Objectives
The aim of this study was to systematically evaluate the diagnostic value of cell free DNA (cfDNA) for breast cancer.
Results
Among 308 candidate articles, 25 with relevant diagnostic screening qualified for final analysis. The mean sensitivity, specificity and area under the curve (AUC) of SROC plots for 24 studies that distinguished breast cancer patients from healthy controls were 0.70, 0.87, and 0.9314, yielding a DOR of 32.31. When analyzed in subgroups, the 14 quantitative studies produced sensitivity, specificity, AUC, and a DOR of 0.78, 0.83, 0.9116, and 24.40. The 10 qualitative studies produced 0.50, 0.98, 0.9919, and 68.45. For 8 studies that distinguished malignant breast cancer from benign diseases, the specificity, sensitivity, AUC and DOR were 0.75, 0.79, 0.8213, and 9.49. No covariate factors had a significant correlation with relative DOR. Deek's funnel plots indicated an absence of publication bias.
Materials and Methods
Databases were searched for studies involving the use of cfDNA to diagnose breast cancer. The studies were analyzed to determine sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, diagnostic odds ratio (DOR), and the summary receiver operating characteristic (SROC). Covariates were evaluated for effect on relative DOR. Deek's Funnel plots were generated to measure publication bias.
Conclusions
Our analysis suggests a promising diagnostic potential of using cfDNA for breast cancer screening, but this diagnostic method is not yet independently sufficient. Further work refining qualitative cfDNA assays will improve the correct diagnosis of breast cancers.
... The early detection of breast cancer is a desirable objective as it allows the initiation of effective therapies against tumor cells which have accumulated fewer oncogenic events. Many studies have focused on quantifying cfDNA levels in order to distinguish benign from malignant disease [256]. Although in most studies significantly increased levels of cfDNA were found in patients with breast cancer compared to healthy controls, the absolute amounts of cfDNA were highly variable and overlapping. ...
The analysis of cell-free circulating tumor DNA (ctDNA) is a very promising tool and might revolutionize cancer care with respect to early detection, identification of minimal residual disease, assessment of treatment response, and monitoring tumor evolution. ctDNA analysis, often referred to as “liquid biopsy” offers what tissue biopsies cannot—a continuous monitoring of tumor-specific changes during the entire course of the disease. Owing to technological improvements, efforts for the establishment of preanalytical and analytical benchmark, and the inclusion of ctDNA analyses in clinical trial, an actual clinical implementation has come within easy reach. In this chapter, recent advances of the analysis of ctDNA are summarized starting from the discovery of cell-free DNA, to methodological approaches and the clinical applicability.
... Using a validated protocol for cfDNA isolation and quantification [21], we analysed cfDNA levels in patients with moderate to severe renal impairment, including patients requiring dialysis. Consistent with previous data, cfDNA levels in patients with CKD stages 2-5, including dialysis dependent patients, were equivalent to those reported for healthy controls [27][28][29][30] and no association between eGFR and cfDNA concentrations was detected (Figure 1(b)). In addition, apoptotic DNA fragments were not present, suggesting that the elevated cardiovascular risk associated with CKD is not driven by processes that drive apoptosis or that the amount of apoptosis is insufficient to overwhelm clearance mechanisms [24,31]. ...
Patients with chronic kidney disease (CKD) are at increased risk of cardiovascular disease. Circulating free nucleic acids, known as cell-free DNA (cfDNA), have been proposed as a novel biomarker of cardiovascular risk. The impact of renal impairment on cfDNA levels and whether cfDNA is associated with endothelial dysfunction and inflammation in CKD has not been systematically studied. We analysed cfDNA concentrations from patients with varying degrees of CKD. In addition, to determine whether there is a relationship between cfDNA, inflammation, and endothelial dysfunction in CKD, levels of proinflammatory cytokines and von Willebrand Factor (vWF) were measured in patients treated with the peroxisome proliferator-activated receptor gamma agonist rosiglitazone or placebo for 8 weeks. cfDNA levels were not increased with renal impairment or associated with the degree of renal dysfunction (P = 0.5). Treatment with rosiglitazone for 8 weeks, but not placebo, was more likely to lead to a reduction in cfDNA levels (P = 0.046); however, the absolute changes in cfDNA concentrations during treatment were not statistically significant (P > 0.05). cfDNA levels correlated with markers of endothelial dysfunction (hsCRP P = 0.0497) and vWF (P = 0.0005). In conclusion, cell-free DNA levels are not influenced by renal impairment but do reflect endothelial dysfunction in patients with CKD.
Eighty seven women with benign breast lesion, 120 patients with breast cancer (BC) and one hundred controls were included in the study. Quantification of mtDNA and nDNA was done by qPCR. Global DNA methylation was measured using ELISA. Circulating cell-free nDNA and mtDNA were significantly elevated in BC and benign breast lesions patients. Global methylation was significantly low in BC patients. Combining the studied parameters in one panel, nDNA/mtDNA/hypomethylation, improved their sensitivity in detecting BC to reach 92.5%. Circulating cell-free nDNA, mtDNA and global DNA hypomethylation can be used as diagnostic and prognostic markers for BC.
Circulating cellfree DNA (cfDNA) is characterized as extracellular DNA that may be present in the blood of healthy individuals in low concentrations. CfDNA is released by apoptosis or necrosis into the bloodstream. Increased levels are found in pathological conditions, such as inflammation, autoimmune diseases, or stress. Significant increase of cfDNA is particularly evident in patients with malignancies, especially in the advanced stages of the disease. In this case, the tumor specific cfDNA is released by necrosis from the cells of primary tumor and metastases. Recently, many studies concentrate on the so called liquid biopsies that allow detection of circulating tumor cells and circulating nucleic acids from peripheral blood for tumor diagnostics. Quantitative methods and detection of genetic and epigenetic alternations of cfDNA in patients with different malignancies have potential applications in molecular diagnosis, prognosis, monitoring of disease progression and response to treatment. This review focuses on potential utility of cfDNA as a blood biomarker in selected solid tumors and hematologic malignancies.Key words: circulating cellfree DNA - tumor marker - solid tumors - hematological malignanciesThis study was supported by grant of Internal Grant Agency of the Czech Ministry of Health NT14575.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE "uniform requirements" for biomedical papers.Submitted: 13. 3. 2015Accepted: 18. 5. 2015.
Cell-free DNA (cfDNA) in the plasma of patients with both malignant and benign breast lesions was analyzed to determine whether the findings may have diagnostic and prognostic implications and to analyze the association between the levels of cfDNA and prognostic parameters.
Plasma samples were obtained from 99 subjects; 42 with breast cancer (BC), 30 with benign breast lesions, and 27 healthy women as normal controls. Circulatory cfDNA was extracted from the plasma samples and quantified using a real-time quantitative PCR method. Immunohistochemistry was done on formalin-fixed paraffin-embedded sections to evaluate the status of hormonal receptors (estrogen receptor [ER] and progesterone receptor [PR]), and the protein expression of both Her2/neu and Topoisomerase IIα.
The level of cfDNA in the BC group was significantly higher than in the benign lesions and control groups. cfDNA level was associated with malignant tumor size, lymph node involvement, stage, and grade as well as Her2/neu and Topoisomerase IIα expression, while it was not associated with ER or PR status.
The present study suggests that the level of cfDNA can be easily quantified using plasma samples. Thus, level of plasma cfDNA might constitute an important noninvasive diagnostic and prognostic valuable tool in cancer breast patients' management.
Purpose:
We undertook the current study with untreated breast cancer to (1) role the variations in the plasma levels of cfDNA and the size distribution in early stage, (2) determine the frequency in plasma of methylation of three candidate genes, RASSF1A, MAL, and SFRP1, and (3) to determine whether detection of cfDNA variations and methylation changes in plasma might have specific clinical utility.
Methods and materials:
Thirty-nine patients woman patients (median age 64 years; range, 36-90 years) who underwent surgery for primary BR and 49 healthy females' subjects (control group without any breast lesion) were evaluated. The cfDNA levels were analyzed using quantitative real-time polymerase chain reaction of β-globin. Based on the ALU repeats, the cfDNA was considered as either total (fragments of 115 bp, ALU115) or tumoral (fragments of 247 bp, ALU247). The association between the levels of the ALU247, ALU115 repeat, and ALU 247/115and the pathologic tumor characteristics was analyzed. Used methylight qPCR method, cfDNA from plasma samples of healthy donors and patients with breast cancer were evaluated for the diagnotic value of the methylation status of three genes (RASSF1A, MAL, SFRP1) frequently methylated in breast cancer.
Results:
The baseline levels of cfDNA were significantly higher in the patients with cancer, and the level of ALU247 was the most accurate circulating cfDNA marker in discriminating the cancer from non-cancer subjects. A high statistical significance was found by considering the T stage and patients with regional LN metastasis positive cancers showed significantly higher cfDNA level of ALU247. Moreover, patients with methylation of at least one of the gene under investigate showed a higher quantity of cfDNA ALU115 (p< 0.0001) and ALU247 level (p< 0.0001).
Conclusions:
We observed that necrosis could be a potential source of circulating tumour-specific cfDNA ALU247; and that cfDNA ALU247 and methylated cfDNA (RASSF1A, MAL and SFRP1) are both a phenotypic feature of tumour biology.
Solid tumors derived from epithelial tissues (carcinomas) are responsible for 90% of all new cancers in Europe, and the main four tumor entities are breast, prostate, lung, and colon cancer. Present tumor staging is mainly based on local tumor extension, metastatic lymph node involvement, and evidence of overt distant metastasis obtained by imaging technologies. However, these staging procedures are not sensitive enough to detect early tumor cell dissemination as a key event in tumor progression. Many teams have therefore focused on the development of sensitive assays that allow the specific detection of single tumor cells or small amounts of cell-free tumor DNA in the peripheral blood of cancer patients. These methods allow the detection and characterization of early metastatic spread and will provide unique insights into the biology of metastatic progression of human tumors, including the effects of therapeutic interventions.
DNA, mRNA and microRNA are released and circulate in the blood of cancer patients. Changes in the levels of circulating nucleic acids have been associated with tumour burden and malignant progression. In the past decade a wealth of information indicating the potential use of circulating nucleic acids for cancer screening, prognosis and monitoring of the efficacy of anticancer therapies has emerged. In this Review, we discuss these findings with a specific focus on the clinical utility of cell-free nucleic acids as blood biomarkers.
Cancers acquire resistance to systemic treatment as a result of clonal evolution and selection. Repeat biopsies to study genomic evolution as a result of therapy are difficult, invasive and may be confounded by intra-tumour heterogeneity. Recent studies have shown that genomic alterations in solid cancers can be characterized by massively parallel sequencing of circulating cell-free tumour DNA released from cancer cells into plasma, representing a non-invasive liquid biopsy. Here we report sequencing of cancer exomes in serial plasma samples to track genomic evolution of metastatic cancers in response to therapy. Six patients with advanced breast, ovarian and lung cancers were followed over 1-2 years. For each case, exome sequencing was performed on 2-5 plasma samples (19 in total) spanning multiple courses of treatment, at selected time points when the allele fraction of tumour mutations in plasma was high, allowing improved sensitivity. For two cases, synchronous biopsies were also analysed, confirming genome-wide representation of the tumour genome in plasma. Quantification of allele fractions in plasma identified increased representation of mutant alleles in association with emergence of therapy resistance. These included an activating mutation in PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha) following treatment with paclitaxel; a truncating mutation in RB1 (retinoblastoma 1) following treatment with cisplatin; a truncating mutation in MED1 (mediator complex subunit 1) following treatment with tamoxifen and trastuzumab, and following subsequent treatment with lapatinib, a splicing mutation in GAS6 (growth arrest-specific 6) in the same patient; and a resistance-conferring mutation in EGFR (epidermal growth factor receptor; T790M) following treatment with gefitinib. These results establish proof of principle that exome-wide analysis of circulating tumour DNA could complement current invasive biopsy approaches to identify mutations associated with acquired drug resistance in advanced cancers. Serial analysis of cancer genomes in plasma constitutes a new paradigm for the study of clonal evolution in human cancers.
Background:
The management of metastatic breast cancer requires monitoring of the tumor burden to determine the response to treatment, and improved biomarkers are needed. Biomarkers such as cancer antigen 15-3 (CA 15-3) and circulating tumor cells have been widely studied. However, circulating cell-free DNA carrying tumor-specific alterations (circulating tumor DNA) has not been extensively investigated or compared with other circulating biomarkers in breast cancer.
Methods:
We compared the radiographic imaging of tumors with the assay of circulating tumor DNA, CA 15-3, and circulating tumor cells in 30 women with metastatic breast cancer who were receiving systemic therapy. We used targeted or whole-genome sequencing to identify somatic genomic alterations and designed personalized assays to quantify circulating tumor DNA in serially collected plasma specimens. CA 15-3 levels and numbers of circulating tumor cells were measured at identical time points.
Results:
Circulating tumor DNA was successfully detected in 29 of the 30 women (97%) in whom somatic genomic alterations were identified; CA 15-3 and circulating tumor cells were detected in 21 of 27 women (78%) and 26 of 30 women (87%), respectively. Circulating tumor DNA levels showed a greater dynamic range, and greater correlation with changes in tumor burden, than did CA 15-3 or circulating tumor cells. Among the measures tested, circulating tumor DNA provided the earliest measure of treatment response in 10 of 19 women (53%).
Conclusions:
This proof-of-concept analysis showed that circulating tumor DNA is an informative, inherently specific, and highly sensitive biomarker of metastatic breast cancer. (Funded by Cancer Research UK and others.).
Cell-free DNA reflects both normal and tumor-derived DNA released into the circulation through cellular necrosis and apoptosis. The authors sought to determine the role of preoperative total plasma cell-free DNA levels in predicting clinical outcome in patients with ovarian cancer.
After institutional review board consent, DNA was extracted from plasma of 164 women with invasive epithelial ovarian carcinoma (EOC), 49 with benign ovarian neoplasms, and 75 age-matched controls. The samples were randomly divided into training (n = 144) and validation (n = 144) sets. Quantification of cell-free DNA was performed using real-time polymerase chain reaction for β-globin, and the number of genome equivalents (GE) per milliliter of plasma was determined. Cell-free DNA was correlated with clinicopathologic parameters.
The training and validation sets were similar in terms of demographic features. In the training set, EOC patients had a median preoperative cell-free DNA level of 10,113 GE/mL, compared with patients with benign ovarian neoplasms (median, 2365 GE/mL; P < .0001) and controls (median, 1912 GE/mL, P < .0001). Cell-free DNA >22,000 GE/mL was significantly associated with decreased patient survival (P < .001). After adjusting for other clinical variables, preoperative cell-free DNA >22,000 GE/mL was an independent predictor (P = .02) for disease-specific survival. Analysis of the validation set confirmed significantly higher cell-free DNA levels in EOC (median, 13,672 GE/mL) and that cell-free DNA >22,000 GE/mL was associated with a 2.83-fold increased risk of death from disease (P < .001).
Preoperative plasma total cell-free DNA levels are significantly elevated in patients with EOC. Elevated plasma cell-free DNA is an independent predictor for death from disease in ovarian cancer. Cancer 2010.
Cell-free DNA reflects both normal and tumor-derived DNA released into the circulation through cellular necrosis and apoptosis. The authors sought to determine the role of preoperative total plasma cell-free DNA levels in predicting clinical outcome in patients with ovarian cancer.
After institutional review board consent, DNA was extracted from plasma of 164 women with invasive epithelial ovarian carcinoma (EOC), 49 with benign ovarian neoplasms, and 75 age-matched controls. The samples were randomly divided into training (n = 144) and validation (n = 144) sets. Quantification of cell-free DNA was performed using real-time polymerase chain reaction for beta-globin, and the number of genome equivalents (GE) per milliliter of plasma was determined. Cell-free DNA was correlated with clinicopathologic parameters.
The training and validation sets were similar in terms of demographic features. In the training set, EOC patients had a median preoperative cell-free DNA level of 10,113 GE/mL, compared with patients with benign ovarian neoplasms (median, 2365 GE/mL; P < .0001) and controls (median, 1912 GE/mL, P < .0001). Cell-free DNA >22,000 GE/mL was significantly associated with decreased patient survival (P < .001). After adjusting for other clinical variables, preoperative cell-free DNA >22,000 GE/mL was an independent predictor (P = .02) for disease-specific survival. Analysis of the validation set confirmed significantly higher cell-free DNA levels in EOC (median, 13,672 GE/mL) and that cell-free DNA >22,000 GE/mL was associated with a 2.83-fold increased risk of death from disease (P < .001).
Preoperative plasma total cell-free DNA levels are significantly elevated in patients with EOC. Elevated plasma cell-free DNA is an independent predictor for death from disease in ovarian cancer.
Circulating cell-free DNA in the blood of cancer patients harbors tumor-specific aberrations. Here, we investigated whether this DNA might also reflect the presence of circulating tumor cells (CTC).
To identify the source of cell-free DNA in blood, plasma derived from 81 patients with prostate cancer was examined for CTCs and cell-free DNA. An epithelial immunospot assay was applied for detection of CTCs, and a PCR-based fluorescence microsatellite analysis with a panel of 14 polymorphic markers was used for detection of allelic imbalances (AI).
The plasma DNA levels significantly correlated with the diagnosis subgroups of localized (stage M0, n = 69) and metastasized prostate cancer (stage M1, n = 12; P = 0.03) and with the tumor stage of these patients (P < 0.005). AI was found on cell-free DNA in plasma from 45.0% and 58.5% of M0 and M1 patients, respectively. Detection of CTCs showed that 71.0% or 92.0% of the M0 and M1 patients harbored 1 to 40 CTCs in their blood, respectively. The occurrence of CTCs correlated with tumor stage (P < 0.03) and increasing Gleason scores (P = 0.04). Notably, significant associations of the number of CTCs with the AI frequencies at the markers D8S137 (P = 0.03), D9S171 (P = 0.04), and D17S855 (P = 0.02) encoding the cytoskeletal protein dematin, the inhibitor of the cyclin-dependent kinase CDKN2/p16 and BRCA1, respectively, were observed.
These findings show, for the first time, a relationship between the occurrence of CTCs and circulating tumor-associated DNA in blood, which, therefore, might become a valuable new source for monitoring metastatic progression in cancer patients.