Review
Circulating free DNA in plasma or serum as biomarker of carcinogenesis: Practical aspects and biological significance

https://doi.org/10.1016/j.mrrev.2006.11.002Get rights and content

Abstract

The presence of small amounts of tumor DNA in cell free DNA (CFDNA) circulating in the plasma or serum of cancer patients was first demonstrated 30 years ago. Since then, overall plasma DNA concentration in cancer patients and genetic or epigenetic alterations specific to tumor DNA have been investigated in patients diagnosed with different types of cancer. The proportion of patients with altered CFDNA varies with the pathology and the nature of the marker. However, several studies have reported the presence of altered CFDNA in over 50% of cancer patients, suggesting that this marker may be common and amenable for a variety of clinical and epidemiological studies. Because the mechanisms and timing of CFDNA release in the blood stream are poorly understood, only few studies have addressed the use of CFDNA for early cancer detection or as a biomarker for mutagenesis and tumourigenesis in molecular epidemiology. In this review, we discuss the technical issues involved in obtaining, using and analyzing CFDNA in cancer or healthy subjects. We also summarize the literature available on the mechanisms of CDNA release as well as on cross-sectional or prospective studies aimed at assessing the clinical and biological significance of CFDNA. These studies show that, in some circumstances, CFDNA alterations are detectable ahead of cancer diagnosis, raising the possibility of exploiting them as biomarkers for monitoring cancer occurrence. Testing these hypotheses will require well-designed studies, assessing multiple markers with quantitative and sensitive methods, with adequate follow-up of subjects, and we provide recommendations for the development of such studies.

Introduction

The presence of abnormally high levels of free circulating DNA (CFDNA) in the plasma/serum of cancer patients was demonstrated in 1977 [1]. However, it is only recently that CFDNA in cancer has attracted attention and that its possible use as a marker for diagnosis or prognosis has been investigated. Mutations in CFDNA have been characterized in a large variety of cancer types and sites, including, for example, colorectal, pancreas, lung, bladder, head and neck and liver cancers [2], [3], [4], [5], [6], [7]. Various types of DNA alterations have been reported in CFDNA, including point mutations, DNA hypermethylations, microsatellite instabilities (MI) and losses of heterozygosity (LOH). In many instances, these alterations were identical to the ones found in the primary tumor tissue of the patient, supporting the tumoral origin of altered CFDNA. Occurrence of alterations in CFDNA, as well as increase in the overall level of CFDNA, is not restricted to any particular tumor site, type or grade. However, there is tendency for significantly larger amounts of CFDNA in patients with late stage disease and metastasis. Thus, CFDNA may provide a very valuable source of genetic material as a surrogate for molecular analysis in cancer and pre-cancer patients.

There are multiple potential uses for CFDNA in cancer diagnosis and prognosis. For example, CFDNA can be used to detect somatic alterations when biopsies are not available. It may also represent a valuable source of tumor DNA when the exact position of a suspected primary lesion is not clearly defined. Moreover, it may provide a useful way to detect early relapse during post-treatment follow-up of patients with defined mutations in the primary lesion. Aside from these clinical applications, CFDNA may also represent a very important source of biomarkers in population-based studies. The fact that CFDNA can be obtained without invasive or painful procedures makes it particularly suitable for studies on normal subjects performed outside a clinical set-up.

Molecular epidemiology is interested in measuring a number of biological processes that occur between exposure of the population to risk factors and the final outcome in some individuals, of the disease. Fig. 1 illustrates the possible applications of CFDNA as a source of biomarker in molecular epidemiology of cancer. With respect to carcinogenesis, these processes include a series of defined steps. First, the internal dose of particular carcinogens to which individuals have been exposed may be assessed, at least in some instances, by measuring the level of the carcinogen or its derivatives in blood, urine or tissues of exposed subjects [8]. Second, mutagenesis (represented by a yellow box in Fig. 1) involves carcinogen metabolism, formation of DNA damage, correction by DNA repair, eventually resulting in the fixation of DNA alterations. The rate of mutation formation is influenced by genetic susceptibility. Each step in the mutagenesis process offers several biomarkers, such as measurement of metabolic or detoxifying enzymatic activities, levels of specific forms of DNA damage, efficiency of DNA repair or detection of acquired, somatic mutations in “reporter” genes. Third, selection of cells with mutations (blue box) occurs through expression of functional changes that drive clonal expansion. This step can be monitored by the detection of mutations in specific, “cancer” genes, which become detectable by virtue of their increase in copy numbers resulting from clonal expansion. This process may lead to pre-neoplastic lesions which can be monitored using a range of molecular pathology and clinical markers and, eventually, to cancer. In this complex process, CFDNA may be useful as a biomarker at several steps, including the late stages of mutagenesis, clonal expansion, early detection of preneoplastic lesions and monitoring of cancer.

In this review, we briefly describe the characteristics of CFDNA, the current hypotheses for its origin and the technical requirements and limitations for its detection and analysis. We also provide a discussion on its possible use in molecular epidemiology, based on recent studies. The implications of CFDNA for molecular pathology have been addressed in several reviews [9], [10], [11].

Section snippets

Occurrence of CFDNA

CFDNA is present in healthy subjects at concentrations between 0 and 100 ng/ml of blood with an average of 30 ng/ml [9]. Assuming that the DNA content of a normal cell amounts to 6.6 pg, these values represent an average of 0–15,000 genome equivalents per ml of blood with an average of 5000 genomes per ml. In systemic lupus erythematosus (SLE) (OMIM# 152700), as well as in arthritis, hepatitis and cancer patients, CFDNA often occurs at higher levels than in normal subjects. In cancer patients, the

Plasma or serum

In the now increasing literature on CFDNA, plasma or serum are often used indifferently. Isolation of serum or plasma on different days after blood withdrawal has demonstrated that serum DNA was contaminated by blood cell DNA and that the amount of contamination increased with the number of days between blood withdrawal and serum isolation [27], [28]. We have measured CFDNA concentrations in the plasma of healthy subjects who were recruited in 23 centers in Europe [29]. In this study, the

Associations between altered CFDNA and disease in cross-sectional studies

The differences in concentrations between cancer patients and healthy subjects have raised the possibility that levels of CFDNA could be used as a tool for early cancer detection and/or prognosis. In a large cross-sectional study including different neoplastic and non-neoplastic patients, and healthy subjects, Chang et al. have shown that there was a highly significant statistical difference in CFDNA concentration between the different groups [39]. However, they could not determine a cut-off

Conclusion and perspectives

In the past 10 years, there has been an explosion in the number of studies analyzing CFDNA in cancer, establishing it as a credible surrogate tissue and a possible tool for the management of cancer patients. However, the definition of its applications in clinical and population studies awaits better knowledge of the mechanism of DNA release in blood and the timing of its occurrence with respect to disease progression. At present, the main advantages of CFDNA as a biomarker is its availability

Acknowledgments

The authors of this paper are partners of European Cancer Risk, Nutrition and Individual Susceptibility (ECNIS), a network of excellence operating within the European Union 6th Framework Program, Priority 5: “Food Quality and Safety” (Contract No. 513943). The work on plasma DNA at IARC was partially supported by the Compagnia di San Paolo (Torino, Italy), the Lega Italiana per la lotta contro I tumori and the European Community (GENAIR programme, QL4-1999-000927).

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