Source of Cell-Free DNA
A number of sources appear to contribute to circulating cell-free DNA. A baseline level of circulating DNA is generally present, even in healthy individuals, indicating normal turnover of nucleated blood cells such as lymphocytes may play a role (Anker et al, 1999; Leon et al, 1977). The elevated levels of circulating DNA observed in cancer patients by researchers may be related to neoplastic cells, however this was not verified until the 1990's, when several reports were published showing that circulating DNA exhibited various tumour-like alterations. Sorenson et al., 1994 identified a mutated K-ras sequence in the plasma of pancreatic cancer patients whose tumours also contained the mutated K-ras gene. Papers published by Chen et al. 1996 and Nawroz et al. 1996 demonstrated that DNA from the plasma of cancer patients contained the same microsatellite alterations as DNA from the patients' primary tumours. Another study published by de Kok et al. 1997 found corresponding K-ras mutations in the primary tumours and plasma of several colorectal cancer patients.
Additionally, circulating DNA from cancer patients were found to have decreased strand stability, oncogene and tumour suppressor alterations, microsatellite alterations, p53 mutations, and many other characteristics in common with neoplastic cells (Anker et al., 2003; Anker et al., 1999). While levels of circulating DNA may be related to natural cell turnover and cell lysis of healthy cells and neoplastic cells, it is unlikely all circulating DNA can be attributed to cell lysis/apoptosis. Approximately ten thousand tumour cells would need to be circulating per millilitre of blood in order to produce the reported elevated cell-free DNA levels, and this number of cells has never been seen. It has been observed that normal cells in culture can spontaneously release a nucleoprotein complex, and cancer cells release more DNA than normal cells. The increased DNA levels seen in cancer patients may therefore be due to an excretion/leakage process by cancerous/diseased cells (Anker et al., 1999).
There are appears to be two main clinical avenues to pursue in the use of circulating DNA. The first use of circulating DNA would be to monitor for disease recurrence or to establish disease free status of an individual. The second use would be as a prognostic tool to detect early stage cancers that may be amenable to treatment or surgical resection and cure. However, it is clear that more work and larger-scale studies are essential to evaluate circulating cell-free DNA.
While no correlation has yet been found between circulating DNA levels and the size or location of the primary tumour, circulating DNA has already been proposed by scientists for diagnostic and prognostic applications (Anker et al., 2003; Chan et al. 2003). And one recent review predicted that analysis of circulating nucleic acids will be incorporated into the diagnosis and monitoring of cancer patients in less than five years (Goessl 2003). Wu et al. (2002) found that elevated serum cell-free DNA is usually detected in specimens containing elevated tumour markers, such as CA 15-3 (a marker for breast cancer) and PSA (a marker for prostate cancer). These and other tumour markers are currently used to screen for cancer, monitor disease progression, evaluate treatment efficacy, and to predict disease recurrence (Chatterjee & Zetter 2005). Circulating DNA may therefore have the potential to be a useful tumour marker, and could complement established markers.
One promising new development involves the detection of Epstein-Barr virus (EBV) DNA in serum, which has been shown to be highly correlated with the nonsquamous type of nasopharyngeal carcinoma, NPC (Hsiao et al. 2002). Using 50-cycle PCR analysis, Hsiao et al. (2002) detected EBV DNA in the serum of 75% of patients with primary NPC, 36.5% of patients with NPC in clinical remission, and only 10.7% of control patients. This study also demonstrated that longitudinal tracking of patients allowed for detection of recurrent disease with high sensitivity using the circulating EBV DNA approach. Thus, circulating DNA may be used in follow-up monitoring of patients who have already been diagnosed with certain cancer types, to monitor for disease recurrence or to establish disease free status. Analysis of specific DNA sequences in the blood, as well as total circulating DNA concentrations, are important for the future of cancer diagnosis, prognosis, and follow-up tracking.
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