Circulating nucleic acids in plasma and serum as a noninvasive investigation for cancer: Time for large‐scale clinical studies?

A noninvasive blood test to detect sporadic cancer has been seen as somewhat of a holy grail by clinicians with an interest in cancer, and its delivery as a quest for many researchers. For this reason, detection of cell-free circulating DNA in the plasma and serum of cancer patients, which has genetic characteristics identical to those of the primary tumour, has resulted in substantial interest and over 200 publications in the medical literature. Interest stems not only from the fact that a blood-based diagnostic and screening test for cancer is an elegant and attractive concept in its own right but also from the fact that conventional diagnostic cancer tests tend to be imperfect.1 As an example, colorectal cancer screening presently relies on faecal occult blood testing, which is both insensitive and nonspecific. In contrast, flexible sigmoidoscopy is sensitive and specific for early distal disease but both invasive and insensitive for proximal disease. Furthermore, barium enema is relatively sensitive and specific but requires colonic preparation, radiation and a day off work, while total colonoscopy is highly sensitive and specific but also invasive and expensive. The situation appears little better for other cancers. No reliable test is available for early detection of lung cancer, with computerised tomography being the most reliable tool. In addition, although several studies indicate that mammographic screening might be a useful strategy for reducing breast cancer mortality, there remains considerable controversy regarding the value of population screening programs.2 Finally, development of conventional tumour markers, e.g., CEA, AFP and the widely used PSA, was driven largely by the introduction of new methods for quantifying small amounts of circulating proteins. However, sensitivity and specificity shortcomings with these assays remain to be overcome.3 The introduction of PCR-based technology in the late 1980s and refinements over the past 10 years have allowed us to detect and quantify extremely small amounts of nucleic acids. This has led to the identification of large numbers of novel molecular targets that may eventually become clinically useful cancer markers. Additionally, many of these markers have been detected in tumourderived nucleic acids (DNA and RNA) extracted from serum and plasma samples. However, it is over 30 years since initial studies indicated that plasma-based nucleic acids might assist in cancer diagnosis. Studies performed in the early 1970s initially showed that increased quantities of DNA could be found in the plasma of patients suffering from different malignancies,4 but it was not until the 1990s that this circulating DNA was shown to exhibit tumourrelated alterations, including decreased strand stability,5 Ras and p53 mutations, microsatellite alterations, aberrant promoter hypermethylation of several genes, rearranged immunoglobulin heavy chain DNA, mitochondrial DNA mutations and tumour-related viral DNA.6,7 Over this period, it was also shown that tumourrelated circulating DNA was not confined to any particular cancer type but appeared to be a ubiquitous finding across the cancer spectrum. Thus, mutant plasma DNA has been found in colorectal, pancreatic, biliary tree, skin, head-and-neck, lung, breast, kidney, ovarian, nasopharyngeal, liver, bladder, gastric, prostate and cervical cancers as well as in haematologic malignancies including lymphomas. The results obtained in plasma/serum DNA in many cancers are opening new research areas and indicate that plasma/ serum may eventually be a suitable source for the development of noninvasive diagnostic, prognostic and follow-up tests for cancer. The diagnostic value of plasma DNA testing appears promising in a number of cancers including melanoma,8 B-cell malignancies9 and NPC.10 However, perhaps the closest to achieving clinical significance is an assay for EBV DNA, which is closely associated with NPC in southern Asia. Using real-time PCR, EBV DNA is detectable in 95% of NPC cases compared 5% of healthy controls. In addition, following diagnosis, the test appears to be useful for determining prognosis and monitoring disease response to treatment. Overall, the absolute levels of EBV DNA at presentation are of considerable prognostic value as are levels following treatment, in so far as a high level is suggestive of the presence of residual disease. It appears likely that in the next few years estimation of EBV DNA will become a routine part of the staging procedure for NPC and will directly influence therapeutic options for this tumour. The prognostic value of plasma/serum tumor DNA has also been established for other cancers, with high levels also indicative of a poor prognosis.6,7 Surprisingly, using essentially similar methodology, cell-free mRNA can also be detected in plasma and should, at least in theory, permit plasma-based expression profiling.11 Studies with RNA markers are particularly promising due to their close association with malignancy. In this short review, we emphasise studies on the most widespread malignancies, colorectal, pancreatic, lung, breast and prostate cancers, which need a simple test that could become clinically available in the not too distant future.