Circulating nucleic acids in plasma and serum (CNAPS): applications in oncology.

The data reviewed in the present article suggest that the clinical value of cfNAs circulating in plasma is already more than a theoretical idea, since the characterization and the quantitation of such nucleic acids (NAs) have been shown to be complementary tools in the diagnosis, prognosis, and management of cancer patients ( Figure 2 ). However, some important questions remain unanswered, largely because most studies have been underpowered as well as because there has been no adequate standardization of laboratory techniques, which complicates the comparison of results from different groups. 3 , 9 , 10 Thus, it appears necessary to drive initiatives directed toward validating and verifying laboratory methods and procedures for required molecular tests before their use in clinical testing. For this, it might be useful to network, as has been demonstrated by successful previous initiatives. 167 Methods based on sequencing (genome-wide sequencing, exome sequencing, etc) appear to be the definitive tool for making the analysis of cfDNA for cancer monitoring useful, since it allows for personalized assays. 26 , 78 However, plasma cfNAs have yet many enigmatic aspects. Elucidation of these might lead us to a revision in the way the pathobiology of cancer is conceived. It is likely that many molecular pathways are involved in the origin of such NAs, since various results point in non-exclusive different directions. It has been postulated that the cfNAs circulating in plasma are not merely biological waste, but that they may be directly involved in the development of metastases, possibly through transfection-like uptake by susceptible cells. 168 This hypothesis, the “genometastasis theory,” is supported by strong evidence – namely, the observation that plasma from cancer patients can transfect and oncogenically transform cultured cells. 169 , 170 Regardless, no conclusive explanation of the circulation mechanism of cfNAs yet exists. Several groups have suggested a relationship between the presence of cfNAs in plasma and the presence of exosomes. 121 , 171 Thus, it appears feasible that at least part of plasma cfNAs circulates within exosomes. This finding correlates with the idea that plasma cfNAs might have a role in tumor progression, since it has been proven that exosomes are able to transfer their RNA content to cells, and that this RNA can be functional in its new location. 18 It has even been demonstrated that miRNAs are transferred during immune synapsis and are able to modulate gene expression in recipient cells. 172 Moreover, cross-talk between tumor-derived exosomes and host cells, such as bone marrow progenitor cells, appears undeniable. 173 Thus, transfer of genetic material from exosomes to cells might be involved in the recruitment and metastatic conversion of host cells. Conversely, it might be possible to exploit this phenomenon for therapeutic purposes and, perhaps, it will be proper to focus the development of clinical tools on the detection and analysis of NAs containing microparticles. Certainly, at this moment, a robust body of research on exosomes and microvesicles exists and increased understanding of such particles in future will shed light on cancer pathobiology and offer promising perspectives on clinical tools. Thus, although the origin and the circulation mechanism of plasma cfNAs remain unclear, there is strong evidence to suggest that such NAs will become useful biomarkers for the diagnosis and monitoring of cancer disease and that they might be essential to tumor development and progression.

The origin of the cfRNA circulating in plasma, its role, and its release mechanisms are yet unknown. The existence of cfRNA in blood was reported many years ago when, in 1987, Wieczorek et al 120 found RNA in proteolipid complexes in the serum of cancer patients. 121 However, the potential clinical value of the detection of cfRNA in plasma did not attract the attention of researchers until 1999, when two parallel studies reported the detection of tyrosinase mRNA in patients with metastatic melanoma 122 and mRNA associated with EBV in patients with nasopharyngeal carcinoma. 123 Many studies have subsequently reported the presence of specific mRNA in plasma from patients with a variety of cancers including colon, 124 – 126 breast, 127 , 128 prostate, 129 , 130 melanoma, 122 lung, 131 and thyroid. 132 It was theorized that the fragility of cfRNA in serum or plasma – due to the fast enzymatic degradation it undergoes, which is increased in cancer patients 133 – might make its detection difficult. However, it was found that the molecules are more stable than it was presumed, 134 possibly due to protection by vesicle-like structures. The origin of cfRNA remains less clear than that of cfDNA. 21 , 24 It has been proposed that apoptosis might be involved in the release of cfRNA, and that its association with apoptotic bodies might explain the resistance to nucleases in blood. 31 However, other hypotheses for the circulation of cfRNA have also been put forward, such as it occurring within lipoprotein complexes 24 or being in other actively-released particles, such as exosomes. 121 Ng et al 135 examined the particle-associated nature of circulating cfRNA by filtering plasma samples from healthy subjects and cancer patients through material with different pore sizes. They found greater amounts of particle-associated mRNA in cancer patients than in healthy subjects, suggesting that most of the cfRNA not associated with particles had degraded. 135 The use of cfRNA as a biomarker has several advantages including the ease with which plasma or serum samples for testing can be obtained, which makes it feasible for the monitoring of metastatic disease and even for wide screenings. However, RNA molecules are fragile, so high sensitivity techniques with simultaneous internal controls are necessary. 136 One of the most analyzed mRNAs in plasma is that of the human transcriptase reverse telomerase (hTERT), which corresponds to a ribonucleoprotein involved in the repair and lengthening of telomeres in eukaryotic cells. This mRNA is overexpressed in a variety of tumors. 124 , 126 , 128 , 129 , 137 – 140 However, cfRNA is not specific to cancer patients and can also be found in healthy volunteers or in those suffering trauma. 46 , 141 In breast cancer patients, using PCR-based techniques, hTERT mRNA has been detected in serum, even in patients with localized disease. 128 Silva et al 127 observed that analysis of specific mRNA epithelium (cytokeratin 19) in plasma, in combination with mammaglobin, facilitates the detection of a greater number of positive cases than does analysis of tumor cfDNA (73% versus [vs] 29%). Moreover, detection of epithelial mRNA has been found to be related to tumor size and proliferation rate. 127 In addition, El-Abd et al have suggested the utility of the detection of metastasin mRNA in serum as a survival marker, with high sensitivity (85%) and specificity (100%). 142 In colon cancer patients, an adequate correlation between hTERT levels in plasma and tumor stage has been observed, which has led such a quantitation to be proposed as a tool for screening, monitoring, 137 and response to treatment. 140 With respect to lung cancer, Miura et al 138 analyzed plasma samples from 112 patients and 80 healthy subjects to detect and quantify hTERT and epidermal growth factor receptor (EGFR) mRNAs. They found that the sensitivity and specificity in lung cancer diagnosis were, respectively, 89% and 73% for hTERT mRNA, and 71% and 80% for EGFR mRNA. Moreover, they found that the number of copies of hTERT mRNA significantly decreased after surgical treatment. 138 These data led the researchers to suggest that hTERT mRNA, especially when combined with EGFR mRNA, may be an excellent biomarker for pulmonary malignancies to diagnose and assess clinical stage. 138 In addition, it has been suggested that the detection of heterogeneous nuclear ribonucleoprotein-B1 mRNA and HER2/neu-specific mRNA might have diagnostic value in lung cancer. 143 In esophagus cancer patients, the detection of squamous cell carcinoma-antigen mRNA (SCC-Ag mRNA) by realtime PCR was shown to be the best predictive factor for recurrence in patients. 144 In hepatocellular cancer, the detection of hTERT mRNA in serum has been suggested to be of diagnostic value, with a sensitivity of 88% and a specificity of 70%. 139 Moreover, the researchers reported a good correlation between the levels of hTERT mRNA in plasma and clinicopathological parameters, such as degree of differentiation. 139 However, other studies have shown no correlation with clinicopathological variables such as tumor size. 145 cfRNA can be detected in other body fluids such as saliva and urine, and satisfactory results have been reported for its detection in these as a marker for the diagnosis of mouth and urological cancers, respectively. 136 The discovery of miRNA in 1993 was a milestone in cell biology research. From that moment, the number of identified miRNAs steadily increased and, to date, more than 900 human miRNAs have been discovered. “miRNAs” are small noncoding molecules of 18–22 nucleotides that regulate gene expression, acting at a post-transcriptional level. It is known that a unique miRNA is able to regulate multiple different mRNAs, so that it might potentially alter the function of multiple channels. 13 miRNAs have been demonstrated to play important roles in control of cell proliferation, cell differentiation, and apoptosis, thus their dysregulation contributes to tumor development. Moreover, it has been demonstrated that miRNAs can function as potential oncogenes or oncosuppressor genes, depending on the cellular context and on the target genes they regulate. 146 Aberrant expression of miRNAs has been associated with multiple types of cancer including colon, 147 – 150 lung, 151 – 154 breast, 155 prostate, 156 gastric, 157 and esophagus, 158 as well as lymphoma. 159 miRNAs have been detected in the plasma and serum of animals and humans, and in both healthy subjects and cancer patients. The first evidence that miRNAs might serve as serological biomarkers of solid tumors was provided by Mitchell et al 156 and Chen et al. 147 miRNAs circulating in plasma have some advantages as clinical markers compared with other forms of cfRNA, since they have a remarkable resistance to endogenous and exogenous ribonuclease activity, extreme pH conditions, and freeze–thaw cycles, whereas synthetic miRNAs are promptly degraded. 160 Circulating miRNAs have been found packaged into various membrane-bound vesicles such as exosomes, 18 microvesicles and apoptotic bodies (reviewed by Zandberga et al), 160 and also in lipoprotein complexes. 141 This is probably the main reason for the high resistance of plasma miRNAs, which makes their detection by PCR-based techniques easy. In addition, miRNAs are tissue specific, which allows the development of panels to help in the diagnosis of tumors of unknown origin. 156 , 161 , 162 Moreover, they are not only detectable in the bloodstream but also in other body fluids. 18 , 163 Thus, the use of miRNAs as serological biomarkers is a very attractive option and research in this field continues to increase. miR-21, probably the most analyzed miRNA, is often overexpressed in many types of tumors. 164 Nonetheless, a number of miRNAs have been demonstrated to have potential value as serological biomarkers in cancer, including miR-1, miR-10b, miR-17-92, miR-24, miR-92a, miR-122, miR-141, miR-155, miR-195, miR-221, and miR-375. 165 The diagnostic value of miRNA panels appears to be superior to that of individual miRNAs. 160 Specific expression patterns of serum miRNAs have been identified for lung cancer, colorectal cancer, and diabetes, providing evidence that serum miRNAs contain fingerprints for a variety of diseases. 147 It has been demonstrated that colorectal cancer patients have an miRNA serum profile significantly different to that of healthy subjects. Specifically, 69 miRNAs have been detected in sera from colorectal cancer patients but not in those from healthy subjects. 147 Moreover, colorectal cancer patients have been found to share a large number of serum miRNAs with lung cancer patients. 147 In addition, Ng et al 148 analyzed plasma samples from 90 colorectal cancer patients and 50 healthy subjects and found that miR-17-3p and miR-92 were overexpressed in patients, which suggested the potential diagnostic value of such plasma levels, with a sensitivity of 89% and specificity of 70%. Subsequent studies have supported Ng et al’s findings, including that by Huang et al, 149 who showed that levels of miR-29a and miR-92a in plasma discriminate colorectal cancer, with 83% sensitivity and 85% specificity. In a recent revision, the prediction value of three miRNAs (miR-221, miR-141, and miR-29a) has been highlighted as 3 independent factors of wrong predictions in different clinical stages. 166 The increased expression of miR-21, miR-106, and miR-15 has been observed in breast cancer patients compared with that observed in healthy subjects, in both tissues and serum, and those levels have been found associated with tumor stage and the presence of lymph node metastases. 155 With respect to lung cancer, Wang et al 152 and Liu et al 153 observed high levels of miR-21 in the plasma of cancer patients compared with that in healthy individuals. These levels were also associated with TNM stage and the presence of lymph node metastasis. Further, Roth et al 154 suggested that miR-361-3p and miR-625 might have a protective effect on the development of non-small cell lung cancer, while its quantification in serum might have the diagnostic potential to detect that cancer, particularly in smokers. The most comprehensive search to date for circulating miRNAs with prognostic significance was performed by Hu et al, 151 who analyzed a large cohort of 303 patients and found that four plasma miRNAs (miR-486, miR-30d, miR-1, and miR-449) were independent predictors of average survival. In prostate cancer, Mitchell et al 156 were able to distinguish patients with cancer from healthy individuals by serum levels of miR-141. Finally, Lawrie et al demonstrated that serum levels of miR-21 were associated with the disease-free interval in patients with diffuse B-cell lymphoma. 159 In future, the search for circulating miRNAs of clinical value is likely to be carried out at the same time as the study of many unknown aspects, such as the role of circulating miRNAs, the cell types that secrete them, and the regulation of this secretion.

According to the World Health Organization, cancer mortality will increase by 45% from 2007 to 2030 due to the demographic increase and population aging. 1 For that reason, many efforts have been made to find sensitive and specific biomarkers for early diagnosis, prognosis, and management of patients during treatment and follow-up. A variety of tumors secrete proteins into the bloodstream that are routinely used as tumor markers in clinical practice, such as the prostate-specific antigen in prostate cancer, the alpha-fetoprotein in hepatocarcinoma, the carcinoembryonic antigen (CEA) in colon cancer, the cancer antigen (CA) 15.3 in breast cancer, and the CA19.9 in pancreatobiliary tumors. 2 However, those techniques might lack sensitivity in nonsecretory tumors, and might give positive results due to inflammatory processes or benign illnesses, limiting their specificity. In addition, there are no known serum markers for most tumors, which highlights the need for extensive study of the biology of tumors to propose new clinical tools. Indeed, it is expected that an improvement in the knowledge of the molecular biology of cancer will lead to earlier diagnosis and more effective treatments. Cancer is produced and progresses as a consequence of complex and gradual processes, in which a variety of genetic and epigenetic alterations are involved (eg, mutations, hypermethylations), and which mainly result in uncontrolled cell growth. Those molecular alterations can be found in the bloodstream, 3 which suggests that we could find understanding of the molecular biology of a specific cancer in plasma ( Figure 1 ). This was the start point for the study of the so-called cell-free nucleic acids in plasma and serum (CNAPS) as cancer biomarkers. Although in the last decades the CNAPS concept has generated a considerable interest in the research community, the concept was first suggested many years before this. Specifically, Mandel and Métais 4 first reported the existence of cell-free DNA (cfDNA) in plasma in the mid-twentieth century. However, their study had scarce impact until 30 years later, when Leon et al 5 demonstrated that cancer patients had higher cfDNA levels than normal people and that patients with persistently high levels of cfDNA after treatment had a worse prognosis than those in whom cfDNA levels decreased. Some years later, Stroun et al 6 suggested that at least some of the cfDNA in serum or plasma was derived from the primary tumor, but this hypothesis was only confirmed later by two nearly parallel studies that described the presence of KRAS mutations in plasma from patients with pancreas neoplasm 7 and acute myelogenous leukemia. 8 Those discoveries were the milestones that opened new pathways for cancer biology research and the search for new clinical tools. The term “CNAPS” refers to different types of cell-free nucleic acids (cfNAs), such as genomic DNA (gDNA), mitochondrial DNA (mitDNA), viral DNA and RNA, messenger (m)RNA, and microRNA (miRNA), which have recently been reported to be present in plasma. 3 , 9 – 13 In spite of the increasing number of studies focused on CNAPS, and of the technological improvements, nowadays, some important aspects of CNAPS biology remain unknown, such as their release mechanisms, 14 method of circulation, 15 – 18 and biological role in cancer progression. 19 Limiting factors in the study of CNAPS have mainly been the lack of harmonization of laboratory techniques, the heterogeneity of the disease progression, and the small number of recruited patients in most of studies, which is for some authors 9 an example of poor translation of basic research into clinical practice.