Origins, structures, and functions of circulating DNA in oncology.

While various clinical applications especially in oncology are now in progress such as diagnosis, prognosis, therapy monitoring, or patient follow-up, the determination of structural characteristics of cell-free circulating DNA (cirDNA) are still being researched. Nevertheless, some specific structures have been identified and cirDNA has been shown to be composed of many "kinds." This structural description goes hand-in-hand with the mechanisms of its origins such as apoptosis, necrosis, active release, phagocytosis, and exocytose. There are multiple structural forms of cirDNA depending upon the mechanism of release: particulate structures (exosomes, microparticles, apoptotic bodies) or macromolecular structures (nucleosomes, virtosomes/proteolipidonucleic acid complexes, DNA traps, links with serum proteins or to the cell-free membrane parts). In addition, cirDNA concerns both nuclear and/or mitochondrial DNA with both species exhibiting different structural characteristics that potentially reveal different forms of biological stability or diagnostic significance. This review focuses on the origins, structures and functional aspects that are paradoxically less well described in the literature while numerous reviews are directed to the clinical application of cirDNA. Differentiation of the various structures and better knowledge of the fate of cirDNA would considerably expand the diagnostic power of cirDNA analysis especially with regard to the patient follow-up enlarging the scope of personalized medicine. A better understanding of the subsequent fate of cirDNA would also help in deciphering its functional aspects such as their capacity for either genometastasis or their pro-inflammatory and immunological effects.

Circulating DNA in higher organisms cancer detection brings back to life an ignored phenomenon.

The concept of circulating DNA is derived from the early transformation experiments on bacteria. We describe first experiments done with graft hybrids which could be due to circulating DNA. Work on uptake of foreign DNA by eukaryotic cells is then reported. This work led us to discover the phenomenon of transcession in plants and animals where we showed that bacterial DNA can spontaneously enter cells of eukaryotes and be transcribed. We then outline the fact that living cells spontaneously release DNA within a homeostatic mechanism. Finally we describe in more detail some experiments suggesting that an exchange of DNA between T and B lymphocytes may have immunological implications. In this work, nude mice injected with DNA excreted by antigen stimulated human T lymphocytes produced specific antibodies expressing human characteristics.

Alu repeat sequences are present in increased proportions compared to a unique gene in plasma/serum DNA: evidence for a preferential release from viable cells?

Small amounts of DNA circulate freely in plasma or serum, but the mechanism of release is not known. To determine if DNA is actively excreted from viable cells, we utilized real-time PCR to measure the proportion of Alu repeat sequences compared to the beta-globin gene in serum and lymphocyte DNA in 27 cancer patients and 22 healthy controls. The proportion of Alu compared to beta-globin was significantly greater in serum DNA than in lymphocyte DNA both in control subjects (p = 0.003) and in cancer patients (p < 0.001). Overall, the proportion was similar in cancer and control patients (p = 0.79). Further experiments showed that the beta-globin gene was not more vulnerable to degradation by nuclease action than Alu sequences. Our results lead us to conclude that active DNA release is likely to play a significant role in the origin of circulating DNA.

About the possible origin and mechanism of circulating DNA apoptosis and active DNA release.

BACKGROUND: In addition to cell lysis, apoptosis has been advanced as the origin of circulating DNA on the basis of several observations. Plasma or serum DNA often presents a ladder pattern reminiscent of that displayed by apoptotic cells when subjected to electrophoresis. However, the phenomenon of active release of DNA from cells might also be expected to result in a ladder pattern on electrophoresis. Non-dividing cells, such as lymphocytes, frog auricles and cultured cell lines including HL-60, spontaneously release a nucleoprotein complex within a homeostatic system in which newly synthesized DNA is preferentially released. CONCLUSION: In relation to DNA synthesis, the phenomenon of extracellular DNA in different culture conditions favors apoptosis or spontaneous active DNA release.

Circulating nucleic acids in plasma or serum.

BACKGROUND: Nucleic acids can be found in small amounts in healthy and diseased human plasma/serum. Higher concentrations of DNA are present in the plasma of cancer patients sharing some characteristics with DNA of tumor cells. Together with decreased strand stability, the presence of specific oncogene or tumor-suppressor gene mutations, microsatellite alterations, Ig rearrangements and hypermethylation of several genes may be detected. Moreover, tumor-related mRNA has been found circulating in the plasma/serum. CONCLUSIONS: The results obtained in many different cancers have opened a new research area indicating that circulating nucleic acids might eventually be used for the development of noninvasive diagnostic, prognostic and follow-up tests for cancer.

Microsatellite alterations and TP53 mutations in plasma DNA of small-cell lung cancer patients: follow-up study and prognostic significance.

BACKGROUND: Small-cell lung cancer (SCLC), one of the major types of lung cancer, is associated with many different somatic molecular genetic changes. These alterations, observed in tumor DNA, have also been identified in the plasma DNA of patients. We undertook the present study to make a prospective investigation into the correlation between abnormal plasma DNA and patient survival. PATIENTS AND METHODS: Thirty-five patients with SCLC were selected after histological diagnosis. Polymorphic markers (ACTBP2, UT762 and AR) were chosen for their reported high rate of alterations in SCLC and analyzed in tumor tissue, normal blood cells and plasma DNA. Furthermore, we looked for mutations of the TP53 gene in tumor and plasma DNA. RESULTS: In 25 patients (71%) at least one molecular change precisely matching that of the primary tumor was detected in the plasma DNA. No difference in survival was observed between patients with aberrant plasma DNA and patients without plasma DNA alterations. However, patients with microsatellite modifications and TP53 mutations concomitantly, showed a significant difference (P = 0.02) in survival compared with patients bearing only one of these molecular changes. In 15 cases it was possible to find a correlation either between tumor response and disappearance of abnormal plasma DNA, or tumor progression and persistence of plasma DNA alterations. CONCLUSIONS: Free plasma DNA with molecular alterations is present to a high degree in plasma DNA of SCLC patients and may have a role as a prognostic factor.

Telomerase RNA as a detection marker in the serum of breast cancer patients.

Tumor-derived circulating DNA has been found in the plasma of cancer patients. Alterations include decreased strand stability, mutations of oncogenes or of tumor suppressor genes, microsatellite alterations, and hypermethylation of several genes. RNA has also been found circulating in the plasma of normal subjects and cancer patients. Tyrosinase mRNA has been extracted from the serum of melanoma patients and subjected to RT-PCR. Moreover, the presence of cell-free EBV-associated RNA has been reported in the plasma of patients with nasopharyngeal carcinoma. Human telomerase comprises two RNA subunits, telomerase RNA template (hTR) and its catalytic component, telomerase reverse transcriptase protein (hTERT). Expression of these subunits correlates with telomerase activity. Using RT-PCR, we investigated whether these RNA subunits were present in the serum of 18 patients with breast cancer, 2 patients with benign breast disease, and 21 normal subjects. The presence of amplifiable RNA was confirmed in all tissue and serum samples using RT-PCR of glyceraldehyde-3-phosphate dehydrogenase RNA. hTR was found in 17 of 18 tumors (94%) and 5 of 18 serum samples (28%). hTERT was also detected in 17 of 18 tumors (94%) and in 4 of 16 available serum samples (25%). hTR and hTERT were undetectable in tissues and sera taken from 2 patients with benign disease and in the sera of 21 normal subjects. We conclude that RNA is detectable in the serum of breast cancer patients and that tumor-derived mRNA can be extracted and amplified using RT-PCR, even in patients with localized disease. This may have implications for cancer diagnosis and follow-up in the future.

Circulating DNA in plasma or serum.

Small amounts of DNA circulate in both healthy and diseased human plasma/serum, and increased concentrations of DNA are present in the plasma of cancer patients. Characteristics of tumor DNA have been found in genetic material extracted from the plasma of cancer patients. These features include decreased strand stability, the presence of specific oncogene or tumor suppressor gene mutations, microsatellite alterations, Ig rearrangements and hypermethylation of several genes. The results obtained in many different cancers have opened a new research area indicating that plasma DNA might eventually be a suitable target for the development of noninvasive diagnostic, prognostic and follow-up tests for cancer. Following the discovery of tumor derived DNA in plasma or serum, cell-free fetal DNA has also been found in maternal plasma and serum. This discovery provides an easily accessible source of fetal genetic material for prenatal diagnosis.

Circulating DNA in plasma or serum

Small amounts of DNA circulate in both healthy and diseased human plasma/serum, and increased concentrations of DNA are present in the plasma of cancer patients. Characteristics of tumor DNA have been found in genetic material extracted from the plasma of cancer patients. These features include decreased strand stability, the presence of specific oncogene or tumor suppressor gene mutations, microsatellite alterations, Ig rearrangements and hypermethylation of several genes. The results obtained in many different cancers have opened a new research area indicating that plasma DNA might eventually be a suitable target for the development of noninvasive diagnostic, prognostic and follow-up tests for cancer. Following the discovery of tumor derived DNA in plasma or serum, cell-free fetal DNA has also been found in maternal plasma and serum. This discovery provides an easily accessible source of fetal genetic material for prenatal diagnosis.

Circulating DNA in plasma or serum.

Small amounts of DNA circulate in both healthy and diseased human plasma/serum, and increased concentrations of DNA are present in the plasma of cancer patients. Characteristics of tumor DNA have been found in genetic material extracted from the plasma of cancer patients. These features include decreased strand stability, the presence of specific oncogene or tumor suppressor gene mutations, microsatellite alterations, Ig rearrangements and hypermethylation of several genes. The results obtained in many different cancers have opened a new research area indicating that plasma DNA might eventually be a suitable target for the development of noninvasive diagnostic, prognostic and follow-up tests for cancer. Following the discovery of tumor derived DNA in plasma or serum, cell-free fetal DNA has also been found in maternal plasma and serum. This discovery provides an easily accessible source of fetal genetic material for prenatal diagnosis.

Detection of circulating tumour DNA in the blood (plasma/serum) of cancer patients.

Small amounts of free DNA circulate in both healthy and diseased human plasma/serum, and increased concentrations of DNA are present in the plasma of cancer patients. Characteristics of tumour DNA have been found in genetic material extracted from the plasma of cancer patients. These features include decreased strand stability and the presence of specific oncogene, tumour suppressor gene and microsatellite alterations. Point mutations of the ras genes have been detected in the plasma DNA of patients suffering from haematopoetic malignancies, colorectal and pancreatic cancer, sometimes prior to clinical diagnosis. Rearranged immunoglobulin heavy chain DNA has been found in the plasma of patients with non-Hodgkins lymphoma and acute B cell leukaemia. Microsatellite instability, expressed either as a new allele or a loss of one allele (LOH) occurs in the plasma and serum DNA of patients suffering from head and neck, lung and renal cell cancer. The results obtained in many different cancers have opened a new research area indicating that plasma DNA might eventually be a suitable target for the development of non-invasive diagnostic, prognostic and follow-up tests for cancer.

Detecting tumor-related alterations in plasma or serum DNA of patients diagnosed with breast cancer.

Chromosomal abnormalities are associated with the development of breast cancer, and widespread allelic loss or imbalance is frequently found in tumor tissues taken from patients with this disease. Using different markers, we studied a total of 61 patients (divided into three groups) for the presence of microsatellite instability and loss of heterozygosity (LOH) in plasma or serum DNA. Of the initial 27 patients, 35% of the tumor samples displayed LOH, whereas 15% had identical alterations in the corresponding plasma samples. In addition, the adjacent normal breast tissue of two patients also displayed LOH. In a second group of 11 patients, 45% of the tumors displayed LOH, and 27% displayed identical plasma DNA alterations; one case displayed an identical LOH in adjacent nontumor tissue. In a third series of 23 patients also studied with tetranucleotide repeats, 81% of the tumor samples displayed LOH, whereas 48% had LOH in the corresponding serum samples. The fact that small tumors (T1) of histoprognostic grade 1 or in situ carcinomas could present DNA alterations in the plasma/serum at an early stage, allied to the widely increased range of available microsatellite markers, suggests that plasma or serum DNA may become a useful diagnostic tool for early and potentially curable breast cancer.

Microsatellite analysis of plasma DNA from patients with clear cell renal carcinoma.

Deletions of DNA sequences on chromosome 3p [loss of heterozygosity (LOH)] are characteristic of clear cell renal carcinoma, which accounts for about 80% of all renal malignancies. Comparing tumor DNA to DNA from normal cells, LOH analysis of microsatellite sequences has aided in molecular diagnosis of renal carcinoma. Because clinically useful tumor markers do not exist for this cancer entity, the aim of the present study was to detect chromosome 3p microsatellite alterations (LOH and microsatellite instability) in plasma DNA from patients with clear cell renal carcinoma. Four chromosome 3p microsatellites (D3S1307, D3S1560, D3S1289, and D3S1300) were amplified by fluorescent PCR using DNA isolated from normal blood cells and plasma of 40 patients. Corresponding tumor DNA was available from 21 patients. Analyzing PCR products on an automated DNA sequencer, we found LOH in at least one locus in 25 plasma samples (63%), and 14 plasma samples (35%) exhibited LOH at more than one locus. Microsatellite instability of plasma DNA was detectable in one patient (3%). No significant association of advanced (>T2N0M0) tumor stages with LOH in plasma DNA could be demonstrated. If present, modifications of plasma DNA and tumor DNA were identical. No alterations of plasma DNA were found in healthy controls. Analysis of plasma DNA from patients with clear cell renal carcinoma reveals tumor-specific microsatellite alterations and may therefore have diagnostic potential as a molecular tumor marker.

A prospective study of K-ras mutations in the plasma of pancreatic cancer patients.

K-ras mutations are frequently found in primary pancreatic adenocarcinomas. In this prospective study, we looked for K-ras mutations in the plasma of patients with pancreatic cancer. We isolated plasma DNA from 21 pancreatic cancer patients using a simple and rapid extraction technique and detected K-ras alterations with a PCR assay and subsequent product sequencing. Patients were followed up to determine their clinical outcome. We found K-ras mutations in the plasma of 17 patients (81%). In cases in which both plasma and pancreatic tissue were available, DNA mutations were similar in corresponding plasma and tissue samples. Plasma DNA alterations were found 5-14 months before clinical diagnosis in four patients. Mutant DNA was not found in the plasma of two patients with chronic pancreatitis or in five healthy controls. Our results indicate that K-ras mutations are often found in DNA isolated from the plasma of pancreatic cancer patients and that a noninvasive plasma-based assay may provide qualitative diagnostic information to clinicians in the future. Larger studies are required to further assess the relevance of our findings to clinical practice.

The use of genetic instability as a clinical tool for cancer diagnosis.

Human tumors exhibit two fundamentally important characteristics, extensive genetic alteration and clonality. Although it is still unclear to what extent tumors have an elevated mutational burden as compared with normal tissue, their clonality results in their ready detection. Thus, assaying tissues for clonal alterations at frequently mutated microsatellite loci represents a viable approach to cancer diagnosis. The most remarkable extension of this concept is that not only can cancer cells be detected in biological samples, but tumor DNA can also be directly detected in the serum or plasma of patients with some forms of cancer. This recent finding is currently being explored but may represent an important contribution to future diagnostic strategies.

K-ras mutations are found in DNA extracted from the plasma of patients with colorectal cancer.

BACKGROUND & AIMS: Circulating DNA can be isolated from the plasma of healthy subjects and from patients with cancer. The aim of this study was to detect K-ras mutations in DNA extracted from the plasma of patients with colorectal cancer. METHODS: Tumor and plasma DNA were extracted from 14 patients with colorectal cancer (stages A-D), and K-ras alterations were detected using a polymerase chain reaction assay that uses sequence-specific primers to amplify mutant DNA. These results were confirmed with another polymerase chain reaction assay that creates an enzyme restriction site in the absence of a K-ras mutation followed by direct sequencing and additional cloning techniques. RESULTS: Seven patients (50%) had a codon 12 K-ras mutation within their primary tumor, and identical mutations were found in the plasma DNA of 6 patients (86%). Mutant DNA was not detected in the plasma specimens of 7 patients whose tumors tested negative for K-ras alterations or in healthy control subjects. Similar results were obtained using all three molecular biological techniques. CONCLUSIONS: K-ras abnormalities can be detected in circulating DNA extracted from the plasma specimens of patients with colorectal cancer. If these results are confirmed in larger studies, genetic analysis of plasma DNA may have clinical applications in the future.

Microsatellite alterations in plasma DNA of small cell lung cancer patients.

Microsatellite instability is an important characteristic of many tumor types especially those associated with hereditary non-polyposis colorectal carcinoma (HNPCC) syndrome. Microsatellite alterations in 50% of primary small cell lung carcinoma (SCLC) have been found. These alterations were also found in the sputum. Because neoplastic characteristics such as decreased strand stability9 and ras mutations have been found in the plasma DNA of cancer patients, we looked for microsatellite alterations in the plasma of SCLC patients. A microsatellite alteration was present in 16 out of 21 (76%) SCLC tumors and in 15 out of 21 (71%) plasma samples. In one case, the alteration was present only in the plasma DNA. If confirmed in larger studies, microsatellite analysis of plasma DNA might constitute a new tool for tumor staging, management and, possibly, detection.