The development of noninvasive methods to detect and monitor tumors continues to be a major challenge in oncology. We used digital polymerase chain reaction-based technologies to evaluate the ability of circulating tumor DNA (ctDNA) to detect tumors in 640 patients with various cancer types. We found that ctDNA was detectable in >75% of patients with advanced pancreatic, ovarian, colorectal, bladder, gastroesophageal, breast, melanoma, hepatocellular, and head and neck cancers, but in less than 50% of primary brain, renal, prostate, or thyroid cancers. In patients with localized tumors, ctDNA was detected in 73, 57, 48, and 50% of patients with colorectal cancer, gastroesophageal cancer, pancreatic cancer, and breast adenocarcinoma, respectively. ctDNA was often present in patients without detectable circulating tumor cells, suggesting that these two biomarkers are distinct entities. In a separate panel of 206 patients with metastatic colorectal cancers, we showed that the sensitivity of ctDNA for detection of clinically relevant KRAS gene mutations was 87.2% and its specificity was 99.2%. Finally, we assessed whether ctDNA could provide clues into the mechanisms underlying resistance to epidermal growth factor receptor blockade in 24 patients who objectively responded to therapy but subsequently relapsed. Twenty-three (96%) of these patients developed one or more mutations in genes involved in the mitogen-activated protein kinase pathway. Together, these data suggest that ctDNA is a broadly applicable, sensitive, and specific biomarker that can be used for a variety of clinical and research purposes in patients with multiple different types of cancer.

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Detection of Circulating Tumor DNA in Early- and Late-Stage Human Malignancies

http://image.sciencenet.cn/olddata/kexue.com.cn/upload/blog/file/2010/11/20101123154018731904.pdf

Presence of fetal DNA in maternal plasma and serum

1described the presence of cell-free nucleic acid (cfNA) in human blood for the first time. This attracted little attention in the scientific community and it was not until 1994 that the importance of cfNA was recognized as a result of the detection of mutated RAS gene fragments in the blood of cancer patients 2,3 (TIMELINE). In 1996, microsatellite alterations on cell-free DNA (cfDNA) were shown in cancer patients 4

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Cell-free nucleic acids as biomarkers in cancer patients

Increased levels of DNA fragments have frequently been found in the blood plasma of cancer patients. Published data suggest that only a fraction of the DNA in blood plasma is derived from cancer cells. However, it is not known how much of the circulating DNA is from cancer or from noncancer cells. By quantitative methylation-specific PCR of the promoter region of the CDKN2A tumor suppressor gene, we were able to quantify the fraction of plasma DNA derived from tumor cells. In the plasma samples of 30 unselected cancer patients, we detected quantities of tumor DNA from only 3% to as much as 93% of total circulating DNA. We investigated possible origins of nontumor DNA in the plasma and demonstrate here a contribution of T-cell DNA in a few cases only. To investigate the possibility that plasma DNA originates from apoptotic or necrotic cells, we performed studies with apoptotic (staurosporine) and necrotic (staurosporine plus oligomycin) cells in vitro and with mice after induction of apoptotic (anti-CD95) or necrotic (acetaminophen) liver injury. Increasing amounts of DNA were found to be released in the supernatants of cells and in the blood plasma samples of treated animals. A clear discrimination of apoptotic and necrotic plasma DNA was possible by gel electrophoresis. The same characteristic patterns of DNA fragments could be identified in plasma derived from different cancer patients. The data are consistent with the possibility that apoptotic and necrotic cells are a major source for plasma DNA in cancer patients.

https://cancerres.aacrjournals.org/content/canres/61/4/1659.full.pdf

DNA Fragments in the Blood Plasma of Cancer Patients: Quantitations and Evidence for Their Origin from Apoptotic and Necrotic Cells

Background The management of metastatic breast cancer requires monitoring of the tumor burden to determine the response to treatment, and improved biomarkers are needed. Biomarkers such as cancer antigen 15-3 (CA 15-3) and circulating tumor cells have been widely studied. However, circulating cell-free DNA carrying tumor-specific alterations (circulating tumor DNA) has not been extensively investigated or compared with other circulating biomarkers in breast cancer. Methods We compared the radiographic imaging of tumors with the assay of circulating tumor DNA, CA 15-3, and circulating tumor cells in 30 women with metastatic breast cancer who were receiving systemic therapy. We used targeted or whole-genome sequencing to identify somatic genomic alterations and designed personalized assays to quantify circulating tumor DNA in serially collected plasma specimens. CA 15-3 levels and numbers of circulating tumor cells were measured at identical time points. Results Circulating tumor DNA was successfully detected in 29 of the 30 women (97%) in whom somatic genomic alterations were identified; CA 15-3 and circulating tumor cells were detected in 21 of 27 women (78%) and 26 of 30 women (87%), respectively. Circulating tumor DNA levels showed a greater dynamic range, and greater correlation with changes in tumor burden, than did CA 15-3 or circulating tumor cells. Among the measures tested, circulating tumor DNA provided the earliest measure of treatment response in 10 of 19 women (53%). Conclusions This proof-of-concept analysis showed that circulating tumor DNA is an informative, inherently specific, and highly sensitive biomarker of metastatic breast cancer. (Funded by Cancer Research UK and others.)

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Analysis of Circulating Tumor DNA to Monitor Metastatic Breast Cancer

About one third of patients with various malignant diseases were found to have extractable amounts of DNA in their plasma whereas no DNA could be detected in normal controls. Using the test establishe

Neoplastic characteristics of the DNA found in the plasma of cancer patients.

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

Oncogene mutations are frequently found in several tumour types and, among these, point mutations of the ras gene are particularly significant. A predominance of N-ras mutations has been found in the bone marrow DNA of patients with myelodysplastic syndrome (MDS) or acute myelogenous leukaemia (AML). On the other hand, increased levels of plasma DNA have previously been observed in patients suffering from various malignant diseases. In the present work we have investigated, by polymerase chain reaction (PCR), point mutations of the N-ras gene in the DNA of plasma, blood cells and bone marrow of 10 patients suffering from AML or MDS. The different ras mutations detected in five cases were always present in the plasma DNA while sometimes absent in the DNA of peripheral blood cells or bone marrow. This indicates that a bone marrow biopsy or aspiration does not necessarily contain all the malignant clones involved in the disease. Plasma could thus prove to be an easily accessible and useful material for detection and monitoring of myeloid disorders.

Point mutations of the N-ras gene in the blood plasma DNA of patients with myelodysplastic syndrome or acute myelogenous leukaemia.

Although it is evident that DNA circulates freely in blood plasma both in disease and in health, the source of this DNA remains enigmatic. It can be presumed that circulating DNA in healthy subjects derives from lymphocytes or other nucleated cells. Yet, it is not known why cancer patients have such large quantities of plasma DNA. There is no doubt that a proportion seems to originate from nucleated blood cells since wild-type DNA has been detected in the plasma of all cancer patients studied as well as in that of healthy controls. However, a substantial proportion of plasma DNA in cancer patients derives from tumor cells. This latter concept is supported by both quantitative and qualitative observations. Plasma DNA levels are not only greater in cancer patients than in normal subjects,1–5 but also correlate inversely with outcome and tend to fall with effective treatment.1,5 In addition, the ability to detect LOH in plasma DNA6–13 suggests that tumor-related DNA is the predominant subtype in at least some cancer patients. What is responsible for the presence of tumor DNA shed in the bloodstream? It could be due to lysis of circulating cancer cells or of micrometastases, to DNA leakage resulting from tumor necrosis or apoptosis, or to a new mechanism of active release.

C. Lederrey

Papers

Neoplastic characteristics of the DNA found in the plasma of cancer patients.

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

Point mutations of the N-ras gene in the blood plasma DNA of patients with myelodysplastic syndrome or acute myelogenous leukaemia.

The origin and mechanism of circulating DNA.

Isolation and characterization of DNA from the plasma of cancer patients.