Maurice Stroun

Neoplastic Characteristics of the DNA Found in the Plasma of Cancer Patients

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 established by one of us (M.B.), which is based on decreased strand stability of cancer cell DNA, we have found that several plasma DNA originate from cancer cells.

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.

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.

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

Summary. 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 myelodysplatic 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.

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

UNLABELLED Ten out of 37 patients with advanced malignant diseases were found to have extractable amounts of DNA in their plasma whereas no DNA could be detected in 50 normal controls. After its purification from the original nucleoprotein complex, DNA plasma levels ranging from 0.15 to 12 micrograms/ml were measured, the lowest concentration detectable with our method being 0.1 microgram/ml. Knowing from recovery experiments performed with 32P-DNA that the loss of DNA during the extraction procedure is about 65%, the real concentration of DNA in the plasma corresponds to about 3 times the given figures. The purified DNA was shown to be double-stranded and composed of fractions ranging from 21 kb to less than 0.5 kb, as determined by agarose gel electrophoresis. All these fractions hybridized with a 32P-labelled human DNA probe indicating the human origin of the bulk of the circulating DNA. IN CONCLUSION the finding of extractable amounts of DNA in the plasma of 27% of the investigated cancer patients, and its absence from the controls, suggests some correlation with malignancy.

The origin and mechanism of circulating DNA.

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.

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.

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.

Plasma DNA microsatellite panel as sensitive and tumor-specific marker in lung cancer patients

The majority of lung cancer patients have tumor‐derived genetic alterations in circulating plasma DNA that could be exploited as a diagnostic tool. We used fluorescent microsatellite analysis to detect alterations in plasma and tumor DNA in 34 patients who underwent bronchoscopy for lung cancer, including 11 small cell lung cancer (SCLC) and 23 nonsmall cell lung cancer (NSCLC) (12 adenocarcinomas, 11 squamous cell carcinomas) and 20 controls. Allelotyping was performed with a selected panel of 12 microsatellites from 9 chromosomal regions 3p21, 3p24, 5q, 9p, 9q, 13q, 17p, 17q and 20q. Plasma DNA allelic imbalance (AI) was found in 88% (30 of 34 patients), with a similar sensitivity in SCLC and NSCLC. In the 24 paired available tumor tissues, 83% (20 of 24) presented at least 1 AI. Among these patients, 85% (17 of 20) presented also at least 1 AI in paired plasma DNA, but the location of the allelic alterations in paired plasma and tumor DNA could differ, suggesting the presence of heterogeneous tumor clones. None of the 20 controls displayed plasma or bronchial DNA alteration. A reduced panel of six markers (at 3p, 5q, 9p, 9q) showed a sensitivity of 85%. Moreover, a different panel of microsatellites at 3p and 17p13 in SCLC and at 5q, 9p, 9q and 20q in NSCLC patients could be specifically used. Analysis of plasma DNA using this targeted panel could be a valuable noninvasive test and a useful tool to monitor disease progression without assessing the tumor.

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.