Mini-ReviewMutation-based detection and monitoring of cell-free tumor DNA in peripheral blood of cancer patients
Introduction
Although significant progress has been made in the development of new therapy approaches, cancer remains the leading cause of death worldwide [1]. Despite the availability of a number of screening schemes, in most cases cancer remains undetected until its advanced stages [2], [3]. In a typical course of disease development, defective cellular adhesion allows malignant cells to be released and to travel to nearby structures or even migrate through the lymphatic or blood system to form malignant formations. If unnoticed, such micrometastases pose a serious risk for disease progression already in early stages of the primary tumor. Surgical treatment resulting in removal of the primary tumor, therefore, might not avert dissemination and generalization of the disease in the long term. Follow-up of cancer patients typically relies on computed tomography (CT),1 positron emission tomography (PET)–CT, or magnetic resonance imaging in combination with monitoring of serum tumor markers [4], [5]. It is known that imaging methods typically spot objects on a millimeter scale (containing tens to hundreds of millions of cells). In addition, the widely established utility of tumor markers is sometimes inefficient due to sensitivity and specificity issues [6], [7]. Therefore, there is great expectation in finding new diagnostic markers for better management of all major cancers. Among the few alternatives, there is growing interest in molecular diagnostics directed at nucleic acids released directly from the tumor and circulating in peripheral blood of patients.
The classic article on the occurrence of nucleic acids in human plasma was published back in 1948 by Mandel and Metais [8], followed by works of Bendich and coworkers in 1965 [9], Koffler and coworkers in 1973 [10], and Leon and coworkers in 1977 [11], who identified the importance of circulating tumor DNA as a vehicle of oncogenesis. With the use of then emerging methods such as radioimmunoassay, the presence of higher cell-free DNA (cfDNA) concentrations in serum in patients with carcinoma compared with healthy persons was observed along with a decrease after the administration of chemotherapy [10], [11]. It was soon recognized that the circulating DNA could serve as a viable tool to monitor the efficiency of anticancer therapies by monitoring its levels in advanced cancers [12], [13], [14]. With the subsequent rapid development of modern polymerase chain reaction (PCR)-based techniques and their widespread availability, the interest in detecting circulating nucleic acids is steadily increasing, with colorectal, prostate, and lung cancers being the main focus of many published studies [15], [16], [17], [18], [19].
Circulating nucleic acids, often referred to as cell-free DNA, emphasize their exogenous nature in comparison with DNA originating from nuclei of the blood cells. Whereas cfDNA detection is currently at the forefront of molecular oncology community interest, the detail mechanism of cfDNA release from its native cell is yet to be fully elucidated. In 2001, a study by Jahr and coworkers revealed a combined contribution of apoptotic and necrotic processes to the overall production of cfDNA [20]. The idea was extended in further detail by Diehl and coworkers [21]. These authors considered that DNA fragments present in the circulation originate from the necrotic neoplastic cells phagocytized by macrophages, and these also engulf nontumor (apoptotic) cells, which is the reason why a particular level of nontumor cfDNA occurs in healthy individuals, as confirmed by others [22]. The two proposed hypothetical mechanisms for necrotic and apoptotic release of DNA are illustrated in Fig. 1. Fig. 1A depicts a mucous membrane of the colon affected by a growing tumor with a layer of necrotic cells on the surface. The necrotic tumor cell is released, and its fragments are captured by the macrophage pseudopodia. An engulfed fragment forms a phagosome, which fuses with lysosome to form a phagolysosome. Subsequently, the ingested particles, including tumor DNA fragment of various lengths, are released into the environment. Fig. 1B shows an alternative mechanism with the mucous membrane of the colon with a normal epithelium layer releasing a cell undergoing apoptosis. The cell forms apoptotic particles captured by the macrophage pseudopodia. The engulfed particle forms a phagosome, which fuses with lysosome to form a phagolysosome. Subsequently, the ingested particles, including equally sized DNA, are released into the environment.
Necrotic cells arise in invasive tumors, where tissue deterioration occurs as a result of hypoxia [23]. Benign tumors do not have this property, and the amount of fragmented DNA produced is minimal [24], [25]. This implies that malignity of the tumor leads to a higher degree of necrosis with a corresponding increase in circulating tumor DNA. Necrosis, however, affects surrounding nontumor cells as well, leading to a parallel release of nontumorous cfDNA into the circulation, resulting in an increase in concentrations of both tumorous and nontumorous DNA in plasma. Thus, the fact that in patients with malignant disease the volume of free DNA is increased, regardless of its origin, can in some circumstances be used for monitoring of cancer.
To successfully detect the presence of tumor cfDNA in plasma or serum, a suitable methodology needs to be selected. This consists of several essential steps. The first step is to process collected blood while avoiding the rupture of blood cell membranes (i.e., hemolysis) and subsequent plasma contamination with DNA derived from the blood cell nuclei. The next step is cfDNA isolation when it is necessary to select the most appropriate method to gain a sufficient amount of quality DNA for further analysis. Here, the essential tool is PCR, and the detection of amplified products can be done either directly in real time (real-time PCR) or following amplification by electrophoresis on a slab gel or capillary format.
Blood samples collected in an anticoagulant solution must be processed within 2 h after the collection to avoid damage to nucleated blood cells and release of their DNA [26]. In some studies sampling was performed in heparinized test tubes [27], [28], and in others it was performed in tubes containing EDTA (ethylenediaminetetraacetic acid) solution [29]. Immediately after blood collection, plasma needs to be separated from the blood cells by centrifugation. The centrifugal speed must not be too high (to avoid causing cell lysis), and recentrifugation may be performed following primary elimination of blood cells at lower speeds [27]. Alternatively, plasma may be extracted by filtration through membranes with a 0.45-μm pore size [29]. Extracted plasma may be stored at −20 °C for extended periods of time before subsequent processing. Some authors have reported the use of serum rather than plasma [30]. It was noted that serum is a less suitable material because it becomes readily contaminated with DNA from the leukocytes when blood coagulum is formed [26].
The basis for successful cfDNA detection is selection of an isolation method that ensures extraction of a sufficient amount of quality DNA. A classic phenol–chloroform extraction or commercial kits based on the principle of membrane columns can be applied. The advantage of the phenol–chloroform method is an unlimited amount of input material; thus, the yield can be higher with an increased volume of isolated plasma, whereas in commercial kits the amount of input material is limited. However, the use of commercial kits is considerably easier, and special kits designed specifically for cfDNA isolation are already available. Kuang and coworkers [28] compared three isolating kits: QIAamp DNA Micro Kit (Qiagen), NucleoSpin Plasma XS (Macherey–Nagel), and Wizard (Promega). In this study, the authors used a method described previously [31] based on a principle that the majority of tumor DNA in plasma derived from necrotic cells occurs in unequally sized fragments ranging from 185 to 926 bp, whereas the DNA from apoptotic (i.e., nontumor) cells is usually present in relatively uniform sizes ranging from 185 to 200 bp. Based on these facts, they examined the amounts of both cfDNA types using real-time PCR by amplifying two different length fragments of Alu sequences, namely, 115 and 247 bp. Alu 115 captured the concentration of short DNA fragments derived from apoptotic cells as well as DNA fragments from tumor cells, whereas Alu 247 captured only the concentration of tumor DNA. From a subsequent Alu 247/115 ratio, they calculated the concentration of DNA derived only from tumor cells. This led to a finding that although the greatest concentration of total cfDNA was obtained using the NucleoSpin Plasma kit, for extracting fragments derived from tumor cells, the QIAamp DNA Micro Kit was more suitable. The results are summarized in Table 1. In another study, a QIAamp MinElute Virus Vacuum Kit (Qiagen) was used for cfDNA extraction. Its advantage was a greater input volume of isolated plasma [29]. In some other studies, column kits were used for isolation from blood [32]. Their disadvantage was a loss of small cfDNA fragments through membrane pores, leading to reduced detection sensitivity [33].
Section snippets
Analysis of cfDNA based on tumor-specific mutations
There are two basic approaches to cfDNA analysis: quantitative analysis and analysis based on DNA-specific mutations. The first approach is based solely on quantification of cfDNA, including both tumor and nontumorous cfDNA [34]. Increased DNA levels in plasma of cancer patients compared with healthy controls indicate the presence of tumor cfDNA. The actual cfDNA amount is typically determined by amplification of Alu sequences or other specific markers (e.g., β-globin, β-actin) [35]. This
Conclusions
The need for efficient tools to detect early stages of cancer progression prompts further development of molecular tests directed at cfDNA. The main factor is minimal invasivity, which is often key in long-term follow-up of patients undergoing challenging anticancer therapy. The routine adaptation of these tests, now frequently referred to as “liquid biopsy”, is expected in the near future. The pace of such adaptation will depend on the availability of easily adoptable and low-cost
Acknowledgments
This work was supported by the Czech Ministry of Health Internal Grant Agency (Project NS 9809). This is contribution number 7 from the Center for Applied Genomics of Solid Tumors (CEGES).
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