1. Introduction
Gastric cancer is one of the most common cancers in the world [1]. In 2021, it is estimated that there will be 26,560 new cases in the United States and that 11,580 patients will die from the disease [2]. At the time of diagnosis, the tumour is confined to the stomach, in 25% of the cases, in 20% has already metastasised to the regional lymph nodes (locally advanced) and in 34% of them it has given distant metastases. Gastric cancer occurs more frequently in male patients, mainly in the Asian and African-American people. The average age of diagnosis is 69 years, with the highest percentage of new cases occurring between the ages of 75–84 years. The overall five-year survival of all patients with gastric cancer at any stage reaches 27.7%. The incidence has declined over the past decades, but the causes are not fully explained. Tumour-related DNA in the bloodstream may be either free or into circulating tumour cells (CTCs) [7, 10]. Although its origin and its biological significance remain largely unknown, cfDNA is a valuable source of biologic material for the development of biomarkers with high sensitivity and specificity for the non-invasive diagnosis of cancer [8].
Several studies have shown that cfDNA carries genetic and epigenetic changes similar to those present in the DNA of the cancer cells from which they originated. This means that cfDNA is mostly derived from the primary tumour cells [9]. However, the number of these cells is very small compared with that of normal blood cells [11]. Technologies with the necessary sensitivity and reproducibility for the detection of CTCs have been particularly developed in recent years, and therefore useful conclusions can be drawn for their biological and clinical utility. Although, the clinical value of CTCs has been proved in the prognosis and follow-up of cancer patients, the number of these cells per microlitre (mL) of blood decreases their sensitivity to a large extent [12]. By developing highly sensitive PCR techniques, detection of gene mutations or epigenetic changes, such as methylation of DNA in a small number of CTCs, will make early detection of cancer more reliable. However, the cost of these examinations, called liquid biopsies, is still quite high, limiting their widespread use in clinical practice [13]. Although its origin, as well as its biological significance, remains largely unknown, cfDNA is a valuable source of biological material for the development of cancer biomarkers with high sensitivity and specificity to enable non-invasive diagnosis of cancer [14]. It was shown by several studies that the concentration of cfDNA is particularly high in the plasma of patients with metastatic cancer, and its levels decrease rapidly after radiotherapy. Also, persistently high levels of cfDNA are associated with poor response to chemotherapy. The above remarks indicate the potentially high diagnostic value of cfDNA and its use as a biomarker. In several studies, plasma cfDNA has been shown to contain genetic and epigenetic changes similar to those present in DNA molecules by the cancer cells from which they are derived.
This means that the cfDNA molecules are derived from primary tumour cells and not from the lymphocytes likely to react with the tumour cells [15].
Epigenetic changes play an important role in the development and progression of gastric cancer and include DNA methylation, histone modifications, etc. Epigenetic alterations affect gene expression without modifying the DNA sequence and therefore are potentially reversible [16]. It has been shown that promoter methylation of several genes is significantly associated with a decrease in progression-free survival and overall survival, compared with that of patients lacking the methylation of these genes [17]. The above results show that cfDNA detection could provide a lot of information about the course of the disease and the evaluation of response to treatment.
2. Materials and methods
2.1. Study design
The study material consisted of blood samples obtained from gastric cancer patients suffering from metastatic disease. Additionally, blood samples taken from healthy individuals were used as a control group. All these control samples were taken from healthy individuals and non-blood-related family members of patients treated in the Department of Medical Oncology of the University Hospital of Alexandroupolis. The majority of them were age-matched with the patient population and received no medical care at the time of the sample collection.
2.2. Sample collection and Isolation of Cell-Free DNA
Blood was collected in plasma collection tubes. Plasma was obtained immediately through centrifugation at 3,000 rpm for 10 minutes and stored at −80°C until DNA extraction. Cell-free DNA from serum samples was isolated using the QIAamp DNA Blood mini kit (Qiagen, Germany) according to the manufacturer’s protocol. DNA concentration was determined by a real-time PCR method using GAPDH gene as an amplifying target. Three microlitres (μL) of DNA elution were used as a template for the Sybr-green-based real-time PCR analysis. The cell-free DNA concentration was calculated according to reproducible standard dilution curves using a known concentration of MCF-7 genomic DNA, as previously described [18].
Table 1
Clinicopathological characteristics of the patients.
| Patient characteristics | n (70) |
|---|---|
| Gender | |
| Females | 27 |
| Males | 43 |
| Age | |
| ≤ 60 years | 19 |
| > 60 years | 51 |
| Tumour differentiation | |
| Intermediate | 27 |
| Poor | 26 |
| Good | 17 |
| Histology | |
| Intestinal | 32 |
| Diffuse | 23 |
| Other | 15 |
2.3. Statistical analysis
Statistical analysis of the data was performed using the Statistical Package for the Social Sciences (SPSS), version 21.0 (IBM). All tests were two-tailed and statistical significance was considered for p-values < 0.05.
3. Results
The mean concentration of cfDNA in patient group (n = 70), was estimated to be 69.1 ng/μL, whereas the mean cfDNA concentration in the healthy control group (n = 20), was estimated to be 34.8 ng/μL. In the patient group, 23 subjects had cfDNA concentration ≤ 50 ng/μL and 47 subjects had cfDNA concentration > 50 ng/μL. In the patient group with a cfDNA concentration of > 50 ng/μL, mean overall survival was 33.7 months, while in patients with cfDNA concentration ≤ 50 ng/μL mean overall survival was 49.4 months and this was statistically significant (p = 0.004). The cut-off value was set at 50 ng/mL, because according to several measurements that were taken during the experimental procedure, this value was considered to give statistically significant results. Similar results were seen regarding progression free survival. In patients with a cfDNA concentration of > 50 ng/μL, PFS was 30 months, while in patients with cfDNA concentration ≤ 50 ng/μL, PFS was 40 months (p = 0.009) (Figures 1, 2).
Figure 1 Correlation of cfDNA concentration with OS in patients with metastatic gastric cancer.
Figure 2 Correlation of cfDNA concentration with PFS in patients with metastatic gastric cancer.
4. Discussion
Gastric cancer is an aggressive tumour. Although the incidence of the disease varies considerably across countries, it does not cease to be a public health problem worldwide. Its aetiology has not yet been sufficiently elucidated. However, it is commonly accepted that gastric cancer represents a multistage process involving genetic and epigenetic modifications such as oncogene activation, overexpression of growth factors and their receptors, and the inactivation of tumour suppressor genes. However, despite advances in diagnosis and treatment, the outcome of advanced gastric cancer remains unfavourable, with a five-year survival rate of 3.9% [3]. Several studies have shown that the risk of developing gastric cancer decreases in people consuming fruits and vegetables. Increased salt intake and nitrosamines found in fried foods and processed meats contribute to the development of gastric cancer [4]. Helicobacter pylori, a helical Gram-negative bacterium located in the gastric mucosa, is associated with the development of gastritis and peptic ulcer and its presence increases the risk of gastric cancer. Eradication of this bacterium appears to reduce the risk of gastric cancer [5, 6]. The accumulation of genetic and epigenetic changes triggers cancer development.
Particularly, in gastric cancer, epigenetic changes play an important role in the onset and progression of the disease and include changes in DNA methylation, histone modifications, chromatin remodelling, and non-coding RNA. In contrast to classical genetic changes (mutations), epigenetic changes affect gene expression without modifying the DNA sequence and are therefore potentially reversible. It is thought that cfDNA is largely derived from apoptotic cancer cells, since apoptosis occurs to a large extent in primary and metastatic tumours.
Concentration of cfDNA is markedly diminished after radiotherapy, owing to an apoptotic death of cancer cells in this case. It has also been observed that the concentration of cfDNA is increased when cell proliferation in cell cultures increases. Thus, both apoptotic and proliferating cells can be a source of cfDNA in the tumour microenvironment. In contrast to unstable RNA molecules contained in actively secreted exosomes, the nature of cfDNA remains unclear. As negatively charged molecules, cfDNAs are bound to plasma proteins to escape degradation by endonucleases. Unfortunately, the plasma proteins to which cfDNA binds are not yet identified (34). In the present study, the concentration of cfDNA was significantly higher (69.1 ng/μL) in patients than in healthy controls (34.8 ng/μL). It seems that cfDNA in healthy individuals reflects the amount of DNA entering the bloodstream after the normal procedure of apoptosis of several types of cells. The increased cfDNA concentration in patients with gastric cancer indicates that the amount of cfDNA is higher due to the additional destruction of cancer cells in this case (owing to progression of disease) resulting in additional amount of cfDNA to the bloodstream. The release of cfDNA in this case can occur either by apoptosis, by necrosis or by active release of DNA from cancer cells [15]. Patients were divided into two groups, based on the concentration of cfDNA detected in their blood plasma. In patients with a cfDNA concentration ≤ 50 ng/μL, the OS and PFS were substantially higher than that seen in patients with a cfDNA concentration > 50 ng/μL. These results indicate that the amount of cfDNA in the plasma is directly correlated with OS and PFS. The results of our study are in agreement with those of a recent study by Bu et al., in which the concentration of cfDNA in the blood of patients with metastatic gastric cancer was 50.8–8,760.8 ng/mL and the mean OS of patients with lower cfDNA levels was significantly longer than that of the patients with higher cfDNA levels [19].
Thus, the measurement of cf-DNA could serve as a potential prognostic marker for the disease. However, the sample of the study was relatively small, so more studies, with a larger number of patients, are needed to establish the concentration of cDNA as a powerful biomarker for the early diagnosis and prognosis of gastric cancer. In conclusion, DNA-based biomarkers are under development using cell-free DNA (cfDNA) in the patient’s blood to diagnose and predict the response to cancer treatment. Until now, this capability is not available on a large scale.
Recent advances in understanding cancer-related DNA methylation have resulted in the use of this method at the clinical practice by developing molecular indicators for early diagnosis and prognosis of the disease. The ability of the methylation markers to detect cancer has been tested in several biological materials, including blood. Additionally, concentration of cfDNA in the plasma of patients with gastric cancer could be a potential biomarker for the early detection and prognosis of the disease.
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