Summary:
Bladder cancer (BlCa) constitutes the 10th most common form of cancer worldwide and the 2nd most common form of urogenital malignancy in the male population -after prostate cancer - in countries with a high Human Development Index (HDI). The prevalence of the disease is higher in men and the incidence of its diagnosis increases with aging. Bladder cancer is characterized by a high rate of heterogeneity, at cellular and molecular level, since individuals with similar clinicopathological features present differencesin the prognosis of the disease and theirresponse to treatment protocols. Therefore, there is the need for accurate prognosis for the implementation of personalized effective treatment and post-treatment monitoring protocols (follow-up) of patients. However, the prognostic markers that are used in clinical practice, are unable to define accurately the disease passing and consequently the majority of patients undergo invasive treatment as well as long-term postoperative follow-up, through cystoscopy, urinary cytology and CT-MRI imaging methods. This condition has as repercussion the deterioration of patients' quality of life and the cost augmentation of managing bladder cancer, making this malignancy the most costly per patient, burdening the health systems significantly. Hence, the study and identification of new molecular biomarkers, which would contribute to improving the diagnosis, the prognosis of patients with urothelial cancer and can be applied to clinical practice, are of great research interest.
Liquid biopsy methods, and in particular the analysis of cell-free DNA (cfDNA), are under of intense research interest. The analysis of cfDNA in modern molecular diagnostics is favored because of the trivial effect of tumor heterogeneity and its non-invasive nature allows of sequential sampling, avoiding the occurrence of possible complications and reducing the high cost of monitoring patients. Cell-free DNA is released into the circulation through mechanisms of cell death and especially apoptosis, having a short half-life, constituting a molecular snapshot of the organism. Furthermore, biological fluids are enriched with cfDNA from all cells of the organism (normal cells, cancer cells, cells from the tumor microenvironment). Therefore, a portion of cfDNA is the tumor cfDNA (circulating tumor DNA, ctDNA), on the basis of which analysis, specific mutations to various types of cancer can be detected, having clinical value in prognosis of disease and prediction response to treatment. The range of mutations may involve point mutations, deletions or gains of genetic locus, structural chromosomal abnormalities, and abnormal epigenetic modification. To date, two applications of cfDNA in the modern molecular diagnostics (EGFR Mutation Test v2, Epi proColon), have been approved by the US Food and Drug Administration (FDA).
The aim of the present study is the development of new technologies for cfDNA analysis in bladder cancer patients and its subsequent clinical evaluation prognosis and progression of the disease.
In the present study a total of 170 cancer tissue samples and 100 serum samples of peripheral blood from patients with bladder cancer were utilized. A protocol for isolating cfDNA from serum, quantifying it, and evaluating its quality was developed and optimized. Thereafter, real-time qPCR protocols were developed and optimized, using SYBR Green chemistry and the relative quantification 2-DDCT method, in order to determine the cfDNA integrity and the copy number of the three target genes, CDKN2A,CCND1, MDM2, in both serum and tissue samples of patients with bladder cancer. Clinical evaluation was then performed, via extensive biostatistical analysis, about the amount and integrity index of cfDNA, as well as the detection of copy number variations of the target genes (CDKN2A, CCND1, MDM2).
Based on the results of clinical evaluation, it appears that in bladder cancer with the increase of the disease stage, the pathways of programmed cell death against necrosis are enhanced. In addition, the CDKN2A gene has a greater impact on patients with non-muscle invasive disease, as patients with homozygous or heterozygous deletion of this genetic locus show off better survival possibilities. The MDM2 gene has a greater impact on patients with muscle invasive disease, since patients with amplification of this genetic locus show off having poor survival rates. Levels of CDKN2A and MDM2 copies in serum of patients with superficial and highly differentiated cancer are significantly lower than in patients with advanced and high-grade cancer. Non-muscle invasive, low grade tumors have good prognosis,so the above indications may constitute a good prognostic marker, which needs to be further confirmed by survival analysis in a larger number of serum samples. In conclusion, cfDNA seems to be an immense promising biomolecule in improving cancer management, as its analysis makes it possible to determine the overall molecular profile of tumors in a non-invasive way. Nevertheless, further study is needed to clarify aspects of its biology and how they can be reclaimed in precision medicine.
