ABSTRACT
Malignant melanoma (MM) is known for its abundance of genetic alterations and a tendency for rapid metastasizing. Identification of novel plasma biomarkers may enhance non-invasive diagnostics and disease monitoring. Initially, we examined copy number variations (CNV) in CDK genes (CDKN2A, CDKN2B, CDK4) using MLPA (gDNA) and ddPCR (ctDNA) analysis. Subsequently, low-coverage whole genome sequencing (lcWGS) was used to identify the most common CNV in plasma samples, followed by ddPCR verification of chosen biomarkers. CNV alterations in CDK genes were identified in 33.3% of FFPE samples (Clark IV, V only). Detection of the same genes in MM plasma showed no significance, neither compared to healthy plasmas nor between pre- versus post-surgery plasma. Sequencing data showed the most common CNV occurring in 6q27, 4p16.1, 10p15.3, 10q22.3, 13q34, 18q23, 20q11.21-q13.12 and 22q13.33. CNV in four chosen genes (KIF25, E2F1, DIP2C and TFG) were verified by ddPCR using 2 models of interpretation. Model 1 was concordant with lcWGS results in 54% of samples, for model 2 it was 46%. Although CDK genes have not been proven to be suitable CNV liquid biopsy biomarkers, lcWGS defined the most frequently affected chromosomal regions by CNV. Among chosen genes, DIP2C demonstrated a potential for further analysis.
Subject(s)
Biomarkers, Tumor , DNA Copy Number Variations , Melanoma , Humans , Melanoma/genetics , Melanoma/pathology , Melanoma/diagnosis , Liquid Biopsy/methods , Biomarkers, Tumor/genetics , Biomarkers, Tumor/blood , Male , Female , Middle Aged , Aged , Adult , Skin Neoplasms/genetics , Skin Neoplasms/pathology , Skin Neoplasms/blood , Cyclin-Dependent Kinase Inhibitor p16/genetics , Cyclin-Dependent Kinase 4/genetics , Aged, 80 and over , Whole Genome Sequencing/methods , Cyclin-Dependent Kinase Inhibitor p15/geneticsABSTRACT
BACKGROUND: Nowadays, modern treatment methods for cancer patients are based on targeting specific molecules involved in cellular signaling system associated with tumor initiation and progression. The success of such approach depends on a correctly chosen dia-gnostic test with high sensitivity that identifies the occurrence and level of bio-markers in patients to select those who will respond and benefit from the treatment. The development of new technologies and the upgrades of the known ones contribute to the innovations in molecular characterization of cancer, which allows the detection of patients mutational status with high sensitivity and specificity. PURPOSE: Here, we discuss the utilization of the third-generation type of polymerase chain reaction (PCR), droplet digital PCR (ddPCR), in the molecular dia-gnostics of oncology diseases. According to the studies reported in our review, ddPCR represents a promising tool in genetic profiling of cancer patients. Therefore, the optimization and precise validation may enable gradual implementation of ddPCR into clinical practice in the field of oncology.
Subject(s)
Neoplasms/diagnosis , Neoplasms/genetics , Polymerase Chain Reaction/methods , Humans , Neoplasm, Residual/diagnosis , Neoplasm, Residual/geneticsABSTRACT
Malignant melanoma is an oncological disease characterized by etiologic heterogeneity and it has increasing incidence and mortality in the Slovak Republic. While it is treated surgically in combination with chemotherapy, targeted therapy, and immunotherapy, malignant melanomas can ulcerate and are susceptible to infections. These are highly aggressive cancers with metastasis, and recent studies have shown the presence of mutations in RAC1, PPP6C and STK19 genes in melanoma patients. Mutations in these genes are driver mutations; important in oncogenesis and providing selective advantage to tumor cells. The aim of our study is to establish a method to detect driver mutations in formalin-fixed, paraffin embedded (FFPE) tissue DNA. We applied Sanger sequencing to detect driver somatic mutations in RAC1, PPP6C, STK19 and BRAF genes in patients with malignant melanoma. Confirmation of BRAF V600E mutation was obtained by allele-specific PCR. The BRAF V600E mutation was present in 15 of 113 patients (13.2%) and the driver mutation in 7 of 113 patients (6.2 %). Our results demonstrate that Sanger sequencing analysis detects mutations in FFPE clinical samples. The identification of these somatic driver mutations in samples with verified malignant melanomas enabled development of a molecular classification of melanomas, and our study provides evidence of diversity of novel driver mutations implicated in malignant melanoma pathogenesis. These findings could have very important implications for targeted therapy.
