Supervisors info:
Σίδερης Διαμάντης, Καθηγητής, Τμήμα Βιολογίας, Ε.Κ.Π.Α.
Ράμπιας Θεόδωρος, Ερευνητής Γ’, Κέντρο Βασικής Έρευνας, Ι.ΙΒ.Ε.Α.Α.
Αυγέρης Μαργαρίτης, Αναπληρωτής Καθηγητής, Τμήμα Ιατρικής, Ε.Κ.Π.Α.
Summary:
Head and Neck Squamous Cell Carcinoma (HNSCC) is one of the most common causes of cancer deaths worldwide. Despite the severity of the disease, diagnosis is not effective due to the lack of screening strategies, resulting in most patients being diagnosed at advanced stages. HNSCC is a highly heterogeneous disease and its treatment requires aggressive surgical and radiotherapy approaches with severe side effects for patients. Therefore, there is a need to characterize the molecular pathways involved in HNSCC in order to establish more effective diagnosis and treatment. This study attempts to investigate the molecular pathways characterizing HNSCC through the evaluation and categorization of a large number of patient data from the TCGA database, to evaluate the qPCR method with TaqMan probes for detecting abnormal gene copy numbers and to apply this protocol to HNSCC samples.
The CSMD1 gene (Chr8, p23.2), according to previous studies undergoes reduced expression,
mutations and methylation in various cancer types. It is considered a candidate tumor suppressor
gene and is being studied in HNSCC mostly about copy number alteration. In this study, TCGA database data for numerous cancer types were analyzed for detecting deletions in CSMD1 gene. It was observed that HNSCC presents the highest percentage of deletions in this gene (22.63%). Considering the frequent presence of deletions in the 8p region in many cancers, it was chosen to study a larger area of chromosome 8 around CSMD1 gene in order to evaluate possible deletions in other genes in the 8p23.3- 8p12 region. Analysis of 496 samples of HNSCC patients from TCGA was performed for the detection of deletions in this region. It was observed that CSMD1 gene shows the highest deletions percentage when compared to any other gene in this region. Α gradual decrease of deletions after CSMD1 gene region (p23.2) towards the p12 region was also remarkable. These data were used to categorize patients into subgroups showing common deletion patterns. The group of patients with deletions only in CSMD1 gene (ONLY CSMD1 DELETED group), the group with deletions in genes of the p23.3 - p23.1 region of chromosome 8 (p23.3 - p23. 1 DELETED group) and the group without deletions (NOT DELETED group) were further studied, using the molecular characterization comparison tools provided by the cBioPortal platform. The groups of patients were analyzed for the presence of mutations in BRCA2, RAD52, ATM and BRCA1 genes, involved in homologous recombination repair as well as in HRAS, SOS2, SOS1, BRAF genes, associated with the HRAS molecular pathway. The results showed that the ONLY CSMD1 DELETED group of patients had clearly increased mutation rates in genes involved in homologous recombination repair compared to the other groups. BRCA2 and RAD52 genes showed statistically significant differences compared to the other groups. Regarding the HRAS molecular pathway, it was observed that the p23.3 - p23.1 DELETED group of patients showed increased frequency of mutations compared to the other two groups. The most significant differences were shown to HRAS and SOS2 genes. Overall, through the meta-analysis of patient data from the TCGA database it was concluded that probably different biological mechanisms are associated with different deletion profiles in this chromosomal region. Patients with targeted deletion only in CSMD1 gene showed increased presence of mutations in homologous recombination genes while patients with extensive deletions at 8p arm showed increased mutations in genes involved in the HRAS molecular pathway.
Quantitative real-time polymerase chain reaction (qPCR) was evaluated using TaqMan probes to
detect copy number alterations (CNAs). Assays were selected to detect copy numbers of DLGAP2,
CSMD1 and PINX1 genes of the p23.3 - p23.1 region of chromosome 8. An evaluation of this method
was performed to identify CNAs in cancer cell lines with available data from clinical studies on
cBioPortal platform about the presence of deletions in these genes. Cancer cell lines DLD1, A549,
HCT116, BB49, SKMEL28, SKOV3 without deletions at 8p, CAL33, RT112, SCC25, PC3 with deletions in
DLGAP2, CSMD1, PINX1 genes and TCCSUP, A375, SF268 with deletions only in CSMD1 gene were
analyzed. It was observed that the copy numbers of cell lines with deletions were found to be
statistically significantly smaller than the ones of cell lines with normal copy number for all the three
genes of interest. This fact demonstrates that in all cases the method was successful in effectively
detecting gene deletions in this area and separating them from the samples with normal gene copy
numbers. The method was applied to samples of HNSCC patients with known mutations in BRCA1, BRCA2, ATM, RAD50, BLM genes involved in homologous recombination as well as different mutations in HRAS gene. A total of twelve patient samples were analyzed, six patients for each of the above categories. The method showed in both cases that one out of six patients had targeted deletion in CSMD1 in the case of patients with mutations in homologous recombination or deletions in all three genes DLGAP2, CSMD1, PINX1 in the case of patients with mutations in the HRAS pathway. This result is consistent with the findings of the meta-analysis of data from TCGA and potentially reveals a biological link between the presence of mutations in homologous recombination or HRAS molecular pathway genes and the generation of a specific deletion profile at 8p. Future application of the method to a wider range of samples is required for more valid results. Understanding the molecular pathways that characterize HNSCC and categorizing patients based on their unique molecular characteristics may allow early and accurate diagnosis and personalized therapeutic interventions to respond to patients' specific molecular profiles.
Keywords:
Head and Neck Cancer, CNAs, Deletion Profiles, CSMD1 gene, Chromosome 8, Replication Stress
