Supervisors info:
Ευρύκλεια Λιανίδου, Καθηγήτρια Ε.Κ.Π.Α. Επιβλέπουσα, Γεωργία Σωτηροπούλου, Αναπληρώτρια Καθηγήτρια, Πανεπιστήμιο Πατρών, Χρήστος Κρούπης, Επίκουρος Καθηγητής, Ε.Κ.Π.Α
Summary:
Mutations in DNA are associated with the biology of cancer and therefore their
detection is one of the most important issues in this cancer research.
In the present study, an attempt was made to choose a method for the detection
of mutations, characterized by high sensitivity and specificity, among a wide
range of methodologies that are currently available in literature. The next
step was to detect mutations in PIK3CA gene which has been implicated in
carcinogenesis in general and particularly in breast cancer.
First, samples of genomic DNA (gDNA) isolated from circulating tumor cells
(CTC), samples of cDNA and samples of cell free DNA (cfDNA) were tested for the
presence of the mutation in exon 20 of the PIK3CA gene. These samples were
isolated from peripheral blood of patients with operable breast cancer (60, 56,
25, respectively), patients with metastatic breast cancer (34, 10, 45,
respectively) and healthy donors (24, 20, 12, respectively). The methodology
used, was previously developed in our laboratory and is based on High
Resolution Melting Analysis (HRMA)1. In cfDNA this method detected PIK3CA
mutations in exon 20 in 20% of samples of patients with metastatic breast
cancer as well as in 12% of samples of patients with operable breast cancer.
Additionally, PIK3CA mutation on exon 20 was detected in 3% of samples of gDNA
isolated from CTCs in patients with operable cancer. No mutation was detected
in all samples of cDNA.
We then developed two new methodologies based on ARMS-PCR and ARMS-HRMA in
order to detect mutations in very low percentages. The application of ARMS-PCR
and additionally its combination with HRMA in the presence of an internal
control was selected. After the optimization of all steps, the methodologies
were evaluated in terms of accuracy by applying them in DNA isolated from FFPE
samples, which were previously analyzed by DNA Sequencing. ARMS-PCR and
ARMS-PCR-HRMA were evaluated in terms of sensitivity by using a number of
mixtures of normal and mutant allele at different rates
Keywords:
PIK3CA, HRMA, ARMS-PCR, CTCs, cfDNA
