Ανάπτυξη και κλινική εφαρμογή μεθόδων ανίχνευσης μεταλλάξεων των γονιδίων BRAF και PIK3CA με συνδυασμό ARMS PCR και υψηλής διακριτικότητας ανάλυση καμπύλων τήξης DNA

Postgraduate Thesis uoadl:1316044 539 Read counter

Unit:
Κατεύθυνση Κλινική Χημεία
Library of the School of Science
Deposit date:
2012-11-08
Year:
2012
Author:
Ευσταθίου Αντωνία
Supervisors info:
Ευρύκλεια Λιανίδου Καθηγ. (Επιβλέπουσα), Κουππάρης Μιχαήλ Καθηγ., Σωτηροπούλου Γεωργία Αναπλ. Καθηγ. Πανεπιστήμιο Πατρών
Original Title:
Ανάπτυξη και κλινική εφαρμογή μεθόδων ανίχνευσης μεταλλάξεων των γονιδίων BRAF και PIK3CA με συνδυασμό ARMS PCR και υψηλής διακριτικότητας ανάλυση καμπύλων τήξης DNA
Languages:
Greek
Summary:
Somatic mutations in the gene encoding for the phosphatidyl-inositol 3-kinase
(PI3K) catalytic subunit, PIK3CA and in the gene encoding for BRAF, have been
discovered in many different human cancers.
The aim of this study was to evaluate a method taken from literature [187] for
the presence of BRAF mutation V600E in DNA samples FFPEs (Formalin-fixed,
paraffin-embedded) of patients with melanoma and colorectal cancer which had
been already verified by DNA sequencing. Real-time PCR was performed in
triplicate for all samples in the LightCycler (Roche), in the presence of
LC-Green Plus saturating dye (Idaho Technology Inc.USA). High-resolution
melting curves were obtained with HR-1 High Resolution Melter (Idaho Technology
Inc.USA). Mutation V600E in BRAF gene was identified in 9/11 FFPEs samples from
melanoma and colorectal cancer patients.

The second aim of our study was to develop an ARMS- PCR method coupled with
high-resolution melting curve analysis (HRMA) for the detection of the hotspot
mutation 1633 G>A in the exon 9 of PIK3CA gene and evaluate this methodology
for its presence in circulating tumor cells (CTCs), from plasma cell-free DNA
(cfDNA) of breast cancer patients and in healthy individuals. DNA from CTCs was
isolated from plasma of 87 patients with operable breast cancer, 69 patients
with metastatic breast cancer and 25 healthy donors. RNA from CTCs was isolated
from plasma of 49 patients with breast cancer after mRNA purification and
complementary DNA synthesis (cDNA). Cell free DNA was isolated from plasma of
62 patients with metastatic breast cancer. After DNA extraction, real-time PCR
was performed in triplicate for all samples and high-resolution melting curves
were obtained with the LightScanner32 (Idaho USA), in the presence of LC-Green
Plus saturating dye (Idaho Technology Inc. USA). Mutation 1633 G>A in exon 9 of
the PIK3CA gene was identified in 6/69 (8,7%) DNA samples from metastatic
breast cancer patients, in 4/87 (4,6%) breast cancer patients with operable
breast cancer and in 1/62 (2%) cell free DNA samples from metastatic breast
cancer patients. PIK3CA mutations were not detected neither in any of the 49
cDNA samples nor in any 25 samples from healthy donors, that were used as
negative controls.
Samples Mutation 1633 G>A in exon 9 %
Healthy donors 0/25 0%
CTCs (early breast cancer) 4/87 4,6%
CTCs (metastatic breast cancer) 6/69 8,7%
cDNA from CTCs 0/49 0%
Cell free DNA (metastatic breast cancer) 1/62 2%
Keywords:
PIK3CA, BRAF, ARMS-PCR, HRMA, CTCs
Index:
Yes
Number of index pages:
9
Contains images:
Yes
Number of references:
173
Number of pages:
137
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