A novel high-resolution chipCE assay for rapid detection of EGFR gene mutations and amplifications in lung cancer therapy by a combination of fragment analysis, denaturing CE and MLPA.
There is a growing interest in evaluating molecular markers as predictors of response to new generation of targeted cancer therapies. One of such areas is biological therapy targeting epidermal growth factor receptor gene (EGFR) in lung cancer. The testing of tumor tissue is focused on specific EGFR mutations and EGFR gene amplification, since tumors exhibiting positivity of either of the two marker types are highly sensitive towards the treatment. Although traditional methods of DNA sequencing and fluorescence in situ hybridization are still in use for the detection of EGFR mutations and gene amplification, respectively, there is a need for new dedicated techniques with the primary emphasis on simplicity, sensitivity, speed and cost effectiveness. The main purpose of this work was to integrate diverse assays for both EGFR tests onto a single platform to eliminate the need for different instruments and separate processing. We demonstrate a chip capillary electrophoresis (chipCE) application for EGFR mutation detection by a combination of fragment analysis and denaturing CE along with multiplex ligation-dependent probe amplification (MLPA) for evaluation of EGFR amplification. All separations are carried out in denaturing sieving polymer on a modified Bioanalyzer 2100 chipCE instrument running at temperatures of up to 65°C. The main strength of the resulting high-resolution chipCE application is in its simplicity, speed of analysis and minimal amount of sample required for complete testing of EGFR status. Such an approach could potentially fit medium throughput laboratories providing molecular pathology services for clinical oncologists with fast turnaround times and limited consumption of tissue material.
Laboratory for Molecular Genetics and Oncology, Genomac International, Prague, Czech Republic. mminarik@email.com
Marek
M
Minarik
Marcus
M
Gassman
Barbora
B
Belsanova
Milos
M
Pesek
Jan
J
Schouten
Richard
R
Chudoba
Bohuslav
B
Gas
Lucie
L
Benesova
D046209:Electrophoresis, Microchip / Q000379:methods*; D066246:ErbB Receptors / Q000235:genetics; D005784:Gene Amplification; D018773:Genes, erbB-1; D015316:Genetic Therapy; D006801:Humans; D016014:Linear Models; D008175:Lung Neoplasms / Q000201:enzymology / Q000235:genetics* / Q000628:therapy; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*; D013997:Time Factors
C512478:EGFR protein, human; D066246:ErbB Receptors