OPTIMIZATION OF THE METHOD OF THE MULTI-TARGET SINGLE-BASE ELONGATION FOR THE DETECTION OF SOMATIC MUTATIONS IN MALIGNANCIES

DOI: https://doi.org/10.29296/24999490-2019-02-06

A.A. Musaelyan(1), I.V. Chistyakov(1), V.D. Nazarov(1), S.V. Lapin(1), M.V. Sogoyan(1), S.E. Khalchitsky(2), W.L. Emanuel(1), T.W. Lobachevskaya(1), A.L. Akopov(1) 1-I.P. Pavlov First Saint-Petersburg State Medical University, Lva Tolstogo Str. 6–8, Saint Petersburg, 197022, Russian Federation; 2-Turner Scientific Research Institute for Children’s Orthopedics, Parkovaya, 64–68, Pushkin, Saint Petersburg, 196603, Russian Federation E-mail: [email protected]

Background. Genotyping of the tumor samples is necessary for a personalized approach in the treatment of patients with malignancies. The identification of somatic mutations in EGFR and KRAS genes can help to predict the course of the disease and to individualize the therapy. One of the methods for detecting somatic point mutations in EGFR and KRAS genes is the multi-target single-base elongation (MSE). Objective. To optimize MSE for the detecting clinically significant aberrations in the EGFR and KRAS genes in the tumor samples. Material and methods. DNA was isolated from 31 samples of non-small cell lung cancer, 9 of which were formalin-fixed and paraffin embedded (FFPE). Laboratory characteristics of the MSE were compared with such one of real-time PCR, digital PCR, and Sanger sequencing. Results. Protocol for verification the aberrations in the EGFR and KRAS genes has been created. The sensitivity of MSE was determined (48 copies/μl of the mutant DNA). The laboratory characteristics of the method were shown to be comparable with such one of real-time PCR. MSE is able to detect aberrations in FFPE tissue samples. MSE can detect more point mutations in comparison with Sanger sequencing: 26 vs 3%, respectively.
Keywords: 
Conclusion. MSE is a highly sensitive and relatively rapid multiparametric method of genotyping a tumor tissue
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