DETECTION OF SOMATIC MUTATIONS IN THE BRAF GENE BY PYROSEQUENCING

Introduction. Detection of somatic mutations in the BRAF gene can be used in clinical oncology to clarify the diagnosis, select therapy and assess the prognosis of the disease. Pyrosequencing technology makes it possible to identify both already known and new mutations, as well as to determine the mutant allele ratio in the sample.

The aim of the study was to develop the pyrosequencing-based method for detecting mutations in 592–601 codons of the BRAF gene.

Material and Methods. The nucleotide sequences were obtained using «PyroMark Q24» instrument. The sensitivity and specificity of the method were estimated using dilutions of plasmid DNA samples containing the intact BRAF gene fragment mixed with sequence containing one of the mutations V600E, V600R, V600K, V600M, and K601E. The clinical testing was performed on 200 samples from thyroid nodules.

Results. The developed method makes it possible to determine samples containing 2 % of the mutant allele for mutations V600K and V600R, 3 % for V600E and V600M, and 10 % for K601E. The pyrogram signal values for samples without mutations ranged from 0 to 19.5 % for different mutations. An analysis algorithm was developed to confirm the presence and differentiation of mutations in the 600 codon at a low proportion of the mutant allele based on the signals ratio on the pyrogram. The 47 clinical samples with mutations were found, 45 with V600E and 1 with V600_K601>E, for one sample, the type of mutation in the 600 codon could not be determined. The proportion of the mutant allele was 3.5–45 %. The concentration of extracted DNA less than 10 copies per mkl was obtained in 47 samples, of which 8 samples were found to have the mutations.

Conclusion. The pyrosequencing-based method was developed for the detection of somatic mutations in 592–601 codons of the BRAF gene. The technique provided sufficient sensitivity to detect frequent mutations in the 600 codon and allowed the detection of rare mutations. Extraction of DNA from clinical samples obtained by fine-needle aspiration biopsy in most cases provided a sufficient concentration of DNA, which made it possible to use the technique in combination with cytological analysis without additional sampling. This approach can be applied to determine somatic mutations in DNA fragments of same length for other oncogenes. 

1. Zaman A., Wu W., Bivona T.G. Targeting Oncogenic BRAF: Past, Present, and Future. Cancers (Basel). 2019; 11(8): 1197. doi: 10.3390/ cancers11081197.

2. Tate J.G., Bamford S., Jubb H.C., Sondka Z., Beare D.M., Bindal N., Boutselakis H., Cole C.G., Creatore C., Dawson E., Fish P., Harsha B., Hathaway C., Jupe S.C., Kok C.Y., Noble K., Ponting L., Ramshaw C.C., Rye C.E., Speedy H.E., Stefancsik R., Thompson S.L., Wang S., Ward S., Campbell P.J., Forbes S.A. COSMIC: the Catalogue Of Somatic Mutations In Cancer. Nucleic Acids Res. 2019; 47(D1): D941–D947. doi: 10.1093/nar/gky1015.

3. Dumaz N., Jouenne F., Delyon J., Mourah S., Bensussan A., Lebbé C. Atypical BRAF and NRAS Mutations in Mucosal Melanoma. Cancers (Basel). 2019 Aug 8; 11(8): 1133. doi: 10.3390/cancers11081133.

4. Ducreux M., Chamseddine A., Laurent-Puig P., Smolenschi C., Hollebecque A., Dartigues P., Samallin E., Boige V., Malka D., Gelli M. Molecular targeted therapy of BRAF-mutant colorectal cancer. Ther Adv Med Oncol. 2019 Jun 18; 11: 1758835919856494. doi: 10.1177/1758835919856494.

5. Cibas E.S., Ali S.Z. The 2017 Bethesda System for Reporting Thyroid Cytopathology. Thyroid. 2017 Nov; 27(11): 1341–1346. doi: 10.1089/thy.2017.0500.

6. Beltsevich D.G., Mudunov A.M., Vanushko V.E., Rumyantsev P.O., Melnichenko G.A., Kuznetsov N.S., Podvyaznikov S.O., Alymov Yu.V., Polyakov A.P., Fadeev V.V., Bolotin M.V., Sevryukov F.E., Krylov V.V., Fedenko A.A., Bolotina L.V., Zharov A.A., Falaleeva N.A., Filonenko E.V., Nevol’skikh A.A., Ivanov S.A., Khailova Zh.V., Gevorkyan T.G. Clinical Guidelines: Differentiated Thyroid Cancer. 2020. 47 p. [Internet]. URL: http://cr.rosminzdrav.ru/#!/recomend/977 (cited: 20.10.2020). (in Russian).

7. Patel K.N., Yip L., Lubitz C.C., Grubbs E.G., Miller B.S., Shen W., Angelos P., Chen H., Doherty G.M., Fahey T.J.3rd, Kebebew E., Livolsi V.A., Perrier N.D., Sipos J.A., Sosa J.A., Steward D., Tufano R.P., McHenry C.R., Carty S.E. The American Association of Endocrine Surgeons Guidelines for the Definitive Surgical Management of Thyroid Disease in Adults. Ann Surg. 2020 Mar; 271(3): e21–e93. doi: 10.1097/ SLA.0000000000003580.

8. Haugen B.R., Alexander E.K., Bible K.C., Doherty G.M., Mandel S.J., Nikiforov Y.E., Pacini F., Randolph G.W., Sawka A.M., Schlumberger M., Schuff K.G., Sherman S.I., Sosa J.A., Steward D.L., Tuttle R.M., Wartofsky L. 2015 American Thyroid Association Management Guidelines for Adult Patients with Thyroid Nodules and Differentiated Thyroid Cancer: The American Thyroid Association Guidelines Task Force on Thyroid Nodules and Differentiated Thyroid Cancer. Thyroid. 2016 Jan; 26(1): 1–133. doi: 10.1089/thy.2015.0020.

9. Poller D.N., Glaysher S. Molecular pathology and thyroid FNA. Cytopathology. 2017 Dec; 28(6): 475–481. doi: 10.1111/cyt.12492.

10. Mironov K.O., Dunaeva E.A., Dribnokhodova O.P., Shipulin G.A. Using genetic analysis system based on pyrosequencing method. Spravochnik zaveduyushchego KDL. 2016; 5: 33–42. (in Russian).

11. Dunaeva E.A., Миронов К.О., Dribnokhodova O.P., Subbotina T.N., Bashmakova E.E., Olhovskii I.A., Shipulin G.A. The quantitative testing of V617F mutation in gen JAK2 using pyrosequencing technique. Klinicheskaia laboratornaia diagnostika. 2014; 59 (11): 60–63. (in Russian).

12. Dribnokhodova O.P., Mironov K.O., Dunaeva E.A., Demidova I.A., Barinov A.A., Voiciehovskaya Ia.A., Markelov M.L., Shipulin G.A. The detection of activating somatic mutations in gene KRAS using pyrosequencing technique. Klinicheskaia laboratornaia diagnostika. 2013; 6: 49–51. (in Russian).

13. Dunaeva E.A., Mironov K.O., Subbotina T.N., Olkhovsky I.A., Shipulin G.A. The development and comparative approbation of methods of increasing sensitivity of detection of mutation V617F in gene JAK2 by pyrosequencing. Klinicheskaia laboratornaia diagnostika. 2017; 62 (2): 125–128. (in Russian).

14. Armbruster D.A., Pry T. Limit of blank, limit of detection and limit of quantitation. Clin Biochem Rev. 2008 Aug; 29 Suppl 1(Suppl 1): S49–52.

15. Hou P., Liu D., Xing M. Functional characterization of the T1799- 1801del and A1799-1816ins BRAF mutations in papillary thyroid cancer. Cell Cycle. 2007 Feb 1; 6(3): 377–9. doi: 10.4161/cc.6.3.3818.