A Complex of Pyrosequencing-Based Methods for Detection of Somatic Mutations in Codons 600 and 601 of the <i>BRAF</i> Gene

A Complex of Pyrosequencing-Based Methods for Detection of Somatic Mutations in Codons 600 and 601 of the BRAF Gene

Dribnokhodova O.P., Esman A.S., Korchagin V.I., Bukharina A.Yu., Dunaeva E.A., Leshkina G.V., Borisova E.V., Voiciehovskaya Ya.A., Daoud A.I., Khlyavich V.N., Mironov K.O.

Key words: pyrosequencing; BRAF; oncogenetics; fine-needle aspiration biopsy; thyroid cancer.

2022, volume 14, issue 2, page 41.

DOI: https://doi.org/10.17691/stm2022.14.2.04

Full text

html pdf

1041

882

Abstract
References
How to cite

The aim of the study is to develop methods for the differentiation of mutations in the BRAF codon 600 and to increase the sensitivity of the K601E mutation detection.

Materials and Methods. The nucleotide sequence of the BRAF codons 592–602 was identified using the PyroMark Q24 genetic analysis system. The mutations search in codon 600 was conducted using the 600-S primer in line with the following order of adding nucleotides: GCTGTCАTCTGCTAGCTAGAC (corresponding to nucleotides 1799–1786). The K601E mutation was detected using the 601-S primer in line with the following order of nucleotide addition: GCTACTCACTGTAG (corresponding to nucleotides 1801–1793). The analytical characteristics of the proposed methods for somatic mutations’ detection were determined using dilutions of plasmid DNA samples containing the BRAF gene region without mutations or with one of the following mutations: V600E, V600R, V600K, V600M, and K601E. Validation was performed on 132 samples of biological material obtained from the thyroid nodules.

Results. The developed methods allow to determine 2% of the V600E or V600M mutations, 1% of the V600K and V600R mutations, and 3% of the K601E mutations in samples with high DNA concentration; it is also possible to confidently detect at least 5% of the mutant allele for all mutations in low concentration samples (less than 500 copies/PCR). During biological material testing, 53 samples with the V600E mutation were detected; the proportion of the mutant allele was 4.9–50.0%.

Conclusion. A complex of methods for determination of the nucleotide sequence of the BRAF codons 592–601 and the algorithm for testing samples and analyzing mutations in the BRAF codons 600–601 was developed. The method provides sufficient sensitivity to detect frequent mutations in codons 600 and 601 and allows them to be precisely differentiated.

Pisapia P., Pepe F., Iaccarino A., Sgariglia R., Nacchio M., Russo G., Gragnano G., Malapelle U., Troncone G. BRAF: a two-faced Janus. Cells 2020; 9(12): 2549, https://doi.org/10.3390/cells9122549.
Frisone D., Friedlaender A., Malapelle U., Banna G., Addeo A. A BRAF new world. Crit Rev Oncol Hematol 2020; 152: 103008, https://doi.org/10.1016/j.critrevonc.2020.103008.
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, https://doi.org/10.1093/nar/gky1015.
Dankner M., Rose A.A.N., Rajkumar S., Siegel P.M., Watson I.R. Classifying BRAF alterations in cancer: new rational therapeutic strategies for actionable mutations. Oncogene 2018; 37(24): 3183–3199, https://doi.org/10.1038/s41388-018-0171-x.
Zaman A., Wu W., Bivona T.G. Targeting oncogenic BRAF: past, present, and future. Cancers (Basel) 2019; 11(8): 1197, https://doi.org/10.3390/cancers11081197.
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; 11: 1758835919856494, https://doi.org/10.1177/1758835919856494.
Bracht J.W.P., Karachaliou N., Bivona T., Lanman R.B., Faull I., Nagy R.J., Drozdowskyj A., Berenguer J., Fernandez-Bruno M., Molina-Vila M.A., Rosell R. BRAF mutations classes I, II, and III in NSCLC patients included in the SLLIP trial: the need for a new pre-clinical treatment rationale. Cancers (Basel) 2019; 11(9): 1381, https://doi.org/10.3390/cancers11091381.
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. III, 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; 271(3): e21–e93, https://doi.org/10.1097/sla.0000000000003580.
Ali S.Z., Cibas E.S. Klassifikatsiya Betesda dlya tsitologicheskoy diagnostiki zabolevaniy shchitovidnoy zhelezy: terminologiya, kriterii i poyasneniya. Per. s angl. Shapiro N.A. [The Bethesda system for reporting thyroid cytopathology: definitions, criteria, and explanatory notes. Shapiro N.A. (translation from English)]. Moscow: Prakticheskaya meditsina; 2020; 240 p.
Bel’tsevich D.G., Mudunov A.M., Vanushko V.E., Rumyantsev P.O., Melnichenko G.A., Kuznetsov N.S., Podvyaznikov S.O., Alymov I.V., Poliakov A.P., Fadeev V.V., Bolotin M.V., Sevriukov 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 Z.V., Gevorkian T.G. Klinicheskie rekomendatsii. Differentsirovannyy rak shchitovidnoy zhelezy [Clinical guidelines. Differentiated thyroid cancer]. Ministry of Health (Russia); 2020; 47 p. URL: https://cr.minzdrav.gov.ru/schema/329_1.
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; 26(1): 1–133, https://doi.org/10.1089/thy.2015.0020.
Malapelle U., Rossi G., Pisapia P., Barberis M., Buttitta F., Castiglione F., Cecere F.L., Grimaldi A.M., Iaccarino A., Marchetti A., Massi D., Medicina D., Mele F., Minari R., Orlando E., Pagni F., Palmieri G., Righi L., Russo A., Tommasi S., Vermi W., Troncone G. BRAF as a positive predictive biomarker: focus on lung cancer and melanoma patients. Crit Rev Oncol Hematol 2020; 156: 103118, https://doi.org/10.1016/j.critrevonc.2020.103118.
Cardus B., Colling R., Hamblin A., Soilleux E. Comparison of Methodologies for the detection of BRAF mutations in bone marrow trephine specimens. J Clin Pathol 2019; 72(6): 406–411, https://doi.org/10.1136/jclinpath-2019-205734.
Cheng L., Lopez-Beltran A., Massari F., MacLennan G.T., Montironi R. Molecular testing for BRAF mutations to inform melanoma treatment decisions: a move toward precision medicine. Mod Pathol 2018; 31(1): 24–38, https://doi.org/10.1038/modpathol.2017.104.
Dunaeva E.A., Mironov 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.
Dribnokhodova O.P., Dunaeva E.A., Leshkina G.V., Yarygina E.A., Bukharina A.Yu., Voiciehovskaya Y.A., Borisova E.V., Bormotova S.K., Daoud A.I., Khlavich V.N., Mironov K.O. Detection of somatic mutations in the BRAF gene by pyrosequencing. Sibirskij onkologiceskij zurnal 2021; 20(5): 75–83, https://doi.org/10.21294/1814-4861-2021-20-5-75-83.
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: 33–43.
Dribnokhodova O.P., Mironov K.O., Dunayeva Ye.A., Demidova I.A., Barinov A.A., Voytsekhovskaya Ya.A., Markelov M.L., Shipulin G.A. The detection of activating somatic mutations in gene KRAS using pyrosequencing technique. Kliniceskaa laboratornaa diagnostika 2013; 6: 49–51.
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. Klinicheskaa laboratornaa diagnostika 2017; 62(2): 125–128.
Armbruster D.A., Pry T. Limit of blank, limit of detection and limit of quantitation. Clin Biochem Rev 2008; 29(Suppl 1): S49–S52.
Ihle M.A., Fassunke J., König K., Grünewald I., Schlaak M., Kreuzberg N., Tietze L., Schildhaus H.U., Büttner R., Merkelbach-Bruse S. Comparison of high resolution melting analysis, pyrosequencing, next generation sequencing and immunohistochemistry to conventional Sanger sequencing for the detection of p.V600E and non-p.V600E BRAF mutations. BMC Cancer 2014; 14: 13, https://doi.org/10.1186/1471-2407-14-13.

Dribnokhodova O.P., Esman A.S., Korchagin V.I., Bukharina A.Yu., Dunaeva E.A., Leshkina G.V., Borisova E.V., Voiciehovskaya Ya.A., Daoud A.I., Khlyavich V.N., Mironov K.O. A Complex of Pyrosequencing-Based Methods for Detection of Somatic Mutations in Codons 600 and 601 of the BRAF Gene. Sovremennye tehnologii v medicine 2022; 14(2): 41, https://doi.org/10.17691/stm2022.14.2.04