Detection of the IDH1/2 gene mutations in tumor samples with low abundance of the mutant allele

Capa

Citar

Texto integral

Acesso aberto Acesso aberto
Acesso é fechado Acesso está concedido
Acesso é fechado Acesso é pago ou somente para assinantes

Resumo

Identification of driver mutations in tumors is an extremely important task in oncology for the choice of treatment strategy and assessment of therapy efficacy. In many cases, especially in disease monitoring, there is a need to detect a small number of copies of the mutant allele against the background of excessive content of wild-type DNA. In this work we investigated the possibilities of highly sensitive detection of mutations in IDH1 and IDH2 genes at suppression of wild-type DNA amplification using oligomers of “locked” nucleic acid (LNA) with subsequent hybridization of fluorescently labeled polymerase chain reaction (PCR) product on a biological microchip (biochip). The limit of detection of mutant DNA is 0.1% in the wild-type DNA background. The effectiveness of this approach is demonstrated by analyzing 26 samples of chondroid tumors and glial brain tumors with low representation of the mutant allele, previously undetected mutations R132C, R132L and R132H were identified in three cases.

Texto integral

Acesso é fechado

Sobre autores

V. Varachev

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: tanased06@rambler.ru
Rússia, ul. Vavilova 32, Moscow, 119991

D. Guskov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: tanased06@rambler.ru
Rússia, ul. Vavilova 32, Moscow, 119991

O. Susova

N.N. Blokhin National Medical Research Center of Oncology, Ministry of Health of the Russian Federation

Email: tanased06@rambler.ru
Rússia, Kashirskoe shosse 23, Moscow, 115478

A. Shekhtman

Russian Children’s Clinical Hospital

Email: tanased06@rambler.ru
Rússia, Leninsky prosp. 117, Moscow, 119117

D. Rogozhin

Russian Children’s Clinical Hospital

Email: tanased06@rambler.ru
Rússia, Leninsky prosp. 117, Moscow, 119117

S. Surzhikov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: tanased06@rambler.ru
Rússia, ul. Vavilova 32, Moscow, 119991

A. Chudinov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: tanased06@rambler.ru
Rússia, ul. Vavilova 32, Moscow, 119991

A. Zasedatelev

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: tanased06@rambler.ru
Rússia, ul. Vavilova 32, Moscow, 119991

T. Nasedkina

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Autor responsável pela correspondência
Email: tanased06@rambler.ru
Rússia, ul. Vavilova 32, Moscow, 119991

Bibliografia

  1. Angulo B., Lopez-Rios F., Gonzalez D. // Exp. Rev. Mol. Diagn. 2014. V. 14. P. 517–524. https://doi.org/10.1586/14737159.2014.910120
  2. Matsuda K. // Adv. Clin. Chem. 2017. V. 80. P. 45–72. https://doi.org/10.1016/bs.acc.2016.11.002
  3. Wilkening S., Hemminki K., Thirumaran R.K., Bermejo J.L., Bonn S., Försti A., Kumar R. // Biotechniques. 2005. V. 39. P. 853–858. https://doi.org/10.2144/000112027
  4. Ogino S., Kawasaki T., Brahmandam M., Yan L., Cantor M., Namgyal C., Mino-Kenudson M., Lauwers G.Y., Loda M., Fuchs C.S. // J. Mol. Diagn. 2005. V. 7. P. 413–421. https://doi.org/10.1016/S1525-1578(10)60571-5
  5. Reckamp K.L., Melnikova V.O., Karlovich C., Sequist L.V., Camidge D.R., Wakelee H., Perol M., Oxnard G.R., Kosco K., Croucher P., Samuelsz E., Vibat C.R., Guerrero S., Geis J., Berz D., Mann E., Matheny S., Rolfe L., Raponi M., Erlander M.G., Gadgeel S.J. // Thorac. Oncol. 2016. V. 11. P. 1690–1700. https://doi.org/10.1016/S1525-1578(10)60571-5
  6. Thomas R.K., Baker A.C., Debiasi R.M., Winckler W., Laframboise T., Lin W.M., Wang M., Feng W., Zander T., MacConaill L., Lee J.C., Nicoletti R., Hatton C., Goyette M., Girard L., Majmudar K., Ziaugra L., Wong K.K., Gabriel S., Beroukhim R., Peyton M., Barretina J., Dutt A., Emery C., Greulich H., Shah K., Sasaki H., Gazdar A., Minna J., Armstrong S.A., Mellinghoff I.K., Hodi F.S., Dranoff G., Mischel P.S., Cloughesy T.F., Nelson S.F., Liau L.M., Mertz K., Rubin M.A., Moch H., Loda M., Catalona W., Fletcher J., Signoretti S., Kaye F., Anderson K.C., Demetri G.D., Dummer R., Wagner S., Herlyn M., Sellers W.R., Meyerson M., Garraway L.A. // Nat. Genet. 2007. V. 39. P. 347–351. https://doi.org/10.1038/ng1975
  7. Mitchell M., Cutler J. // Methods Mol. Biol. 2011. V. 688. P. 17–33. https://doi.org/10.1007/978-1-60761-947-5_3
  8. Botezatu I.V., Kondratova V.N., Shelepov V.P., Mazurenko N.N., Tsyganova I.V., Susova O.Y., Lichtenstein A.V. // Anal. Biochem. 2020. V. 590. P. 113517. https://doi.org/10.1016/j.ab.2019.113517
  9. Li J., Wang L., Mamon H., Kulke M.H., Berbeco R., Makrigiorgos G.M. // Nat. Med. 2008. V. 14. P. 579– 584. https://doi.org/10.1038/nm1708
  10. Nagai Y., Miyazawa H., Huqun, Tanaka T., Udagawa K., Kato M., Fukuyama S., Yokote A., Kobayashi K., Kanazawa M., Hagiwara K. // Cancer Res. 2005. V. 65. P. 7276–7282. https://doi.org/10.1158/0008-5472
  11. Imanishi T., Obika S. // Chem. Commun. (Camb). 2002. V. 16. P. 1653–1659. https://doi.org/10.1039/b201557a
  12. Rahman S.M., Seki S., Obika S., Yoshikawa H., Miyashita K., Imanishi T. // J. Am. Chem. Soc. 2008. V. 130. P. 4886–4896. https://doi.org/10.1021/ja710342q
  13. Latorra D., Campbell K., Wolter A., Hurley J.M. // Hum. Mutat. 2003. P. 22. P. 79–85. https://doi.org/10.1002/humu.10228
  14. Parris B.A., Shaw E., Pang B., Soong R., Fong K., Soo R.A. // Respirology. 2019. V. 24. P. 215–226. https://doi.org/10.1111/resp.13463
  15. Itonaga M., Matsuzaki I., Warigaya K., Tamura T., Shimizu Y., Fujimoto M., Kojima F., Ichinose M., Murata S. // PLoS One. 2016. V. 11. P. e0151654. https://doi.org/10.1371/journal.pone.0151654
  16. Emelyanova M., Ghukasyan L., Abramov I., Ryabaya O., Stepanova E., Kudryavtseva A., Sadritdinova A., Dzhumakova C., Belysheva T., Surzhikov S., Lyubchenko L., Zasedatelev A., Nasedkina T. // Oncotarget. 2017. V. 8. P. 52304–52320. https://doi.org/10.18632/oncotarget.17014
  17. Picca A., Berzero G., Di Stefano A.L., Sanson M. // Expert Rev. Mol. Diagn. 2018. V. 18. P. 1041–1051. https://doi.org/10.1080/14737159.2018.1548935
  18. Pirozzi C.J., Yan H. // Nat. Rev. Clin. Oncol. 2021. V. 18. P. 645–661. https://doi.org/10.1038/s41571-021-00521-0
  19. Tian W., Zhang W., Wang Y., Jin R., Wang Y., Guo H., Tang Y., Yao X. // Front. Pharmacol. 2022. V. 13. P. 982424. https://doi.org/10.3389/fphar.2022.982424
  20. Varachev V.O., Guskov D.A., Shekhtman A.P., Rogozhin D.V., Polyakov S.A., Chudinov A.V., Zasedatelev A.S., Nasedkina T.V. // Russ. J. Bioorg. Chem. 2023. V. 49. P. 1147–1152. https://doi.org/10.1134/S1068162023050205
  21. Nafa K., Hameed M., Arcila M.E. // Methods Mol. Biol. 2016. V. 1392. P. 71–82. https://doi.org/10.1007/978-1- 4939-3360-0_8
  22. Шаманин В.А., Карпов И.В., Писарева Е.Е., Гуткина Н.И., Коваленко С.П. // Сибирский онкологич. журнал. 2018. T. 17. C. 30–35. https://doi.org/10.21294/1814-4861-2018-17-4-30-35
  23. Emelyanova M., Arkhipova K., Mazurenko N., Chudinov A., Demidova I., Zborovskaya I., Lyubchenko L., Zasedatelev A., Nasedkina T. // Appl. Immunohistochem. Mol. Morphol. 2015. V. 23. P. 255–265. https://doi.org/10.1097/PAI.0000000000000084
  24. Kukhtin A.C., Sebastian T., Golova J., Perov A., Knickerbocker C., Linger Y., Bueno A., Qu P., Villanueva M., Holmberg R.C., Chandler D.P., Cooney C.G. // Lab. Chip. 2019. V. 19. P. 1217–1225. https://doi.org/10.1039/c8lc01404c

Arquivos suplementares

Arquivos suplementares
Ação
1. JATS XML
Baixar
2. Fig. 1. Suppression of amplification of the wild-type R132 IDH1 allele in a sample containing ~50% of the mutant R132H allele at different concentrations of LNA oligomer. (a) – Amplification curves of the wild-type R132 allele and the mutant R132H allele: wild-type (wt) allele – curves 1 (0 nM), 2 (50 nM) and 3 (100 nM); mutant allele (R132H) – curves 4 (0 nM), 5 (50 nM) and 6 (100 nM); (b) – difference in the threshold cycle values ​​of the amplification curves of the wild-type (wt) and mutant allele (R132H) allele at different concentrations of LNA compared to zero concentration.

Baixar (97KB)
Metadados
3. Fig. 2. Analysis of melting curves with a TaqMan probe for suppression of the amplification of the wild-type allele R132 IDH1 (wt) in a sample containing 15–20% of the mutant allele (R132H) at different concentrations of LNA oligomer: 1 – wild-type sample (0 nM); samples with the R132H mutation – curves 2 (0 nM), 3 (50 nM), and 4 (100 nM). An increase in the R132H peak is noticeable with increasing LNA oligomer concentration.

Baixar (84KB)
Metadados
4. Fig. 3. Detection of the R132H IDH1 mutation in a sample containing 0.5% of the mutant allele at different concentrations of the LNA oligomer. Hybridization patterns: (a) 0 nM LNA, (b) 50 nM LNA, (c) 100 nM LNA; (d) normalized values ​​of fluorescent signals.

Baixar (71KB)
Metadados
5. Fig. 4. Detection of mutations in the IDH1 gene in tumor samples (chondrosarcoma) with a low content of the mutant allele. The upper part of the figure shows the hybridization patterns on the biochip, the lower part – the normalized values ​​of the fluorescence signals. Sample with the R132C mutation: (a) – 0 nM LNA oligomer in the PCR reaction, (b) – with the addition of 100 nM LNA oligomer. Sample with the R132L mutation: (c) – 0 nM LNA oligomer in the PCR reaction, (d) – with the addition of 100 nM LNA oligomer. Wells with the fluorescent dye Cy5, which act as a marker, are located in the corners of the biochip.

Baixar (119KB)
Metadados

Declaração de direitos autorais © Russian Academy of Sciences, 2024