A joint determination of gene expression and methylation for the diagnosis of clear cell renal cancer

Background. Clear cell renal cell carcinoma is the most frequent and most aggressive kidney cancer. Approximately 30 % at the initial diagnosis reveal distant metastases. This is due to the fact that kidney cancer in the early stages is asymptomatic. Very often (about 50 %), kidney cancer is detected by chance, during a routine examination or during treatment for other reasons. In this regard, the development of new methods of early diagnosis of clear cell renal cell carcinoma is actual.

Materials and methods. The levels of mRNA expression and methylation of a number of genes in the surgical material of paired samples (normal kidney tissue and tumor, 21 clinical cases) were studied. Quantitative determination of gene expression was performed using real-time polymerase chain reaction on a Step One Plus instrument (Applied Biosystems, USA) using TaqMan®Gene Expression Assays (Applied Biosystems, USA). High molecular DNA was isolated from tissue by phenol-chloroform extraction. Methyl-specific polymerase chain reaction was performed on a T100 Thermal Cycler amplifier (Bio-Rad, USA).

Results. As a result of joint analysis of the levels of gene expression and methylation, a system of potentially diagnostic markers has been developed, including the determination of the expression of a number of protein coding genes and the hypermethylation of several miRNA genes in tumor samples. Simultaneous determination of expression and methylation allows for the correct identification of tumors as SCRV with a sensitivity of 95.24 % (95 % confidence interval 76.18–99.88 %) and specificity of 95.24 % (95 % confidence interval 76.18–99.88 %).

Conclusion. According to the results of the study, a system was developed based on the determination of the expression levels of the CA9, HIG2, EGLN3,  NDUF4L2  genes and the methylation of the MIR9-1, MIR34b/c,  MIR124a-3,  MIR129-2.  Depending on the requirements for sensitivity, reliability of determination, or ease of implementation, various combinations of these genes are possible.

1. Muglia V.F., Prando A. Renal cell carcinoma: histological classification and correlation with imaging findings. Radiol Bras 2015;48(3):166—74. DOI: 10.1590/0100-3984.2013.1927. PMID: 26185343.

2. Ljungberg B., Bensalah K., Bex A. et al. Guidelines on Renal Cell Carcinoma. European association of urology 2014:70. DOI: 10.1016/j.eururo.2015.01.005.

3. Caoili E.M., Davenport M.S. Role of per-cutaneous needle biopsy for renal masses. Semin Intervent Radiol 2014;31(1):20—6. DOI: 10.1055/s-0033-1363839. PMID: 24596436.

4. Vetterlein M.W., Jindal T., Becker A. et al. Small renal masses in the elderly: contemporary treatment approaches and comparative oncological outcomes of nonsurgical and surgical strategies. Investig Clin Urol 2016;57(40):231—9. DOI: 10.4111/icu.2016.57.4.231. PMID: 27437532.

5. Marconi L., Dabestani S., Lam T.B. et al. Systematic review and meta-analysis of diagnostic accuracy of percutaneous renal tumour biopsy. Eur Urol 2016;69:660-73. DOI: 10.1016/j.eururo.2015.07.072.

6. Vrba L., Munoz-Rodriguez J.L., Stampfer M.R., Futscher B.W. miRNA gene promoters are frequent targets of aberrant DNA methylation in human breast cancer. PLoS One 2013;8(1):e54398. DOI: 10.1371/journal.pone.0054398. PMID: 23342147.

7. Piletic K., Kunej T. MicroRNA epigenetic signatures in human disease. Arch Toxicol 2016;90(10):2405—19. DOI: 10.1007/s00204-016-1815-7. PMID: 27557899.

8. Baylin S.B., Jones P.A. Epigenetic Determinants of Cancer. Cold Spring Harb Perspect Biol 2016;8(9):a019505. DOI: 10.1101/cshperspect.a019505. PMID: 27194046.

9. Loginov V.I., Beresneva E.V., Kazubskaya T.R. et al. Methylation of 10 miRNA genes in clear cell renal cell carcinoma and their diagnostic value. Onkourologiya = Cancer Urology 2017;13(3):27:33. (In Russ.).

10. Apanovich N.V., Peters M.V., Korotaeva A.A. et al. Molecular genetic diagnostics of clear cell renal cell carcinoma. Onkourologiya = Cancer Urology 2016;12(4):16—20. (In Russ.).

11. Loginov V.I., Dmitriev A.A., Senchenko V.N. et al. Tumor suppressor function of the SEMA3B gene in human lung and renal cancers. PLos One 2015;10(5):e0123369. DOI: 10.1371/journal.pone.0123369. PMID: 25961819.

12. Beresneva E.V., Rykov S.V., Khodyrev D.S. et al. Methylation profile of group of miRNA genes in clear cell renal cell carci¬noma; involvement in cancer progression. Genetika = Genetika 2013;49(3): 366—75. (In Russ.).

13. Lee K.H., Lotterman C., Karikari C. et al. Epigenetic silencing of MicroRNA miR-107 regulates cyclindependent kinase 6 expression in pancreatic cancer. Pancreatology 2009;9(3):293—301. DOI: 10.1159/000186051. PMID: 19407485.

14. Yang C., Cai J., Wang Q. et al. Epigenetic silencing of miR-130b in ovarian cancer promotes the development of multidrug resistance by targeting colony-stimulating factor 1. Gynecol Oncol 2012;124(2): 325—34. DOI: 10.1016/j.ygyno.2011.10.013. PMID: 22005523.

15. Li H.P., Huang H.Y., Lai Y.R. et al. Si-lencing of miRNA-148a by hyper methylation activates the integrin-mediated signaling pathway in nasopharyngeal carcinoma. Oncotarget 2014;5(17):7610—24. DOI: 10.18632/oncotarget.2282. PMID: 25277193.

16. Wang C., Hu J., Lu M. et al. A panel of five serum miRNAs as a potential diagnostic tool for early-stage renal cell carcinoma. Sci Rep 2015;5:7610. DOI: 10.1038/srep07610. PMID: 25556603.

17. Yadav S., Khandelwal M., Seth A. et al. Serum microRNA expression profiling: potential diagnostic implications of a panel of serum microRNAs for clear cell renal cell cancer. Urology 2017;104:64—9. DOI: 10.1016/j.urology.2017.03.013. PMID: 28336290.

18. Wykoff C.C., Beasley N.J., Watson P.H. et al. Hypoxia-inducible expression of tumor-associated carbonic anhydrases. Cancer Res 2000;60:7075—83. PMID: 11156414.

19. Meng Q., Xiang L., Fu J. et al. Transcriptome profiling reveals miR-9-3p as a novel tumor suppressor in gastric cancer. Oncotarget 2017;6(8/23):37321—31. DOI: 10.18632/oncotarget.16310. PMID: 28418879.

20. Pronina I.V., Klimov E.A., Burdennyy A.M. et al. Methylation of the genes for the microRNAs miR-129-2 and miR-9-1, changes in their expression, and activation of their potential target genes in clear cell renal cell carcinoma. Moleculyarnaya Biologiya = Molecular Biology 2017;51(1):61—71. (In Russ.).

21. Cao H., Liu Z., Wang R. et al. miR-148a suppresses human renal cell carcinoma malignancy by targeting AKT2. Oncol Rep 2017;37(1):147—54. DOI: 10.3892/or.2016.5257. PMID: 27878305.

22. Varachev V., Loginov V., Pronina I. et al. Hypermethylated tumor suppressor microRNAs as novel markers of clear cell renal cell carcinoma. FEBS Open Bio 2018;8(1):304—5. DOI: 10.1002/2211-5463.12453.

23. Marconi L., Dabestani S., Lam T.B. et al. Systematic review and meta-analysis of diagnostic accuracy of percutaneous renal tumour biopsy. Eur Urol 2016;69:660—73. DOI: 10.1016/j.eururo.2016.04.027. PMID: 27157997.