Diagnostic informative value of liquid-based cytology optimized with genetic methods for the differential diagnosis of precancerous and malignant diseases of the cervix

Purpose of the study. Was to assess diagnostic informative value of liquid-based cytology optimized with genetic methods for the differential diagnosis of precancerous and malignant diseases of the cervix.

Materials and methods. The study included 381 patients. Cervical pathologies were diagnosed with liquid-based cytology only and liquid-based cytology optimized with genetic methods of assessing the expression of miRNA‑20a, miRNA‑375, miRNA‑21 and –23b. Results of liquid-based cytology and genetic methods were verified by histological examination of the material. Statistical analysis was performed using descriptive statistics methods with the calculation of the mean and standard error of the mean. The mean values were compared with the help of the Mann-Whitney test.

Results. Diagnostic results of liquid-based cytology were consistent with histological results in 107 (73.8 %) of 145 cervical cancer (CC) patients, in 52 (57.1 %) of 91 patients with high-grade squamous intraepithelial lesions (HSIL), and in 30 (65.2 %) of 46 patients with low-grade squamous intraepithelial lesions (LSIL). Optimization of liquid-based cytology by assessing the expression of miRNA‑21 and miRNA‑23b in the cervical epithelium improved the diagnostic sensitivity of the method from 73.8 % to 80 %, and its specificity from 94.1 % to 97.9 %. The diagnostic sensitivity and specificity of liquid-based cytology for differential diagnosis of CC and HSIL was 87 % and 78.8 %, respectively. Optimization of liquidbased cytology by assessing the expression of miRNA‑20a and miRNA‑375 in the cervical epithelium for the differential diagnosis of CC and HSIL improved the diagnostic sensitivity of the method from 87 % to 95.1 %, and its specificity from 78.8 % to 93.9 %.

Conclusions. We revealed the most informative pairs of miRNAs in the cervical epithelium, as an analysis of their expression expanded the possibilities of liquid-based cytology both as a method for diagnosing CC and as a method for the differential diagnosis between CC and HSIL.

1. Williamson A-L. The Interaction between Human Immunodeficiency Virus and Human Papillomaviruses in Heterosexuals in Africa. J Clin Med. 2015 Apr 2;4(4):579–592. https://doi.org/10.3390/jcm4040579

2. Briko NI, Sekacheva MI, Lopukhov PD, Kobzev GS, Kaprin AD, Novikova EG, et al. Clinical and epidemiological features of papillomavirus infection manifestations on the example of cervical cancer and genital warts. Annals of the Russian academy of medical sciences. 2020;75(1):77–82. https://doi.org/10.15690/vramn1212

3. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018 Nov;68(6):394–424. https://doi.org/10.3322/caac.21492

4. Arbyn M, Weiderpass E, Bruni L, de Sanjosé S, Saraiya M, Ferlay J, et al. Estimates of incidence and mortality of cervical cancer in 2018: a worldwide analysis. Lancet Glob Health. 2020 Feb;8(2):e191–203. https://doi.org/10.1016/S2214-109X(19)30482-6

5. The state of cancer care in Russia in 2019. Ed. by A.D.Kaprin, V.V.Starinsky, A.O.Shakhzadova. M.: P.A.Herzen Moscow State Medical Research Institute-branch of the Federal State Budgetary Institution "NMIC of Radiology" of the Ministry of Health of Russia, 2020, 239 p.

6. Elgina SI, Zolotarevskaya OS, Razumova VA, Kratovskiy AYu. Application of liquid-based cytology in diagnosing cervical pathology. Mother and Child in Kuzbass. 2018;(3(74)):46–49. (In Russian).

7. Kazaishvili T. N. Early diagnosis of cervical cancer by liquid cytology. I National Congress "Oncology of the reproductive Organs from prevention and early detection to effective treatment"; 19– 21 May 2016, Moscow. Available at: https://www.rpmj.ru/rpmj/article/viewFile/131/132. Accessed: 01.11.2020. (In Russian).

8. Brase JC, Wuttig D, Kuner R, Sültmann H. Serum microRNAs as non-invasive biomarkers for cancer. Mol Cancer. 2010 Nov 26;9:306. https://doi.org/10.1186/1476-4598-9-306

9. He L, Thomson JM, Hemann MT, Hernando-Monge E, Mu D, Goodson S, et al. A microRNA polycistron as a potential human oncogene. Nature. 2005 Jun 9;435(7043):828–833. https://doi.org/10.1038/nature03552

10. Pritchard CC, Cheng HH, Tewari M. MicroRNA profiling: approaches and considerations. Nat Rev Genet. 2012 Apr 18;13(5):358–369. https://doi.org/10.1038/nrg3198

11. Kroh EM, Parkin RK, Mitchell PS, Tewari M. Analysis of circulating microRNA biomarkers in plasma and serum using quantitative reverse transcription-PCR (qRT-PCR). Methods. 2010 Apr;50(4):298–301. https://doi.org/10.1016/j.ymeth.2010.01.032

12. Kit OI, Vodolazhsky DI, Timoshkina NN. The role of microRNAs in the regulation of melanoma signaling pathways. Molecular Medicine. 2017;15(1):15–23. (In Russian).

13. Solayman MHM, Langaee T, Patel A, El-Wakeel L, El-Hamamsy M, Badary O, et al. Identification of Suitable Endogenous Normalizers for qRT-PCR Analysis of Plasma microRNA Expression in Essential Hypertension. Mol Biotechnol. 2016 Mar;58(3):179–187. https://doi.org/10.1007/s12033-015-9912-z

14. Fang Q-Y, Deng Q-F, Luo J, Zhou C-C. MiRNA-20a-5p accelerates the proliferation and invasion of non-small cell lung cancer by targeting and downregulating KLF9. Eur Rev Med Pharmacol Sci. 2020 Mar;24(5):2548–2556. https://doi.org/10.26355/eurrev_202003_20522

15. Karimkhanloo H, Mohammadi-Yeganeh S, Ahsani Z, Paryan M. Bioinformatics prediction and experimental validation of microRNA-20a targeting Cyclin D1 in hepatocellular carcinoma. Tumour Biol. 2017 Apr;39(4):1010428317698361. https://doi.org/10.1177/1010428317698361

16. Li J, Huang H, Sun L, Yang M, Pan C, Chen W, et al. MiR-21 indicates poor prognosis in tongue squamous cell carcinomas as an apoptosis inhibitor. Clin Cancer Res. 2009 Jun 15;15(12):3998–4008. https://doi.org/10.1158/1078-0432.CCR-08-3053

17. Yao T, Lin Z. MiR-21 is involved in cervical squamous cell tumorigenesis and regulates CCL20. Biochim Biophys Acta. 2012 Feb;1822(2):248–260. https://doi.org/10.1016/j.bbadis.2011.09.018

18. Wang W, Wang Y, Liu W, van Wijnen AJ. Regulation and biological roles of the multifaceted miRNA-23b (MIR23B). Gene. 2018 Feb 5;642:103–109. https://doi.org/10.1016/j.gene.2017.10.085

19. Allah AW, Yahya M, Elsaeidy KS, Alkanj S, Hamam K, El-Saady M, et al. Clinical assessment of miRNA-23b as a prognostic factor for various carcinomas: A systematic review and meta-analysis. Meta Gene. 2020;24:100651. https://doi.org/10.1016/j.mgene.2020.100651

20. Ali OS, Shabayek MI, Seleem MM, Abdelaziz HG, Makhlouf DO. MicroRNAs 182 and 375 Sera Expression as Prognostic Biochemical Markers in Breast Cancer. Clin Breast Cancer. 2018 Dec;18(6):e1373–e1379. https://doi.org/10.1016/j.clbc.2018.07.020

21. Wu S, Chen H. Anti-Condyloma acuminata mechanism of microRNAs-375 modulates HPV in cervical cancer cells via the UBE3A and IGF-1R pathway. Oncol Lett. 2018 Sep;16(3):3241–3247. https://doi.org/10.3892/ol.2018.8983

22. Shen J, Stass SA, Jiang F. MicroRNAs as potential biomarkers in human solid tumors. Cancer Lett. 2013 Feb 28;329(2):125–136. https://doi.org/10.1016/j.canlet.2012.11.001

23. Ruspatent. Registered on 19.12.2019. Certificate of state registration No. RU2709651C1. Kit OI, Timoshkina NN, Pushkin AA, Kutilin DS, Rostorguev EE, Kuznetsova NS. Method for differential diagnosis of gliomas based on analysis of gene expression and micro-RNA. (In Russian).

24. Soloman D. The 1988 Bethesda system for reporting cervical/vaginal cytologic diagnoses: Developed and approved at the national cancer institute workshop in Bethesda, MD, December 12–13, 1988. Diagnostic Cytopathology. 1989;5(3):331–334. https://doi.org/10.1002/dc.2840050318

25. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001 Dec;25(4):402–408. https://doi.org/10.1006/meth.2001.1262

26. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001 May 1;29(9):e45. https://doi.org/10.1093/nar/29.9.e45

27. Mulhollanda R, Yousef HMSA, Laing M, Gupta R, Leung EYL. Comparison of women with possible endocervical and non-cervical glandular neoplasms detected in liquid-based cervical cytology- incidence, clinical characteristics and outcomes: A cohort study. Eur J Obstet Gynecol Reprod Biol. 2021 Feb;257:100– 105. https://doi.org/10.1016/j.ejogrb.2020.12.025

28. Jihad NA, Naif HM. Evaluation of microRNA-20, -21 and -143 expression in human papilloma virus induced premalignant and malignant cervical lesions. Gene Reports. 2020;20:100702. https://doi.org/10.1016/j.genrep.2020.100702

29. Nebgen DR, Lu KH, Bast RC. Novel Approaches to Ovarian Cancer Screening. Curr Oncol Rep. 2019 Jul 26;21(8):75. https://doi.org/10.1007/s11912-019-0816-0