CCND1 and PTEN genes as markers of progression in vulvar cancer

Purpose of the study. Studying the relative copy number of 12 genes: MYC, PTEN, CCND1, PIK3CA, TP53, CDKN2A, MDM2, MCL1, NFKBIA, MTAP, BIRC2, KMT2C, to search for potential prognostic cancer markers in vulvar cancer (VR).

Patients and methods. The study included 50 patients aged 29–80 years with the diagnosis of squamous cell carcinoma of the vulva without metastases – group 1 (n = 25) and with metastases to the lymph nodes – group 2 (n = 25). Total DNA from paraffin blocks of tumor and conditionally healthy vulvar tissue served as the material for the study. The relative copy number of genes (RCQ) was assessed using the RT-qPCR method: MYC, PTEN, CCND1, PIK3CA, TP53, CDKN2A, MDM2, MCL1, NFKBIA, MTAP, BIRC2, KMT2C and reference genes: GAPDH and B2M. Raw RT-qPCR data were analyzed using Bio-Rad CFX Manager software (ver. 2.1). RCQ of the genetic locus was calculated using the formula 2-ΔCt. Analysis of the significance of differences included determination of the OR (CI 95 %), Chi-square, Mann-Whitney U-test. Differences were considered statistically significant at p < 0.05.

Results. Amplification was observed for 9 genes: MYC (48 % of cases), MCL1 (39 %), NFKBIA (46 %), CCND1 (52 %), PIK3CA (48 %), TP53 (62 %), MDM2 (40 %), BIRC2 (54 %), KMT2C (42 %); losses were recorded for 3 genes PTEN (44 % of cases), MTAP (32 %), CDKN2A (36 %). In the 2nd group of patients, a statistically significant 1.7-fold increase in RCQ of the CCND1 gene (p < 0.05) and a 1.9-fold decrease in RCQ of the PTEN gene (p < 0.05) were found.

Conclusion. In the study, CCND1 amplification events and/or PTEN loss were associated with lymph node metastasis in RV patients. Thus, the copy numbers of these genes may serve as prognostic markers of the disease.

1. Cancer Stat Facts: Vulvar Cancer. [(accessed on 21 March 2022)]; Available online: https://seer.cancer.gov/statfacts/html/vulva.html.

2. Kang Y.J., Smith M., Barlow E., Coffey K., Hacker N., Canfell K. Vulvar cancer in high-income countries: Increasing burden of disease. Int J Cancer. 2017 Dec 1;141(11):2174-2186. doi: 10.1002/ijc.30900.

3. Singh N., Gilks C.B. Vulval squamous cell carcinoma and its precursors. Histopathology. 2020 Jan;76(1):128-138. doi: 10.1111/his.13989.

4. Eva L.J., Sadler L., Fong K.L., Sahota S., Jones R.W., Bigby S.M. Trends in HPV-dependent and HPV-independent vulvar cancers: The changing face of vulvar squamous cell carcinoma. Gynecol Oncol. 2020 May;157(2):450-455. doi: 10.1016/j.ygyno.2020.01.029.

5. Zhang J., Zhang Y., Zhang Z. Prevalence of human papillomavirus and its prognostic value in vulvar cancer: A systematic review and meta-analysis. PLoS One. 2018 Sep 26;13(9):e0204162. doi: 10.1371/journal.pone.0204162.

6. Zięba S., Pouwer A.W., Kowalik A., Zalewski K., Rusetska N., Bakuła-Zalewska E., et al. Somatic Mutation Profiling in Premalignant Lesions of Vulvar Squamous Cell Carcinoma. Int J Mol Sci. 2020 Jul 10;21(14):4880. doi: 10.3390/ijms21144880.

7. Singh N., Gilks C.B. Vulval squamous cell carcinoma and its precursors. Histopathology. 2020 Jan;76(1):128-138. doi: 10.1111/his.13989.

8. Hinten F., Molijn A., Eckhardt L., Massuger LFAG, Quint W., Bult P., et al. Vulvar cancer: Two pathways with different localization and prognosis. Gynecol Oncol. 2018 May;149(2):310-317. doi: 10.1016/j.ygyno.2018.03.003.

9. Sand F.L., Nielsen D.M.B., Frederiksen M.H., Rasmussen C.L., Kjaer S.K. Response: Letter regarding "The prognostic value of p16 and p53 expression for survival after vulvar cancer: A systematic review and meta-analysis". Gynecol Oncol Rep. 2019 Sep 3;30:100494. doi: 10.1016/j.gore.2019.100494.

10. Woelber L., Eulenburg C., Choschzick M., Kruell A., Petersen C., Gieseking F, et al. Prognostic role of lymph node metastases in vulvar cancer and implications for adjuvant treatment. Int J Gynecol Cancer. 2012 Mar;22(3):503-8. doi: 10.1097/IGC.0b013e31823eed4c.

11. Polterauer S., Schwameis R., Grimm C., Hillemanns P., Jückstock J., Hilpert F., et al. Lymph node ratio in inguinal lymphadenectomy for squamous cell vulvar cancer: Results from the AGO-CaRE-1 study. Gynecol Oncol. 2019 May;153(2):286-291. doi: 10.1016/j.ygyno.2019.02.007.

12. Zarrei M, MacDonald JR, Merico D, Scherer SW. A copy number variation map of the human genome. Nat Rev Genet. 2015;16(3):172–183. doi: 10.1038/nrg3871.

13. Xu W, Lu J, Zhao Q, Wu J, Sun J, Han B, Zhao X, Kang Y. Genome-Wide Plasma Cell-Free DNA Methylation Profiling Identifies Potential Biomarkers for Lung Cancer. Dis Markers. 2019 Feb 5;2019:4108474. doi: 10.1155/2019/4108474.

14. Petrusenko N.A., Verenikina E.V., Yakubova D.Yu., Timoshkina N.N. Mutations in the BRCA1 / 2 genes in patients in the south of Russia with malignant neoplasms of the ovaries // Yakut Medical Journal. - 2020. - No. 4 (72), p.87-89. DOI 10.25789 / YMJ.2020.72.21.

15. Petrusenko N.A., Nikitina V.P., Spiridonova D.A., Kecheryukova M.M. Changes in the copy number of genes in malignant tumors of the cervix with endophytic and exophytic forms of growth // Modern problems of science and education. - 2019. - No. 3. URL: http://www.science-education.ru/ru/article/view?id=28981 (date of access: 04/26/2021)].

16. Vodolazhsky D.I., Timoshkina N.N., Maslov A.A., Kolesnikov E.N., Tatimov M.Z. CNV of 17 genetic loci in patients diagnosed with gastric adenocarcinoma//. Modern problems of science and education. – 2017. – No. 3. URL: https://science-education.ru/ru/article/view?id=26405 (accessed: 11/16/2022).

17. Macintyre G., Goranova T.E., De Silva D., Ennis D., Piskorz A.M., Eldridge M., et al. Copy number signatures and mutational processes in ovarian carcinoma. Nat Genet. 2018;50(9):1262–1270. doi: 10.1038/s41588-018-0179-8/

18. Zarrei M., MacDonald J.R., Merico D., Scherer S.W. A copy number variation map of the human genome. Nat. Rev. Genet. 2015;16:172–183. doi: 10.1038/nrg3871.

19. Maslov A.A., Chalkhakhyan L.K., Malinin S.A., Kaminsky G.V., Mirzoyan E.A. Genes copy number variation in colorectal cancer patients as a marker of the disease clinical outcome and response to therapy. South Russian Journal of Cancer. 2022;3(2):52-64. https://doi.org/10.37748/2686-9039-2022-3-2-6

20. Genecards. The Human Gene Database. 2016. Available from: www.genecards.org.

21. Ortiz A.B., Garcia D., Vicente Y., Palka M., Bellas C., Martin P. Prognostic significance of cyclin D1 protein expression and gene amplification in invasive breast carcinoma. PLoS ONE. 2017;12(11):e0188068. doi: 10.1371/journal.pone.0188068.

22. Yang L., Ye F., Bao L., Zhou X., Wang Z., Hu P., et al. Somatic alterations of TP53, ERBB2, PIK3CA and CCND1 are associated with chemosensitivity for breast cancers. Cancer Sci. 2019;110(4):1389–1400. doi: 10.1111/cas.13976.

23. Choschzick M., Hess S., Tennstedt P., Holst F., Bohlken H., Gieseking F., et al. Role of cyclin D1 amplification and expression in vulvar carcinomas. Hum Pathol. 2012 Sep;43(9):1386-93. doi: 10.1016/j.humpath.2011.11.014.

24. Shen W.H., Balajee A.S., Wang J., Wu H., Eng C., Pandolfi P.P., et al. Essential Role for Nuclear PTEN in Maintaining Chromosomal Integrity. Cell. 2007;128:157–170. doi: 10.1016/j.cell.2006.11.042.

25. Cancer Genome Atlas Network Genomic Classification of Cutaneous Melanoma. Cell. 2015;161:1681–1696. doi: 10.1016/j.cell.2015.05.044.

26. Zuo Q., Liu J., Huang L., Qin Y., Hawley T., Seo C., et al. AXL/AKT axis mediated-resistance to BRAF in-hibitor depends on PTEN status in melanoma. Oncogene. 2018;37:3275–3289. doi: 10.1038/s41388-018-0205-4.

27. Qin Y., Zuo Q., Huang L., Huang L., Merlino G., Yu Y. PERK mediates resistance to BRAF inhibition in melanoma with impaired PTEN. NPJ Precis. Oncol. 2021;5:68. doi: 10.1038/s41698-021-00207-x.

28. Liu A., Zhu Y., Chen W., Merlino G., Yu Y. PTEN Dual Lipid- and Protein-Phosphatase Function in Tumor Progression. Cancers (Basel). 2022 Jul 28;14(15):3666. doi: 10.3390/cancers14153666.

29. Dibble C.C., Cantley L.C. Regulation of mTORC1 by PI3K signaling. Trends Cell Biol. 2015 Sep;25(9):545-55. doi: 10.1016/j.tcb.2015.06.002.