vestimИзвестия Национальной академии наук Беларуси. Серия медицинских наукProceedings of the National Academy of Sciences of Belarus, Medical series1814-60232524-2350The Republican Unitary Enterprise Publishing House "Belaruskaya Navuka"10.29235/1814-6023-2023-20-4-278-288vestim-934Research ArticleКЛИНИЧЕСКАЯ И ЭКСПЕРИМЕНТАЛЬНАЯ МЕДИЦИНАCLINICAL AND EXPERIMENTAL MEDICINEАнализ взаимосвязи соматических мутаций с формированием лейкоплакии и плоскоклеточного рака слизистой оболочки ротовой полостиAnalysis of the relationship of somatic mutations with the development of leukoplakia and squamous cell carcinoma of the oral mucosaКарпукН. А.KarpukN. A.

Карпук Наталья Анатольевна – кандидат медицинских наук, доцент.

пр-т Фрунзе, 27, 210009, Витебск

Natalia A. Karpuk – Ph. D. (Med.), Associate Professor, Vitebsk State Medical University.

27, Frunze Ave., 210009, Vitebsk

ms.karpuk@mail.ruhttps://orcid.org/0000-0002-7450-3757РубниковичС. П.RubnikovichS. P.

Рубникович Сергей Петрович – член-корреспондент, доктор медицинских наук, профессор, ректор.

пр. Дзержинского, 83, 220116, Минск

Sergey P. Rubnikovich – Corresponding Member, D. Sc. (Med.), Professor, Rector, Belarusian State Medical University.

83, Dzerzhinski Ave., 220116, Minsk

rubnikovich@mail.ruhttps://orcid.org/0000-0002-4912-2880ЖильцовИ. В.ZhyltsouI. V.

Жильцов Иван Викторович – доктор медицинских наук, профессор, заведующий кафедрой.

пр-т Фрунзе, 27, 210009, Витебск

Ivan V. Zhyltsou – D. Sc. (Med.), Professor, Head of the Department, Vitebsk State Medical University.

27, Frunze Ave., 210009, Vitebsk

zhyltsou@tut.byhttps://orcid.org/0000-0002-6093-4548МазурО. Ч.MazurO. C.

Мазур Оксана Чеславовна – научный сотрудник.

ул. Академическая, 27, 220072, Минск

Oksana C. Mazur – Researcher, Institute of Genetics and Cytology of the National Academy of Sciences of Belarus.

27, Akademicheskaya Str., 220072, Minsk

terezia@mail.ruhttps://orcid.org/0000-0001-9991-7035КарпукИ. Ю.KarpukI. Yu.

Карпук Иван Юрьевич – доктор медицинских наук, доцент, профессор, декан.

пр-т Фрунзе, 27, 210009, Витебск

Ivan Yu. Karpuk – D. Sc. (Med.), Associate Professor, Professor, dean, Vitebsk State Medical University.

27, Frunze Ave., 210009, Vitebsk

ikarpuk@mail.ruhttps://orcid.org/0000-0003-4543-2862МихаленкоЕ. П.MikhalenkaA. P.

Михаленко Елена Петровна – кандидат биологических наук, ведущий научный сотрудник.

ул. Академическая, 27, 220072, Минск

Alena P. Mikhalenka – Ph. D. (Biol.), Leading Researcher, Institute of Genetics and Cytology of the National Academy of Sciences of Belarus.

27, Akademicheskaya Str., 220072, Minsk

michalenko75@mail.ruВитебский государственный медицинский университетVitebsk State Medical UniversityБелорусский государственный медицинский университетBelarusian State Medical UniversityИнститут генетики и цитологии НАН БеларусиInstitute of Genetics and Cytology of the National Academy of Sciences of Belarus202302122023204278288Copyright © Карпук Н.А., Рубникович С.П., Жильцов И.В., Мазур О.Ч., Карпук И.Ю., Михаленко Е.П., 20232023Карпук Н.А., Рубникович С.П., Жильцов И.В., Мазур О.Ч., Карпук И.Ю., Михаленко Е.П.Karpuk N.A., Rubnikovich S.P., Zhyltsou I.V., Mazur O.C., Karpuk I.Y., Mikhalenka A.P.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vestimed.belnauka.by/jour/article/view/934

Молекулярно-генетические основы патогенеза лейкоплакий и плоскоклеточного рака слизистой оболочки ротовой полости (СОРП) недостаточно изучены. Исследований, посвященных данной проблеме, мало, а их результаты противоречивы. При этом ранняя диагностика рака СОРП и прогнозирование его развития являются важными проблемами здравоохранения.

Цель исследования ‒ провести анализ взаимосвязи соматических мутаций с формированием лейкоплакии и плоскоклеточного рака слизистой оболочки ротовой полости.

Материалом для исследования являлись 48 образцов измененного эпителия СОРП пациентов с лейкоплакией СОРП (ЛСОРП) (24 образца) и с плоскоклеточным раком СОРП (ПРСОРП) (24 образца).

Выявленные в настоящем исследовании патогенные и вероятно патогенные варианты генов TP53, NRAS и BRAF, как поодиночке, так и в сочетаниях, с высокой вероятностью (ОР 3000‒11000) ассоциированы с ЛСОРП с дисплазией эпителия первой степени, а варианты генов ERCC3, HOXB13, KRAS, MSH3, MSH6, PIK3CA и TP53 с высокой вероятностью (ОР 90‒22 000) ассоциированы с развитием ПРСОРП. Описанные патогенные варианты генов KRAS и TP53, как правило, приводят к формированию ЛСОРП с дисплазией эпителия первой степени, а последующее образование патогенных вариантов генов PIC3CA и/или HOXB13 и MSH3 вызывает злокачественную трансформацию измененных клеток эпителия СОРП ( р = 0,0000048). Данная информация позволяет разработать тест-системы на основе ПЦР и NGS для ранней диагностики ПРСОРП и прогнозирования его развития.

The molecular genetic basis for pathogenesis of leukoplakia and squamous cell carcinoma of the oral mucosa (OM) is not well understood. Few studies are devoted to this problem and their results are incomplete and contradictory. At the same time, the early diagnosis of OM cancer and the prediction of its development are important public health problems.

The aim of the study was to analyze the relationship of somatic mutations with the formation of leukoplakia and squamous cell carcinoma of the oral mucosa.

48 altered OM epithelium samples of patients with OM leukoplakia (OML) (24 samples) and OM squamous cell carcinoma (OMSCC) (24 samples) were taken as material for research.

The pathogenic and probably pathogenic variants of the TP53, NRAS, and BRAF genes identified in this study, both  as one by one and in combination, are associated with high probability (RR 3000‒11 000) with OML with grade 1 epithelial squamous intraepithelial neoplasia. Identified pathogenic and probably pathogenic variants of the ERCC3, HOXB13, KRAS, MSH3, MSH6, PIK3CA, and TP53 genes are associated with a high probability (RR 90‒22 000) with the OMSCC development. The observed pathogenic variants of the KRAS and TP53 genes are highly likely to lead to the formation of OML with grade 1 squamous intraepithelial neoplasia of the epithelium; a subsequent formation of pathogenic variants of the PIC3CA and/or HOXB13 and MSH3 genes leads to malignant transformation of altered OM epithelial cells ( p = 0.0000048). This information allows designing PCR-and NGS-test systems for predicting the development and early diagnosis of OMSCC.

ДНК-секвенированиесоматические мутациилейкоплакия слизистой оболочки ртаплоскоклеточный рак слизистой оболочки ртаDNA sequencingsomatic mutationsleukoplakia of the oral mucosasquamous cell carcinoma of the oral mucosaРабота выполнена в рамках Государственной программы научных исследований «Установить спектр мутаций эпителия у пациентов с лейкоплакией слизистой оболочки рта» (№ ГР 20200246 от 02.03.2020).The work was carried out within the framework of the state research program “To establish the spectrum of epithelial mutations in patients with leukoplakia of the oral mucosa” (No. GR 20200246 dated of 02.03.2020).Referencesvan der Waal, I. Oral leukoplakia: Present views on diagnosis, management, communication with patients, and research / I. van der Waal // Curr. Oral Health Rep. – 2019. – Vol. 6. – P. 9–13. https://doi.org/10.1007/s40496-019-0204-8van der Waal I. Oral leukoplakia: Present views on diagnosis, management, communication with patients, and research. Current Oral Health Reports, 2019, vol. 6, pp. 9–13. https://doi.org/10.1007/s40496-019-0204-8Kalavrezos, N. Mouth cancer for clinicians. Part 1: Cancer / N. Kalavrezos, C. Scully // Dent. Update. – 2015. – Vol. 42, N 3. – P. 250–260. https://doi.org/10.12968/denu.2015.42.3.250Kalavrezos N., Scully C. Mouth cancer for clinicians. Part 1: Cancer. Dental Update, 2015, vol. 42, no. 3, pp. 250–260. https://doi.org/10.12968/denu.2015.42.3.250Detection of TP53-mutations in brush biopsies from oral leukoplakias / C. Scheifele [et al.] // Mund. Kiefer. Gesichtschir. – 2002. – Vol. 6, N 6. – P. 410–414. https://doi.org/10.1007/s10006-002-0425-0Scheifele C., Schlechte H., Bethke G., Reichart P. A. Detection of TP53-mutations in brush biopsies from oral leukoplakias. Mund-, Kiefer- und Gesichtschirurgie, 2002, vol. 6, no. 6, pp. 410–414. https://doi.org/10.1007/s10006-002-0425-0Specific characteristics of rapid diagnosis in periodontology / S. P. Rubnikovich [et al.] // Вес. Нац. aкад. навук Беларусі. Сер. мед. навук. – 2021. – Т. 18, № 2. – С. 196–203. https://doi.org/10.29235/1814-6023-2021-18-2-196-203Rubnikovich S. P., Dedova L. N., Semizhon P. A., Denisova, Yu. L., Kandrukevich, O. V. Specific characteristics of rapid diagnosis in periodontology. Vestsi Natsyyanal’nai akademii navuk Belarusi. Serуya medуtsynskikh navuk = Proceedings of the National Academy of Sciences of Belarus. Medical series, 2021, vol. 18, no. 2, pp. 196–203. https://doi.org/10.29235/1814-6023-2021-18-2-196-203Reddy, V. M. p53 immunoprofiling of potentially malignant oral disorders: a case series analysis / V. M. Reddy, A. Kamath, R. A. Radhakrishnan // Indian J. Cancer. – 2012. – Vol. 49, N 1. – P. 27–32. https://doi.org/10.4103/0019-509X.98913Reddy V. M., Kamath A., Radhakrishnan R. A. p53 immunoprofiling of potentially malignant oral disorders: a case series analysis. Indian Journal of Cancer, 2012, vol. 49, no. 1, pp. 27–32. https://doi.org/10.4103/0019-509X.98913Notch1 mutations are drivers of oral tumorigenesis / E. Izumchenko [et al.] // Cancer Prev. Res. (Phila). – 2015. – Vol. 8, N 4. – P. 277–286. https://doi.org/10.1158/1940-6207.CAPR-14-0257Izumchenko E., Sun K., Jones S., Brait M., Agrawal N., Koch W. [et al.]. Notch1 mutations are drivers of oral tumorigenesis. Cancer Prevention Research (Phila), 2015, vol. 8, no. 4, pp. 277–286. https://doi.org/10.1158/1940-6207.CAPR-14-0257Warnakulasuriya, S. Malignant transformation of oral leukoplakia: a systematic review of observational studies / S. Warnakulasuriya, A. Ariyawardana // J. Oral Pathol. Med. – 2016. – Vol. 45, N 3. – P. 155–166. https://doi.org/10.1111/jop.12339Warnakulasuriya S., Ariyawardana A. Malignant transformation of oral leukoplakia: a systematic review of observational studies. Journal of Oral Pathology and Medicine, 2016, vol. 45, no. 3, pp. 155–166. https://doi.org/10.1111/jop.12339Роль белков NOTCH в процессах канцерогенеза / М. В. Новикова [и др.] // Успехи молек. онкол. – 2015. – Т. 2, № 3. – С. 30–42.Novikova M. V., Rybko V. A., Khromova N. V., Farmakovskaya M. D., Kopnin P. B. The role of notch pathway in carcinogenesis. Uspekhi molekulyarnoi onkologii [Advances in molecular oncology], 2015, vol. 2, no. 3, pp. 30–42 (in Russian).Minichromosome maintenance-2 (MCM2) expression differentiates oral squamous cell carcinoma from pre-cancerous lesions / S. M. Razavi [et al.] // Malays. J. Pathol. – 2015. – Vol. 37, N 3. – P. 253–258.Razavi S. M., Jafari M., Heidarpoor M., Khalesi S. Minichromosome maintenance-2 (MCM2) expression differentiates oral squamous cell carcinoma from pre-cancerous lesions. Malaysian Journal of Pathology, 2015, vol. 37, no. 3, pp. 253–258.Association between p53 Arg72Pro polymorphism and the risk of human papillomavirus-related head and neck squamous cell carcinoma: a meta-analysis / L. Y. Xia [et al.] // Asian Pac. J. Cancer Prev. – 2013. – Vol. 14, N 10. – P. 6127–6130. https://doi.org/10.7314/apjcp.2013.14.10.6127Xia L. Y., Zeng X. T., Li C., Leng W. D., Fan M. W. Association between p53 Arg72Pro polymorphism and the risk of human papillomavirus-related head and neck squamous cell carcinoma: a meta-analysis. Asian Pacific Journal of Cancer Prevention, 2013, vol. 14, no. 10, pp. 6127–6130. https://doi.org/10.7314/apjcp.2013.14.10.6127Genomic profile of oral squamous cell carcinomas with an adjacent leukoplakia or with an erythroleukoplakia that evolved after the treatment of primary tumor: A report of two cases / I. P. Ribeiro [et al.] // Mol. Med. Rep. – 2017. – Vol. 16, N 5. – P. 6780–6786. https://doi.org/10.3892/mmr.2017.7428Ribeiro I. P., Marques F., Barroso L., Rodrigues J., Caramelo F., Melo J. B., Carreira I. M. Genomic profile of oral squamous cell carcinomas with an adjacent leukoplakia or with an erythroleukoplakia that evolved after the treatment of primary tumor: A report of two cases. Molecular Medicine Reports, 2017, vol. 16, no. 5, pp. 6780–6786. https://doi.org/10.3892/mmr.2017.7428Analysis of ras oncogene mutations in human squamous cell carcinoma of the head and neck / F. Núñez [et al.] // Surg. Oncol. – 1992. – Vol. 1, N 6. – P. 405–411. https://doi.org/10.1016/0960-7404(92)90043-kNúñez F., Domínguez O., Coto E., Suarez-Nieto C., Perez P., Lopez-Larrea C. Analysis of ras oncogene mutations in human squamous cell carcinoma of the head and neck. Surgical Oncology, 1992, vol. 1, no. 6, pp. 405–411. https://doi.org/10.1016/0960-7404(92)90043-kVariants in FAT1 and COL9A1 genes in male population with or without substance use to assess the risk factors for oral malignancy / C. M. Chung [et al.] // PLoS ONE. – 2019. – Vol. 14, N 1. – P. e0210901. https://doi.org/10.1371/journal.pone.0210901Chung C. M., Hung C. C., Lee C. H., Lee C. P., Lee K. W., Chen M. K., Yeh K. T., Ko Y. C. Variants in FAT1 and COL9A1 genes in male population with or without substance use to assess the risk factors for oral malignancy. PLoS ONE, 2019, vol. 14, no. 1, p. e0210901. https://doi.org/10.1371/journal.pone.0210901QIAamp® DNA FFPE Tissue Handbook (February 2020) [Electronic resource]. – Mode of access: https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/dna-purification/genomic-dna/qiaamp-dna-ffpe-tissue-kit/. – Date of access: 27.10.2022.QIAamp® DNA FFPE Tissue Handbook (February 2020). Available at: https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/dna-purification/genomic-dna/qiaamp-dna-ffpe-tissue-kit/ (accessed 27.10.2022).Illumina TruSight Oncology 500 Reference Guide [Electronic resource]. – Mode of access: https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/trusight/oncology-500/trusight-oncology-500-reference-guide-1000000067621_07.pdf. – Date of access: 26.05.2021.Illumina TruSight Oncology 500 Reference Guide. Available at: https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/trusight/oncology-500/trusight-oncology-500-reference-guide-1000000067621_07.pdf (accessed 26.05.2021).Экспериментальное обоснование применения мезенхимальных стволовых клеток для восстановления тканей периодонта / Ю. Л. Денисова [и др.] // Мед. вестн. Север. Кавказа. – 2020. – Т. 15, № 3. – С. 333–337.Denisova Yu. L., Sirak S. V., Rubnikovich S. P., Andreeva V. A., Kuz’menko E. V., Khomich I. S., Volotovskii I. D., Vladimirskaya T. E. Experimental substantiation of mesenchymal stem cell application for the restoration of periodontal tissue. Meditsinskii vestnik Severnogo Kavkaza = Medical News of North Caucasusthis, 2020, vol. 15, no. 3, pp. 333–337 (in Russian).Каюмов, А. Р. Молекулярный анализ генома: учеб.-метод. пособие / А. Р. Каюмов. – Казань : Казань, Казан. федер. ун-т. – 2016. – 60 с.Kayumov A. R. Molecular analysis of the genome. Kazan, Kazan Federal University, 2016. 60 p. (in Russian).Next generation sequencing for the detection of actionable mutations in solid and liquid tumors / A. J. Fox [et al.] // J. Vis. Exp. – 2016. – Vol. 115. – P. e52758. https://doi.org/10.3791/52758Fox A. J., Hiemenz M. C., Lieberman D. B., Sukhadia S., Li B., Grubb J. [et al.]. Next generation sequencing for the detection of actionable mutations in solid and liquid tumors. Journal of Visualized Experiments, 2016, vol. 115, p. e52758. https://doi.org/10.3791/52758Assessing statistical significance in multivariable genome wide association analysis / L. Buzdugan [et al.] // Bioinformatics. – 2016. – Vol. 32, N 13. – P. 1990–2000. https://doi.org/10.1093/bioinformatics/btw128Buzdugan L., Kalisch M., Navarro A., Schunk D., Fehr E., Bühlmann P. Assessing statistical significance in multivariable genome wide association analysis. Bioinformatics, 2016, vol. 32, no. 13, pp. 1990–2000. https://doi.org/10.1093/bioinformatics/btw128Evaluation of KRAS concomitant mutations in advanced lung adenocarcinoma patients / V. Aran [et al.] // Medicina (Kaunas). – 2021. – Vol. 57, N 10. – P. 1039–1048. https://doi.org/10.3390/medicina57101039Aran V., Zalis M., Montella T., de Sousa C. A. M., Ferrari B. L., Ferreira C. G. Evaluation of KRAS concomitant mutations in advanced lung adenocarcinoma patients. Medicina (Kaunas), 2021, vol. 57, no. 10, pp. 1039–1048. https://doi.org/10.3390/medicina57101039Lane, D. P. p53: oncogene or anti-oncogene? / D. P. Lane, S. Benchimol // Genes Dev. – 1990. – Vol. 4, N 1. – P. 1–8. https://doi.org/10.1101/gad.4.1.1Lane D. P., Benchimol S. p53: oncogene or anti-oncogene? Genes & Development, 1990, vol. 4, no. 1, pp. 1–8. https://doi.org/10.1101/gad.4.1.1Canaud, G. A review of mechanisms of disease across PIK3CA-related disorders with vascular manifestations / G. Canaud, A. M. Hammill, D. Adams // Orphanet J. Rare Dis. – 2021. – Vol. 16, N 1. – P. 306–316. https://doi.org/10.1186/s13023-021-01929-8Canaud G., Hammill A. M., Adams D. A review of mechanisms of disease across PIK3CA-related disorders with vascular manifestations. Orphanet Journal of Rare Diseases, 2021, vol. 16, no. 1, pp. 306–316. https://doi.org/10.1186/s13023-021-01929-8Oncoprotein HBXIP enhances HOXB13 acetylation and co-activates HOXB13 to confer tamoxifen resistance in breast cancer / B. Liu [et al.] // J. Hematol. Oncol. – 2018. – Vol. 11, N 1. – Art. 26. https://doi.org/10.1186/s13045-018-0577-5Liu B., Wang T., Wang H., Zhang L., Xu F., Fang R. [et al.]. Oncoprotein HBXIP enhances HOXB13 acetylation and co-activates HOXB13 to confer tamoxifen resistance in breast cancer. Journal of Hematology & Oncology, 2018, vol. 11, no. 1, art. 26. https://doi.org/10.1186/s13045-018-0577-5Hoxb13, a potential prognostic biomarker for prostate cancer / A. Ouhtit [et al.] // Front Biosci (Elite Ed.). – 2016. – Vol. 1, N 8. – P. 40–45. https://doi.org/10.2741/E749Ouhtit A., Al-Kindi M. N., Kumar P. R., Gupta I., Shanmuganathan S., Tamimi Y. Hoxb13, a potential prognostic biomarker for prostate cancer. Frontiers in Bioscience (Elite Ed.), 2016, vol. 1, no. 8, pp. 40–45. https://doi.org/10.2741/E749Exome sequencing identifies biallelic MSH3 germline mutations as a recessive subtype of colorectal adenomatous polyposis / R. Adam [et al.] // Am. J. Hum. Genet. – 2016. – Vol. 99, N 2. – P. 337–351. https://doi.org/10.1016/j.ajhg.2016.06.015Adam R., Spier I., Zhao B., Kloth M., Marquez J., Hinrichsen I. [et al.]. Exome Sequencing Identifies biallelic MSH3 germline mutations as a recessive subtype of colorectal adenomatous polyposis. American Journal of Human Genetics, 2016, vol. 99, no. 2, pp. 337–351. https://doi.org/10.1016/j.ajhg.2016.06.015Berger, A. H. A continuum model for tumour suppression / A. H. Berger, A. G. Knudson, P. P. Pandolfi // Nature. – 2011. – Vol. 476, N 7359. – P. 163–169. https://doi.org/10.1038/nature10275Berger A. H., Knudson A. G., Pandolfi P. P. A continuum model for tumour suppression. Nature, 2011, vol. 476, no. 7359, pp. 163–169. https://doi.org/10.1038/nature10275

The authors declare that there are no conflicts of interest present.