From bioinformatic screening of genetic markers to low invasive lymph node metastases diagnosis in patients with cervical cancer

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Abstract

Background. The problem of lymph nodes metastases diagnosing in cervical cancer remains relevant and not fully resolved. The last decade studies results have shown the great potential of molecular markers in lymph nodes metastasis prediction, however, additional studies for their implementation in clinical practice are required.

Aim. Bioinformatic and laboratory screening of molecular markers of cervical tumors regional metastasis for its low invasive diagnosis.

Material and methods. The study was performed on 400 patients with cervical cancer and 40 donors without oncological pathology. To identify potential molecular markers of lymph node metastatic lesions, the Cancer Genome Atlas database was initially analyzed. The identified markers were validated by the Real-Time-polymerase chain reaction in tumor cell samples (extracted using laser microdissection) and extracellular deoxyribonucleic acid (DNA). The Mann–Whitney test was used to assess the differences; the Bonferroni correction was used to account for multiple comparisons.

Results. At the bioinformation stage, the change in the copy number of 5493 genes was analyzed, of which 79 genes were selected that most often change their copy number. During the data validation, it was found that primary tumor cells and tumor cells of metastases from the lymph nodes differ from normal cervix cells in the level of gene copies. The copy number of the CCND1 and PPARGC1A genes has the highest potential for regional lymph nodes metastases diagnosing in patients with cervical cancer; the PIK3CA, SPEN, ERBB3, APC, MUC4, CASP8, HLA-A, IGSF1 and TMTC1 loci have a lower potential. The EP300, TTN, DMD, DST, LAMP3, TORC2, TP53 and FOXO3 genes can be used to diagnose cervical cancer, whether metastatic or not. Additional validation of markers was carried out on extracellular DNA of blood plasma of cervical cancer patients and conditionally healthy donors. The presence of a differential copy number of PIK3CA, SPEN, ERBB3, APC, CCND1, HLA-A, TTN, MUC4, DST, PPARGC1A genes was found in two groups of patients with cervical cancer with and without metastatic lesions of the lymph nodes.

Conclusion. The study made it possible to form a list of potential molecular markers for low invasive diagnostics of cervical cancer in general (EP300, LAMP3, TORC2, FOXO3, TP53) and cervical cancer with metastatic lesions of regional lymph nodes (PIK3CA/DST, APC/PPARGC1A, ERBB3/HLA-A and LAMP3/MUC4).

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About the authors

Denis S. Kutilin

National Medical Research Oncology Center

Author for correspondence.
Email: k.denees@yandex.ru
ORCID iD: 0000-0002-8942-3733
SPIN-code: 8382-4460
Scopus Author ID: 55328886800

Cand. Sci. (Biol.), Leading Researcher

Russian Federation, Rostov-on-Don, Russia

Madina M. Kecheryukova

National Medical Research Oncology Center

Email: adele09161@mail.ru
ORCID iD: 0000-0001-7800-7198
SPIN-code: 8756-7134
Russian Federation, Rostov-on-Don, Russia

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Supplementary files

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1. JATS XML
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2. Рис. 1. Геномное положение амплифицированных (А) и делетированных (Б) областей

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3. Рис. 2. Молекулярный профиль опухолевых клеток шейки матки у пациенток без поражения лимфатических узлов

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4. Рис. 3. А. Относительная копийность генов в опухолевых клетках шейки матки у больных без метастатического поражения лимфатических узлов (n=150); *статистически значимые отличия относительно нормальных клеток (p <0,05). Б. Относительная копийность генов в опухолевых клетках шейки матки у больных с метастатическим поражением регионарных лимфатических узлов (n=150); *статистически значимые отличия относительно нормальных клеток (p <0,05)

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5. Рис. 4. А. Относительная копийность генов в метастатических опухолевых клетках шейки матки (n=150); *статистически значимые отличия относительно нормальных клеток (p <0,05). Б. Визуализация кластеризации генов по выполняемой функции или участию в сигнальных путях

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6. Рис. 5. Показатель относительной копийности генов во внеклеточной ДНК плазмы крови больных раком шейки матки с метастатическим поражением регионарных лимфатических узлов и без него; *статистически значимые отличия от условно здоровых доноров (р <0,05), **статистически значимые различия между двумя группами больных раком шейки матки (р <0,05)

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