Nataliia Starodubtseva (Presenter)
Research Center for Obstetrics and Gynecology
Bio: I work as a Research Scientist at the laboratory of proteomics and metabolomics of human reproduction at the Research Center for Obstetrics, Gynecology and Perinatology Ministry of Healthcare and Social Development of the Russian Federation. My main scientific focus is application of proteomics and metabolomics research in diagnostics using different mass-spectrometry techniques. I participate in FTICR, MALDI-TOF, ambient ionization mass spectrometry methods development; biomarker panel for pregnancy (preeclampsia, intrauterine growth restriction, etc.) and gynecological (endometriosis, uterine fibroids, cancer) pathologies search and in prenatal/postnatal diagnostics; development and application in routine clinical practice of hormone profiling techniques for accurate and sensitive analyses of samples with extremely low hormone concentration.
Authorship: Nataliia Starodubtseva (1,2); Vitalii Chagovets (1); Maria Nekrasova (1); Maka Zardiashvili(1); Àlexey Kononikhin (1,2); Niso Nazarova(1); Vladimir Frankevich (1)
(1) V. I. Kulakov Research Center for Obstetrics, Gynecology and Perinatology, (2) Moscow Institute of Physics and Technology
| Short Abstract Changes in lipid constituent of cervical tissue during neoplasia processes. Development of complex genetic and lipidomic approach for the prediction of the risk and progression of cervical intraepithelial neoplasia in patients with human papillomavirus associated diseases of the cervix uteri. The lipid profiles of the cervical tissues with neoplastic changes and the surrounding healthy tissue from 5 patients with low-grade and 5 - with high-grade squamous intraepithelial lesion were obtained. A group of possible biomarkers for the prediction of evolving neoplastic transformation was proposed. The comprehensive analysis of tissue lipid constituent with molecular genetic markers significantly increases the diagnostic potential of cervical cancer risk assessment. |
Long Abstract
Introduction
Cervical cancer is the third-most common cause of reproductive system cancer in women (WHO, 2014). The process of malignancy, started from HPV infection caused cellular changes, can take from 10 to 40 years, but in rare cases, cervical cancer develops in 1-2 years. Therefore, early diagnosis of cervical cancer and its precancerous stages - cervical intraepithelial neoplasia (CIN) of varying severity with the estimation of malignancy risk. On the other hand, diagnosis of cervical diseases in the early stages will prevent unnecessary aggressive treatment, keeping the women reproductive function and improving the quality of life.
Malignant cell metabolism changes in order to maintain the uncontrolled proliferation. One of the most important features of cancer cells metabolism is fatty acid synthesis increase, necessary for the process of carcinogenesis.
The aim of this research was the development complex genetic and lipidomic approach using mass-spectrometry (MS) for the prediction of the risk and progression of cervical intraepithelial neoplasia in patients with human papillomavirus (HPV)-associated diseases of the cervix uteri.
Methods
On the pilot study we recruited 5 patients with low-grade squamous intraepithelial lesion (LSIL) 5 patients with high-grade squamous intraepithelial lesion (HSIL). The inclusion criteria were: reproductive age, HPV infection (more than 1,5-3 years long), written informed consent, absence of any severe somatic pathologies (including kidney, liver, lung diseases, diabetes), inflammatory diseases, pregnancy, lactation, hormone therapy (in the last 6 months). The patients did not differ in age (30 ± 2 years), racial/ethnic, BMI (21.1 ± 2.2) and the standard of living. The patients’ examination included collection of complaints, history data, gynecological status determination, expanded colposcopy, cytology and morphological study.
The cervical tissues with neoplastic changes and the surrounding healthy tissue were obtained during punch biopsy procedure under extended colposcopy control and frozen in liquid nitrogen immediately, followed by storage at –800 Ñ. All procedures and study methods were approved by the Commission of biomedical ethics at V.I. Kulakov Research Center for Obstetrics, Gynecology and Perinatology.
Lipids were extracted by Folch after milling the tissue sample in liquid nitrogen (the organic phase was dried under dry nitrogen and redissolved in MeOH/IPA, 1:1). Samples were analyzed by direct ESI-MS in positive mode (Q-TOF Maxis Impact, Bruker, Germany) with 5 ppm mass accuracy. Identification was performed by exact mass and MS2 spectra.
The molecular biological studies of the cervical canal transitional epithelium smear involved real-time multiplex PCR assay with quantification and typing of HPV 21 and quantification of the mRNA expression of the genes MKI 67 (KI67), CTSL2, CDKN2A (P16), ESR1, PGR, BCL2, BAX, BAG1, CD68, SCUBE2, and PTEN.
Results
Differences in the lipid profiles of the cervical tissues with neoplastic transformation and the surrounding healthy tissue were analyzed in combination with the morphological features in order to find the specific biomarkers of cervical intraepithelial neoplasia progression.
Lipid tissue extracts mass profiles were analyzed by multivariate analysis (PLS) resulted in three distinct clusters, corresponding to the HSIL, LSIL and healthy tissue samples. VIP components analysis revealed a group of potential cervical epithelial tumor markers (in particular, polar lipids) with statistically different level of abundance in the tissues with neoplasia of different severity level.
The dominant type of HPV in women with CIN in the cervix and anus is the HPV type 16, 68 (ð<0.05). Differences in the mRNA expression level in patients with cervical neoplasia of varying degrees were found. The increased mRNA expression of the genes MKI67 (KI67) and CDKN2A (P16), the decreased expression of PGR, BCL2 in combination with high-risk HPV types, and an abnormal colposcopic pattern correlate with the outcomes of LSIL and may be regarded as possible biomarkers for the prediction of the course of already developed neoplasia.
Conclusions
For the first time the combination of MS approach with genetic study was applied to analyze the changes in lipid constituent of cervical tissue during neoplastic transformation of different severity level in the patients with HPV carriage. A group of possible biomarkers for the prediction of evolving neoplastic transformation of the cervical epithelium was proposed. The comprehensive analysis of tissue lipid profile with molecular genetic markers may significantly increase the diagnostic potential of cervical cancer risk assessment.
References & Acknowledgements:
This work was supported by grant No. 16-14-00029 of RSF, and N.S. acknowledge MERF grant No. ÌÊ-8484.2016.7.
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