MSACL 2019 EU Abstract

Self-Classified Topic Area(s): Proteins & Proteomics

Shortgun Proteomics for Differential Diagnosis of HPV-associated Cervix Transformation

Natalia Starodubtseva (1, 2), Alexey Kononikhin (1, 2), Anna Bugrova (1, 2), Аlexander Brhozovskiy (1), Niso Nazarova (1), Vladimir Frankevich (1), Eugene Nikolaev (1)
(1) V.I. Kulakov National Medical Research Center of Obstetrics, Gynecology and Perinatology, Moscow, Russia (2) Emanuel Institute for Biochemical Physics, Russian Academy of Sciences, Moscow, Russia

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Natalia Starodubtseva (Presenter)
National Medical Research Center of Obstetrics

Presenter Bio: PhD in biophysics “The role of IL-17 in the pathogenesis of psoriatic process” (2013). From April 2013 - Head of 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. Main scientific focus - application of proteomics and metabolomics research in diagnostics using different mass-spectrometry techniques (FTICR, MALDI-TOF, ambient ionization MS). For the “Development and implementation of high-resolution mass-spectrometry methods for non-invasive express-diagnostics of obstetric, gynecological and neonatal pathologies”, the Moscow Government award was awarded in 2016.

Relevant Financial Disclosures (within past 24 months)

Grant/Research Support Russian Science Foundation project no. 18-75-10097

Discussing Products? NO, presenter will NOT present educational content related to products/services of the ineligible company(ies) listed above.

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Abstract

Cervical cancer is preceded by cervical intraepithelial neoplasia of different degrees of severity (low grade squamous intraepithelial lesion, LSIL and high-grade squamous intraepithelial lesion, HSIL). None of the routine tests allow to assess the risk of neoplasia progression which is very important for young women. Cervicovaginal fluid (CVF) is a valuable source of clinical information about the physiological and pathophysiological status of the female reproductive tract. The aim of this study was to investigate the features of the CVF proteome in HPV-associated transformation of the cervical epithelium.
CVF samples were obtained from 73 patients from 21 to 45 years with various forms of HPV-associated cervical lesions (LSIL, HSIL and cancer) and HPV-negative patients (NILM). CVF proteins were reduced; alkylated, precipitated in acetone with TFA and digested with trypsin. Tryptic peptide mixtures were separated on a nano-HPLC Agilent 1100 system using a self-packed capillary column by a 95-min gradient from 3% to 35% of ACN in water at a flow rate of 0,3 mkl/min. Mass-spectrometry analysis was carried out on a 7T LTQ FT Ultra (Thermo Electron, Bremen, Germany) instrument using a nanospray ion source (positive ion mode, 2.3 kV). Raw MS files were processed with MaxQuant software (version 1.1.1.2) against the SwissProt database.
A total of 675 proteins were identified with 1% FDR. PCA resulted in four distinct clusters in full accordance with the clinical diagnosis. Thus, CVF proteome proved to reflect the stage of cervical epithelium neoplastic process. To assess the changes in the proteome composition in HPV associated the neoplastic transformation of the cervix Welch’s t-test with Bonferroni correction (p<0.01) was performed. As a result 94 proteins showed significant changes compared to the NILM group.
The obtained CVF proteomic data was analyzed using the PLS-DA method to build a statistical model, allowing to differentiate severe dysplasia (HSIL, CANCER) from the mild/normal stage (NILM and LSIL). This non-invasive diagnostic approach is particularly important from a clinical point of view as it determines the treatment of patients. In a case of severe dysplasia (HSIL, CANCER) surgery is required, for LSIL periodically repeated examinations are more appropriate. A development set of 40 samples was used for PLS-DA model training. The proteomic composition of CVF demonstrated an excellent ability for mild/severe cervical neoplasia differentiation (R2=0.95, Q2=0.88). ROC analysis of the PLS-DA model built on the validation set of new samples (n=33) resulted in 77% sensitivity and 94% specificity at 0.48 threshold, with AUC equal to 0.87.
This data confirms high diagnostic potential of CVF proteome for cervical epithelium transformation stage determination.
This work was supported by the Russian Science Foundation project no. 18-75-10097.