Shotgun Lipidomics for Differential Diagnosis of HPV-Associated Cervix Transformation

Abstract

A dramatic increase in cervical diseases associated with human papillomaviruses (HPV) in women of reproductive age has been observed over the past decades. An accurate differential diagnosis of the severity of cervical intraepithelial neoplasia and the choice of the optimal treatment requires the search for effective biomarkers with high diagnostic and prognostic value. The objective of this study was to introduce a method for rapid shotgun lipidomics to differentiate stages of HPV-associated cervix epithelium transformation. Tissue samples from 110 HPV-positive women with cervicitis (n = 30), low-grade squamous intraepithelial lesions (LSIL) (n = 30), high-grade squamous intraepithelial lesions (HSIL) (n = 30), and cervical cancers (n = 20) were obtained. The cervical epithelial tissue lipidome at different stages of cervix neoplastic transformation was studied by a shotgun label-free approach. It is based on electrospray ionization mass spectrometry (ESI-MS) data of a tissue extract. Lipidomic data were processed by the orthogonal projections to latent structures discriminant analysis (OPLS-DA) to build statistical models, differentiating stages of cervix transformation. Significant differences in the lipid profile between the lesion and surrounding tissues were revealed in chronic cervicitis, LSIL, HSIL, and cervical cancer. The lipids specific for HPV-induced cervical transformation mainly belong to glycerophospholipids: phosphatidylcholines, and phosphatidylethanolamines. The developed diagnostic OPLS-DA models were based on 23 marker lipids. More than 90% of these marker lipids positively correlated with the degree of cervix transformation. The algorithm was developed for the management of patients with HPV-associated diseases of the cervix, based on the panel of 23 lipids as a result. ESI-MS analysis of a lipid extract by direct injection through a loop, takes about 25 min (including preparation of the lipid extract), which is significantly less than the time required for the HPV test (several hours for hybrid capture and about an hour for PCR). This makes lipid mass spectrometric analysis a promising method for express diagnostics of HPV-associated neoplastic diseases of the cervix.

Keywords: cervical cancer; cervical intraepithelial neoplasia; diagnostics; human papillomavirus; lipidomics; mass spectrometry.

Figure 1

mRNA expression levels in cells of cervical smears with lesions of the cervical epithelium. The medians of the level of gene expression with statistically significant differences in the study groups are presented.

Figure 2

Risk index values for disease progression by group. The medians in the study groups and the interquartile range are presented.

Figure 3

Relative intensity of marker lipids in the mass spectrum of affected tissues. Green corresponds to chronic cervicitis, blue to LSIL, yellow to HSIL, and red to SCC. The diagram shows Q1–1.5*IQR, Q1, Me, Q3, and Q3 + 1.5*IQR. Black dots correspond to outliers.

Figure 4

Spearman’s correlation analysis of the cervical tissues’ lipid profile in CINs and SCC, histological diagnosis, and gene mRNA expression levels at a confidence level of 0.05. Positive correlation is highlighted in blue and negative correlation in red. The degree of correlation is highlighted in color—the stronger the correlation, the darker the color. “X”—correlations with p-value > 0.05. Parameter group shows the degree of cervical epithelium transformation: 0-ChC, 1-LSIL, 2-HSIL, and 3-CC.

Figure 5

Algorithm for management of patients with HPV-associated cervix transformation including the OPLS-DA models, based on ESI-MS data of cervical tissue extracts.