Research Article: γH2Ax Expression as a Potential Biomarker Differentiating between Low and High Grade Cervical Squamous Intraepithelial Lesions (SIL) and High Risk HPV Related SIL

Date Published: January 24, 2017

Publisher: Public Library of Science

Author(s): Konstantinos Leventakos, Sotirios Tsiodras, Theodore Kelesidis, Maria Kefala, Christine Kottaridi, Aris Spathis, Alina-Roxani Gouloumi, Abraham Pouliakis, Asimakis Pappas, Vasileios Sioulas, Charalambos Chrelias, Petros Karakitsos, Ioannis Panayiotides, Marcia Edilaine Lopes Consolaro.

http://doi.org/10.1371/journal.pone.0170626

Abstract

γH2AX is a protein biomarker for double-stranded DNA breakage; its expression was studied in cervical squamous intraepithelial lesions and carcinomas.

Immunostaining for phospho-γH2AX was performed in sections from histologically confirmed cervical SIL and carcinomas, as well as from normal cervices used as controls. In total, 275 cases were included in the study: 112 low grade SIL (LGSIL), 99 high grade SIL (HGSIL), 24 squamous cell carcinoma (SCC), 12 adenocarcinoma and 28 cervical specimens with no essential lesions. Correlation of histological grading, high risk vs. low risk HPV virus presence, activated vs. non-activated status (by high risk HPV mRNA expression) and γH2AX expression in both basal and surface segments of the squamous epithelium was performed.

Gradual increase of both basal and surface γH2AX expression was noted up from normal cervices to LGSIL harboring a low risk HPV type, to LGSIL harboring a high risk virus at a non-activated state (p<0.05). Thereafter, both basal and surface γH2AX expression dropped in LGSIL harboring a high risk virus at an activated state and in HGSIL. γH2AX could serve as a potential biomarker discriminating between LGSIL and HGSIL, as well as between LGSIL harboring high risk HPV at an activated state.

Partial Text

Infection by HPV is responsible for the development of cervical squamous intraepithelial lesions (SIL) and cervical carcinoma, as well as for other squamous cell carcinomas in the anogenital and the head and neck area. There is an ongoing quest for biomarkers predicting the eventual progression of HPV related SIL into carcinoma.

We examined histologically confirmed cervical squamous intraepithelial lesions (SIL), squamous cell carcinomas (SCC) and adenocarcinomas (AdC) available through a prospective HPV registry operating in the “Attikon” University Hospital and maintained by the Departments of Cytopathology and Pathology of the hospital [6]. Moreover, cases within normal limits were included for comparison purposes. Specifically, control and SIL cases concerned specimens received in a 3- year period, i.e. from January 2010 up until December 2012 included; SCC and AdC samples were retrieved from the totality of the files of the Department of Pathology, i.e. from July 2003 up to December 2015 included. The “Attikon” University Hospital Institutional Ethics Review Board approved the study, in compliance with the Helsinki declaration. Written informed consent was obtained from all participating patients.

In total, 275 cases (cervical biopsies or hysterectomy specimens) were included in the study: 112 LGSIL, 99 HGSIL, 24 SCC, 12 AdC and 28 cervical specimens with no essential lesions (S1 Dataset). The mean age of the 275 subjects studied was 38.7±13.3 years (range 18–81 years). Table 1 depicts the association between cytological and histological diagnosis in the study sample. Overall, cytology results correlated well with histology (Table 1). For example, cytological high grade lesions (i.e. ASC-H, HGSIL, SCC, AdC) correlated strongly with histological high grade lesions (OR: 28; 95% CI 13.5–58.6, p<0.001). High risk HPV types were mostly noted in high grade histological lesions. HPV-16 was noted in 35 out 99 (35.4%) in samples from histologically confirmed HGSIL cases vs. 29/112 (25.9%) in histologically confirmed LGSIL cases, p<0.001]. On the other hand, low risk types predominated in histologically confirmed low grade lesions (p = 0.01, data not shown). Mixed infections with multiple HPV types were identified from both low grade [60/112, (53.6%)] and high grade lesions [35/99 (35.4%)]. We assessed the expression of the histone 2AX phosphorylated at serine-139 (γH2AX) protein in cervical histological lesions of increasing grade. We found a gradual increase of both basal and surface γH2AX expression up from normal cervices to LGSIL harboring a low risk HPV type, to LGSIL harboring a high risk virus at a non-activated state. Thereafter, both basal and surface γH2AX expression dropped in LGSIL harboring a high risk virus at an activated state and in HGSIL. Moreover, there was a higher surface γH2AX expression in LGSIL cases with HR HPV infection at a non—activated state (as identified by the mRNA expression) versus LGSIL cases with HR HPV infection at an activated state.   Source: http://doi.org/10.1371/journal.pone.0170626