P16^INK4a expression as a potential prognostic marker in cervical pre-neoplastic and neoplastic lesions

Abstract

An immunohistochemical analysis with monoclonal antibody p16^INK4a was performed in formalin-fixed, paraffin-embedded samples of 60 cases. The aim was to investigate in biopsies the expression of p16^INK4a of normal uterine cervical tissue, pre-cancerous and cancerous lesions, and their relation with human papilloma virus (HPV) and HIV status. Three parameters were evaluated: percentage of p16^INK4a positive cells, reaction intensity, and cell staining pattern. All of these parameters were statistically different when compared among different histological groups. However, logistic regression model showed that the reaction intensity was the best indicator of the expression of p16^INK4a. This expression increases from normal to invasive squamous carcinoma. Sixty-six percent of the patients with CIN grade 1 (CIN1) expressed p16^INK4a (all these cases were infected with high risk HPV). Our study supports the hypothesis that p16^INK4a expression in pre-cancerous lesions and cancers can be used to identify HPV-transformed cells. Of great interest for routine diagnostic use is the fact that immunohistochemical testing for p16^INK4a seems to be capable of identifying HPV-positive cells and potentially recognizing those lesions with an increased risk of progression to high-grade lesions.

Introduction

There is epidemiological evidence that persistent infection, high viral load [8], [24], and integration of oncogenic types of human papilloma virus (HPV) in the host genome [32] play a preponderant role in the development of squamous uterine cervix carcinoma. Although oncogenic HPV has been detected in almost all pre-neoplastic and neoplastic uterine cervical lesions [31] and its presence is considered an important causal factor, other genetic and epigenetic factors may be necessary to increase the risk of transition from cervical infection to cancer [14].

More than 100 types of HPV have already been identified, of which approximately 30 types are associated with ano-genital infections [27]. Of these, 15 types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82) have a high oncogenic risk [17] and can be detected in almost all cervical cancers [31]. HPV 16 and 18 are the most prevalent, representing 59.8% and 15%, respectively, of the viral types involved in invasive cancer [17]. HPV16 has also been demonstrated to be the single most important factor linked with disease progression [27].

HPV DNA integration occurs within the viral E1–E2 region, thereby disrupting E2 and triggering uncontrolled expression of the transforming genes E6 and E7 [26]. HPV E7 interacts with, and neutralizes, the function of pRB pocket proteins, p107 and 130 [5]. This interaction, analogous to cyclin-dependent-kinase-mediated phosphorylation, induces pRB degradation, resulting in liberation of activated E2Fs and stimulation of entrance into the S phase, causing high levels of expression of the cyclin-dependent kinase inhibitor p16^INK4a [6], [16]. The keratinocytes transformed by the oncogenic HPV genes E6 and E7 are not cellular clones with completely malignant phenotypes, but require additional mutagenic agents [4]. Induction of the p16^INK4a protein occurs during immortalization in HPV-positive pre-malignant lesions, an early event resulting in tumorigenesis [18].

Alterations in protein p16^INK4a expression have been described in many cancers [1] due to mutation, homozygotic deletion, or gene hypermethylation [7], [9]. Several papers have evaluated the role of p16^INK4a as a diagnostic marker of cervical neoplasia [9], [10], [12], [13], [18], [19], [22], [23], [28], [29]. Recently, some authors have shown that p16^INK4a expression is associated with disease progression [19], [30].

Morphological criteria are important for diagnosis of cervical intraepithelial neoplasia (CIN) [20], [21], although it is not possible to differentiate those that will regress or persist from those that will progress into invasive cancer. Owing to the limitations of current conventional examinations, there is a need to identify new, specific, and more sensitive markers to determine the presence of high-risk HPV types and functional activity of these viral oncogenes [3], [12], [23], [32].

Our objective was to investigate, through immunohistochemistry, the expression of p16^INK4a in biopsies of normal uterine cervical tissue, pre-cancerous and cancerous lesions, and their relation with HPV and HIV status.