ORIGINAL ARTICLEP16INK4a expression as a potential prognostic marker in cervical pre-neoplastic and neoplastic 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 p16INK4a [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 p16INK4a protein occurs during immortalization in HPV-positive pre-malignant lesions, an early event resulting in tumorigenesis [18].
Alterations in protein p16INK4a 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 p16INK4a as a diagnostic marker of cervical neoplasia [9], [10], [12], [13], [18], [19], [22], [23], [28], [29]. Recently, some authors have shown that p16INK4a 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 p16INK4a in biopsies of normal uterine cervical tissue, pre-cancerous and cancerous lesions, and their relation with HPV and HIV status.
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Materials and methods
Informed consent for this study was obtained from 60 patients selected from women who presented an atypical transformation zone (ATZ) and an atypical cytology result or negative cytology with persistent ATZ (⩾6 months), between October 1999 and March 2001 at the State Center for Oncology – CICAN (State Health Authority and at the Outpatient Clinic of Gynecology (Infectious Diseases/AIDS Unit) of the Professor Edgar Santos Hospital, Federal University of Bahia (UDAI-HUPES-UFBA) in the city of
Results
According to histopathological examination, the 60 cases were classified as follows: 11 patients were normal/negative for neoplasia (including squamous metaplasia), 15 cases were classified as CIN1, 10 cases as CIN2, 15 cases as CIN3, and nine cases as invasive squamous cell carcinoma. Among the cases diagnosed as CIN or carcinoma, 90.6% were HPV-DNA positive (Table 1). Of the patients who tested positive for HPV, two types of low-risk HPV were identified: types 6 and 54, as well as 14 types
Discussion
Using immunohistochemical analysis, we determined the presence of p16INK4a in histologic samples of cervical biopsies from 60 patients with ATZ and atypical cytology or negative cytology, considering the following parameters: percentage of cells positive for p16INK4a, reaction intensity, and cellular reaction pattern.
Our results show that the expression of p16INK4a increases from normal to invasive squamous carcinoma in the uterine cervix and reinforce that it might be a useful prognostic
Acknowledgments
The authors thank MTM Laboratories, Germany, for providing the antibody anti-human p16INK4a/MTS1 (Code MTM-E6H4). Furthermore, we are indebted to Patti Gravitt and Roche Molecular Systems for transferring the HPV PCR detection system.
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