ARTICLE |
Correspondence to: Christiane Pienna Soares, Dept. of Clinical Analysis, Faculty of Pharmaceutical Sciences, University of São Paulo State (UNESP), Rua Expedicionários do Brasil 1612, Centro, 14 801 902 Araraquara, São Paulo, Brazil. E-mail: soarescp@hotmail.com
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Summary |
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Human papillomavirus (HPV) is believed to promote the oncogenic process, and the correlation between viral oncoproteins and dysfunction of p16INK4A tumor suppressor protein in oral lesions is controversial. To test the hypothesis that anogenital HPV types participate in disruption of the regulation of p16INK4A suppressor protein in oral lesions, we analyzed 46 oral biopsy specimens for the presence of HPV 6/11 and 16/18 by in situ hybridization (ISH) and for p16INK4A expression by immunohistochemistry (IHC). Eighteen (39%) of the 46 oral lesions were HPV-positive and 28 (61%) were HPV-negative. HPV 6/11 DNA was found in 5 (11%) and HPV 16/18 in 13 (28%) of 46 biopsies. Nine of the 18 HPV-positive oral lesions (50%), assessed by catalyzed signal amplification coupled to ISH (CSAISH), gave high-intensity p16INK4A immunostaining. Focal and diffuse patterns were observed in 11/13 (77%) lesions with HPV 16/18, focal immunopositivity in 3/5 (80%) with HPV 6/11, and negative or sporadic p16-labeling in 18/28 (64%) without the presence of HPV DNA. These results showed a strong association between overexpression of p16 protein and malignant oral lesions, mainly those infected by HPV 16/18. We can conclude that high-risk HPV types are associated with p16 overexpression, and p16 may serve as a biomarker in oral cancer related to high-risk HPV infection. (J Histochem Cytochem 51:12911297, 2003)
Key Words: HPV, oral cancer, p16INK4A, immunohistochemistry, in situ hybridization
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Introduction |
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ORAL CANCER is the sixth most common malignancy and is a major cause of cancer morbidity and mortality worldwide (
Tumor cells are therefore believed to result from deregulation of two major pathways of cell cycle control: the p53 and pRB pathways mediated by p16INK4A (
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Materials and Methods |
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Tissue Specimens
Forty-six formalin-fixed and paraffin-embedded oral biopsy specimens that had been taken for routine pathological diagnosis were obtained from the School of Dentistry archives, University of São Paulo State (UNESP), Brazil and approved by the Brazilian institutional ethical committee on human experimentation. All the samples were archival specimens graded according to the criteria recently proposed for oral white lesions (
DNADNA In Situ Hybridization
Sections were dewaxed in xylene, passed through an alcohol series, and air-dried. The slides were washed in standard saline citrate (2 x SSC = 0.3 M NaCl and 0.03 M C6H5Na3O7), pH 7.0, 0.2 N HCl, and a second 2 x SSC bath. The sections were incubated with 20 µg/ml proteinase K (GIBCO; Gaithersburg, MD) at 37C for 4 min, washed in fresh 2 x SSC buffer, 0.1 M triethanolamine solution containing 1% acetic anhydride (for 10 min at room temperature), and finally 2 x SSC buffer. ISH was performed with wide-spectrum 6/11 and 16/18 nonradioactively labeled HPV DNA probes (DAKO; Glostrup, Denmark) and a commercially available hybridization kit with a catalyzed system of amplification (CSA), Genpoint (DAKO). The probe solution was dropped on the sections, which were covered with coverslips, and cellular DNA was denatured on a hot plate at 95C for 10 min. The sections were briefly chilled in an iced chamber and incubated at 37C overnight in a humidified chamber. After hybridization, the sections were washed in 0.05 M Tris-HCl buffer (at RT), 2 x SSC, and 0.5 x SSC buffers. Hybridized DNA was detected with the Genpoint system (DAKO) according to the manufacturer's instructions and the sections were incubated in 40 mg of diaminobenzidine and 20 µl of peroxidase in 100 ml of saline PBS. Finally, the sections were rinsed in distilled water, counterstained with Meyer's hematoxylin, and washed in dilute aqueous ammonia and distilled water. After that, the sections were dehydrated in graded alcohol, allowed to dry, and mounted in Entellan mounting medium (Merck; Darmstadt, Germany). For each reaction three controls were used: one positive control (commercial slide in the Genpoint kit), one negative control (commercial slide in the Genpoint kit without DNA probe), and one normal oral tissue control. DNA extraction and ß-globin gene amplification were performed to verify the DNA quality of the tissue samples.
Immunohistochemistry for p16INK4A Protein
Immunostaining Procedures.
The sections were dewaxed in xylene, rehydrated in graded alcohol, and rinsed in water. To inhibit endogenous peroxidase activity, the sections were treated by immersion in five changes of 0.3% H2O2 in absolute methanol (5 min each change) and rinsing in water. For antigen retrieval, the sections were immersed in 10 mM sodium citrate buffer (pH 6.0) and boiled twice for 12 min in a high-intensity microwave oven. To detect p16INK4A protein, an IHC assay was performed using the streptavidinbiotin system (LSAB; DAKO). The slides were incubated with primary monoclonal antibody for p16 protein (p16/Abs4, diluted 1:40; Labvision, Fremont, CA) in a humidified chamber at 4C overnight. The sections were then incubated with biotinylated anti-rabbit antibody and streptavidinperoxidase complex (LSAB system; DAKO) at 37C for 30 min. Careful rinses were performed, with several changes of PBS between each stage of the procedure. The sections were incubated with diaminobenzidine (Gibco), then counterstained lightly with Carrazzi's hematoxylin, dehydrated in graded alcohol, allowed to dry in air, and mounted with Permount mounting medium (Merck).
Controls. Positive controls were included in each assay, comprising cell block sections of HeLa cell line, negative controls in which the primary antibody was replaced with PBS, and a control of normal oral tissue. The immunoreaction cut-off was established by quantifying nuclear and cytoplasmic positivity in normal tissue, which ranged from zero to 5%.
Quantitative Evaluation of p16INK4A Immunostaining
The IHC expression of p16 was quantified in a double-blind protocol and classified according to nuclear and cytoplasmic positivity. The biopsies were scored as positive when more than 5% cells (cut-off) stained positively, and were separately graded as described by
Statistical Analysis
The p16INK4A IHC expression results were analyzed by the 2 probability test, with p<0.05 being considered statistically significant.
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Results |
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The oral lesions used in the present study (Table 1) were most commonly located on the mouth floor (20%), tongue (17%), and palate (12%).
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Table 2 reports the frequency of HPV ISH expression in oral lesions. Eighteen (39%) of the 46 oral lesions were HPV-positive and 28 (61%) were HPV-negative. HPV 6/11 DNA was found in two (14%) of 14 OSPs, two (20%) of 10 OPLs, and one (6%) of 17 OSCCs. HPV 16/18 was found in one (20%) of 5 OHs, three (22%) of 14 OSPs, four (40%) of 10 OPLs, and five (29%) of 17 OSCCs. No oral lesion was simultaneously positive for HPV 6/11 and 16/18. These results show a strong correlation of the premalignant and malignant lesions with HPV 16/18 positivity.
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Table 3 shows the data of p16INK4A IHC expression to histopathological diagnosis in oral biopsies. p16INK4a focal and diffuse patterns of positivity were found mainly in premalignant and malignant oral lesions (OPL and OSCC), whereas sporadic pattern was associated with benign lesions (OH and OSP). These results showed a strong association between overexpression of p16 protein and oral lesions that were malignant or with high malignancy potential (p=5.0 E-03; p<0.05). Interestingly, the OH lesion (n=1) infected with HPV DNA 16/18 gave a diffuse pattern. On the other hand, some premalignant (n=3) and malignant (n=2) lesions, HPV-negative, had negative or sporadic p16 scores. These results may indicate a close association between high-risk HPV infection and specific p16 IHC patterns.
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Table 4 matches p16INK4A expression in oral lesions against infection by HPV. Nine of the 18 HPV-positive oral lesions (50%), assessed by CSA-ISH, gave intense p16INK4A immunostaining in a diffuse pattern and five (28%) a focal pattern of staining. Focal and diffuse patterns were observed in 11 of 13 (77%) HPV 16/18-infected lesions, focal immunopositivity in three of five (80%) with HPV 6/11 DNA, and negative or sporadic p16 labeling in 18 of 28 (64%) with no HPV DNA (Fig 1). These results also demonstrate that different patterns of p16 immunopositivity could be associated with low- or high-risk HPV infection in oral tissues (p=1.0 E-04; p<0.05).
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However, we found focal p16 expression in nine (32%) of the oral lesions that were negative for HPV DNA, of which eight (29%) were diagnosed as OSCC and one (3.5%) as OPL. This result suggests that moderate expression in the absence of viral DNA could be associated with histological type and that p16 gene dysfunction could be caused by other carcinogenic co-factors. On the other hand, we found biopsies with HPV 6/11 and 16/18, respectively, with p16 pattern negative (n=2) or sporadic (n=2). Lesions with HPV 6/11 and 16/18, but p16-negative, were diagnosed as OSP, whereas those combining HPV 6/11 and 16/18 DNA with sporadic p16 expression were classified as, respectively, OSP and OSCC. These results also suggest that, in some lesions, HPV appears not to be involved in p16INK4A deregulation. It is possible that high-risk viral presence alone does not give E6/E7 viral protein expression and therefore results in normal p16 function. Further carcinogenic mechanisms and/or expression of other oncoproteins may therefore have to be considered for a more complete understanding of the rapid cell proliferation or malignancy transformation.
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Discussion |
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The choice of a suitable method for detection of HPV DNA has become increasingly complex (
We also found a close association between low-risk HPV and oral squamous papilloma, whose prognostic behavior is usually benign. However, we found HPV 16/18 in one (20%) of five OHs and in three (22%) out of 14 OSPs, suggesting that HPV can lead benign cells to malignant progression. A high prevalence of HPV 16/18 has been found in OSP, suggesting viral involvement in oral carcinogenesis, and recent studies have shown a close association between oral hyperplasia and/or OSP HPV 6/11 and HPV16/18 (
Abnormalities in various components of the cell cycle regulatory machine and disruption of cell cycle control are common features of many cancers, including oral cancers (
In the present study, p16 positivity patterns were found to correlate with the stage of tumor progression, focal and diffuse features being associated, respectively, with premalignant and malignant oral lesions, and negative or sporadic labeling with benign lesions. Some studies on cervical cancer have demonstrated that p16INK4A may serve as a marker to differentiate neoplastic lesions from hyperplastic or reactive lesions (
We can conclude that high-risk HPV types are involved in p16 overexpression, perhaps due to viral integration and malfunction of this tumor suppressor protein, and probably contribute to the multistep oral carcinogenesis process. Moreover, our results suggest that p16 immunohistochemical expression is a useful marker for high-risk HPV infection and the development of oral cancer. Further studies will attempt to uncover more details of the molecular events, involving p16 and HPV, that drive the oral carcinogenic process.
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Acknowledgments |
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Supported by FUNDUNESP, a Brazilian research-funding organization.
Received for publication February 12, 2003; accepted May 22, 2003.
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