Detection and Quantification of Human Papillomavirus in Benign and Malignant Parotid Lesions

Materials and Methods

Histopathological and clinical data. A total of 79 tumor cases, comprising of 40 patients with benign lesions (SGTs) and 39 patients with malignant lesions who underwent surgery aimed at curative tumor resection, were studied (see Table I for clinical data). Tumor specimens were obtained by a retrospective compilation from the records of the pathology departments of the Hôpital Erasme (M.R., Brussels, Belgium), the CHU Saint-Pierre-Institut Bordet (N.S., Brussels, Belgium) and the CHU Sart-Tilmant (L.D., Liège, Belgium). The Institutional Review Boards of these hospitals approved the study. Hematoxylin- and eosin-stained sections of the 79 tumors were examined by two pathologists to confirm the diagnosis. Paraffin blocks that presented the highest proportion of tumors, avoiding the necrotic area frequently observed in the central part of the tumor, were selected for analysis in the studies.

DNA extraction. The formalin-fixed, paraffin-embedded tissue samples were sectioned (10×5 μm), de-paraffinized, and digested with proteinase K overnight at 56°C. DNA was purified using the QIAamp DNA Mini Kit (Qiagen, Benelux, Belgium), according to the protocol recommended by the manufacturer.

Detection of HPV using PCR amplification. HPV was detected using PCR with GP5+/GP6+ primers (synthesized by Eurogentec, Liege, Belgium). The GP5+/GP6+ primers amplify a consensus region located within the L1 region of the HPV genome. PCR for HPV-L1 DNA amplification was performed in a 25-μl reaction mixture containing 2 μl of extracted DNA, 2.5 μl of 1×PCR buffer, 0.025 U of Taq DNA polymerase (Roche, Mannheim, Germany), 200 μM dNTPs and 0.5 pmol of each primer. The cycling conditions for the PCR were as follows: denaturation was performed at 94°C for 1 min, annealing was performed at 55°C for 1 min and 30 s, and extension was performed at 72°C for 2 min for a total of 45 amplification cycles. The first cycle was preceded by a 7-minute denaturation step at 94°C. The last cycle was followed by an additional 10-min extension step at 72°C. Aliquots (10 μl) of each PCR product were separated via electrophoresis on a 1.8% agarose gel and stained with ethidium bromide to visualize the amplified HPV-L1 DNA fragments.

Real-time quantitative PCR amplification of type-specific HPV DNA. All DNA extracts were tested for the presence of 18 different HPV genotypes using TaqMan-based real-time quantitative PCR that targeted type-specific sequences of the following viral genes: 6 E6, 11 E6, 16 E7, 18 E7, 31 E6, 33 E6, 35 E6, 39 E7, 45 E7, 51 E6, 52 E7, 53 E6, 56 E7, 58 E6, 59 E7, 66 E6, 67 L1, and 68 E7. For the real-time quantitative PCR assays, the analytical sensitivity ranged from 1 to 100 copies and was calculated using standard curves for the 18 type-specific PCRs constructed with plasmids containing the entire genome of the various HPV types. Real-time quantitative PCR for the detection of β-globin was performed in each PCR assay to verify the quality of DNA in the samples and to measure the amount of analyzed DNA (11). The following HPV types tested were considered high risk: 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, and 66.

Immunohistochemistry. All samples were fixed in 4% buffered formaldehyde for 24 h, dehydrated and embedded in paraffin. Immunohistochemistry was performed on 5-μm-thick sections mounted on silane-coated glass slides. Before starting the immunohistochemistry protocol, de-paraffinized tissue sections were placed in a 0.01-M citrate buffer (pH 6.0) and briefly pre-treated in a microwave for 2×5 min at 900 W. The sections were then incubated with a solution of 0.06% hydrogen peroxide for 5 min to block endogenous peroxidase activity, rinsed in phosphate-buffered saline (PBS; 0.04 M Na₂HPO₄, 0.01 M KH₂PO₄ and 0.12 M NaCl, pH 7.4) and were successively exposed to solutions containing avidin (0.1 mg/ml in PBS) and biotin (0.1 mg/ml in PBS) for 5-min periods to avoid false-positive staining reactions resulting from the presence of endogenous biotin. After a thorough washing with PBS, the sections were incubated for 15 min with a solution of 0.5% casein in PBS and sequentially exposed to solutions of the specific primary antibody (anti-p16; Abcam, Cambridge, UK) for 1 h, the corresponding biotinylated secondary antibody (polyclonal goat anti-mouse IgG) for 30 min and the avidin-biotin-peroxidase complex (ABC kit; DakoCytomation, Glostrup, Denmark) for 45 min, all at room temperature. The samples were subjected to thorough washing steps to remove unbound proteins in between incubation steps. The antigen-dependent presence of the peroxidase complex in the sections was visualized following incubation with the chromogenic substrates containing diaminobenzidine and H₂O₂. After rinsing, the sections were counterstained with luxol fast blue and mounted in a synthetic medium. To exclude antigen-independent staining, the incubation step with primary/secondary antibodies was omitted from the protocol in the control samples. In all instances, these controls were negative. Assessment of p16 immunoreactivity was performed by two investigators who were blinded to the clinical details of the patients.