Twenty specimens of esophageal carcinoma were collected from Zhongxiang Chaihu Hospital in the high-incidence area. The average age of the patients was 58 years (range 41-58 years). Twenty patients were all Henan emigrants. Twenty-two specimens were collected from Macheng district. The mean age of patients was 55.7 years (range 33-67 years). Forty specimens were collected from Department of Pathology, Hubei Cancer Hospital. The mean age of patients was 56.9 years (range 37-75 years). The patients were all natives. All the patients were histologically diagnosed as esophageal squamous cell carcinoma.

All the samples were cut into 5-µm thick sections using Leica RM2135 microtome. H-E staining was performed before IHC and ISH. At the same time, all the samples were confirmed by pathologists and 40 normal esophageal mucosa tissue specimens were used as controls. PV and HPV (16/18-E6) antibody and Ultrasensive^TM S-P immunohistochemistry kit were purchased from Maixin Biotechnology Company (Fuzhou, China). Biotinylated HPV (16/18) DNA probes and in situ hybridization detection systems were purchased from DAKO Company.

Immunohistochemistry (Ultrasensive^TM S-P method) for detection of PV and HPV (16/18-E6) was performed following the manufacturer’s instructions. Briefly, paraffin-embedded sections were dewaxed, antigen retrieval was performed by heating the sections in 10mM of citrate buffer (pH 6.0) for 90 s. The tissue sections were treated with 3% hydrogen peroxide in PBS containing 0.01 mol/L sodium phosphate (pH 7.2), then with 0.15 mol/L NaCl to block endogenous peroxidase and normal rabbit serum to block non-specific binding sites. Mouse monoclonal anti- HPV (16/18-E6) antibody was used as the primary antibody at a dilution of 1:100. Rabbit polyclonal anti-PV antibody was used as the primary antibody at dilution of 1:50 and 1:100, respectively. Peroxidase activity was measured with 3, 3’-diaminobenzidine. The primary antibody was absent in negative controls. Sections were counterstained with hematoxylin. The positive control sections were supplied by Maixin Bio Company.

All in situ hybridizations were performed using in situ hybridization detection systems and biotinylated HPV (16/18) DNA probes from DAKO Company. Briefly, paraffin-embedded sections were dewaxed and then digested by immersion in 0.8% pepsin solution in 0.2N HCl at 37°Cfor 10 min. Following digestion, the sections were rinsed 4 times in deionized water, then immersed in 0.3% H₂O₂ for 20 min, and rinsed 5 times in deionized water. The slides were dried in air for 15 min, one drop (approximately 20 µL) of probe was applied to the sections, and covered with coverslips. The probe and HPV target DNA were denatured by placing the slides on a PCR cycler at 90°C for 5 min. Following denaturation, slides were transferred to a pre-warmed humid chamber for hybridization at 37°C for 60 min. Following hybridization, coverslips were removed by immersing the slides in 1×TBST at room temperature. The slides were transferred to fresh TBST bath before strigent washing at 58°C for 30 min followed by rinsing the slides 3 times in 1×TBST, one min each. The slides were placed on a level surface and enough streptavidin-AP reagent was applied to each section to cover the tissue, incubated for 20 min at room temperature and then enough BCIP/NBT substrate solution was applied to each section to cover the tissue. The slides were incubated at room temperature for 60 min, counterstained in nuclear fast red and cover slips were mounted. Purple-blue ISH signals were observed under microscope and photos were taken for analysis. Slides with PV and HPV positive esophageal cancer tissues were used as positive controls. The hybridization solution without probe or streptavidin-AP reagent was used as a negative control.

Without any knowledge of any patient’s clinical and pathological data, all slides were evaluated independently by two pathologists. An evaluated standard of results was established corresponding to the staining intensity of positive cells: - , negative; ＋, 5-25% positive cells; ＋＋, 26-50% positive cells; ＋＋＋～＋＋＋＋, positive cells > 50%. Statistical analyses were performed with SPSS 10.0 software and Fisher’s exact probability test was used to analyze the correlation between HPV expression and clinicopathologic features of ESCC. P < 0.05 was considered statistically significant.

IHC revealed that the positive rate of PV was 75.0% (15/20), 68.18% (15/22) and 72.5% (29/40) respectively and the HPV positive rate was 45.0％ (9/20), 36.36％ (8/22), 37.5% (15/40) respectively in esophageal carcinoma tissues from Henan emigrants, the local citizens, and patients in Hubei Cancer Hospital. No PV and HPV were detectable in all normal esophageal mucosa tissues. Only few samples showed weak staining. No correlation was found between HPV infection in esophageal carcinoma tissues and grades of esophageal carcinoma cells. The positive rate of HPV in three groups of esophageal cancer samples was significantly higher than that in normal mucosa samples (P  <  0.01). The positive rate was not obviously different among the three groups of esophageal carcinoma tissue samples. PV concentration at 1:50 and 1:100 showed the same positive rate (Table 1, Figures 1A-1C).

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Figure 1 Positive expression of PV (A) and HPV(16/18-E6) (B) and negative expression of HPV (16/18-E6) (C) in esophageal carcinoma (IHC×100)

In situ hybridization showed that the HPV (16/18) DNA positive rate was 30.0% (6/20)，31.8％ (7/22)，25.0％ (10/40) respectively in esophageal carcinoma tissues from Henan emigrants, the local citizens, and patients in Hubei Cancer Hospital. Forty normal mucosa tissue specimens showed negative results (Table 2). Among the three kinds of esophageal carcinoma tissue samples, no statistically significant difference was found (P > 0.05), while significant difference was found between normal mucosa tissues and the three kinds of esophageal carcinoma tissue samples (P < 0.05). Positive signals of HPV (16/18) DNA were located in nuclei. Positive cells located in the center of carcinoma were in the shape of small shuttle (Figure 2A). The negative control is shown in Figure 2B.

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Figure 2 Positive (A) and negative (B) expression of HPV(16/18) DNA in esophageal squamous cell carcinomas (ISH×100)