Fresh surgically resected tissue samples from 189 patients who were pathologically diagnosed with esophageal carcinoma were obtained between March 2013 and December 2015; written informed consent was obtained from each specimen donor. All specimen donors were treated in the Pathology Department of Tangshan People's Hospital (Hebei, China); all patients came from the Tangshan area. The mean age was 58 years (range, 40–76 years), of which 136 were male and 53 female. All samples were esophageal squamous cell carcinoma, of which 30 were well differentiated, 104 were moderately differentiated and 55 were poorly differentiated. The samples were separated into 98 early-stage, 63 middle-stage and 28 late-stage specimens, according to the sixth and seventh editions of International Union Against Cancer classification (15); all fresh samples were stored at −80°C prior to the experiments.

The following media were used in the present study: Dulbecco's modified Eagle medium (DMEM) with antibiotics (100 U/ml penicillin/streptomycin), fetal bovine serum, trypsin, and these reagents were provided by BioTek China, Beijing, China. QIAamp DNA Mini kit, Qiagen RNeasy Mini kit and Qiaquick Gel Extraction kit were provided by Qiagen GmbH, Hilden, Germany. Takara Ex Taq kit (including buffer, MgCl_2, dNTPs, Taq DNA polymerase) was provided by Takara Bio, Inc., Otsu, Japan. The 293 cell line obtained from National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention (Beijing, China).

The 293 cell line were cultivated using 10 ml media (DMEM with 100 U/ml penicillin-Streptomycin and 10% fetal bovine serum) in a 10 cm petri dish and maintained at 37°C in humidified atmosphere, containing 5% CO₂/and 95% air. Cell media were changed every 2–3 days.

The 293 cells were washed twice using phosphate buffer saline (PBS) when the 293 cells were allowed to reach 90% confluence. Subsequently, cells were digested using 2 ml 0.25% trypsin (including 0.02% EDTA), which was terminated using 2 ml fresh cell medium, and transferred to 10 ml flasks. Cells were centrifuged at 3,000 × g for 5 min at 4°C and the supernatant was removed. DNA extraction was performed using QIAamp DNA Mini kit, according to the manufacturer's protocol.

The plasmid HPV18/pBR322 (National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, China) DNA, containing the whole HPV18 genome, and DNA from the 293 cell line were stored at −20°C.

DNA was extracted from each tissue specimen (~25 mg) using the QIAamp DNA Mini kit according to the manufacturer's protocol, and each sample was eluted with ~50 µl sterilized distilled water. The extracted DNA was stored at −20°C until further processing.

The quality of the DNA from each tissue sample was assessed using PCR with β-actin primers (16). DNA from the 293 cell line was used as a control to ensure specimen quality.

HPV DNA for each specimen was analyzed by PCR using the primers My09/11 for HPV L1 (My09, 5′-CGTCCMARRGGAWACTGATC-3′; MY11, 5′-GCMCAGGGWCATAAYAATGG-3′, PCR product ~450 bp) (17) and HPV18 E6-specific primers (forward, 5′-GCGCTTTGAGGATCCAACAC-3′ and reverse, 5′-ATTCAACGGTTTCTGGCAC-3′; the PCR product generated was ~335 bp). The HPV18 E6 primers were designed on the basis of the genomic sequence of HPV18 in GenBank (accession number X05015.1).

Each PCR amplification was performed in a volume of 25 µl, containing 1X Ex Buffer (MgCl₂-free), 2.5 mM MgCl₂, 0.2 mM dNTPs, 1 U Ex Taq DNA polymerase, and 5 pmol each of the aforementioned forward and reverse primers. A total of 100 ng extracted DNA template and control DNA were added to the reaction mixture. The following thermal cycling profile was employed: 95°C for 5 min, followed by 31 cycles of 95°C for 30 sec, 55°C for 30 sec and 72°C for 30 sec. The last cycle was followed by a final extension step of 5 min at 72°C.

To confirm the specificity of the HPV18 primers, the HPV18 E6-positive PCR products were purified using a Qiaquick Gel Extraction kit according to the manufacturer's protocol. The products were then ligated into the pMD-18T vector prior to being subjected to sequence analysis by Beijing Rui Bo Xing ke Biological Technology company using DNAman software v6.0 (www.shinegene.org.cn/q2.html).

Total RNA was extracted from each sample using a Qiagen RNeasy Mini kit according to the manufacturer's protocol. RT was performed in a total volume of 25 µl consisting of 4 µl 5X first-strand buffer, 1 µl dNTPs (0.2 mM), 1 µl RNase inhibitor (40 U), 100 ng total RNA, 1 µl (dT)₁₇-p3 (10 pmol primer, GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT), 1 µl SuperScript reverse transcriptase (200 U) and 9.2 µl RNase-free water. The RNA was reverse-transcribed at 42°C for 50 min and deactivated at 70°C for 15 min, and the resultant product was stored at 4°C.

To verify that each tissue sample was harboring HPV with detectable HPV18 E7 mRNA, human GAPDH was employed as a reference gene for RT-PCR using the following primers: GAPDH forward, 5′-CATCACCATCTTCCAGGA-3′ and reverse, 5′-GTCTACCACCCTATTGCA-3′ (13). This approach guaranteed that the integrity of all mRNA extracted from each sample was sufficient for the detection of viral-cell fusion transcripts.

The first PCR was prepared according to the method of Klaes et al (14), in a volume of 25 µl using HPV18 E7-specific forward primers for p1-18 (5′-TAGAAAGCTCAGCAGACGACC-3′) in HPV18 gene sequence 816…836 and reverse primers for p3 (5′-GACTCGAGTCGACATCG-3′); the reaction system consisted of 1X Buffer, 10 pM primers, 2.5 mM MgCl₂, 0.2 mM dNTPs, 100 ng cDNA product and 1 U Ex Taq DNA polymerase. PCR was performed according to the following steps: 95°C for 5 min, followed by 30 cycles at 95°C for 1 min, 56°C for 1 min and 72°C for 3 min. The final cycle consisted of 72°C for 5 min, and the sample was stored at 4°C.

Nested PCR was performed under identical conditions (except for the annealing temperature, which was 67°C) and the primers: the HPV18 E7-specific forward primer p2-18 (5′-ACGACCTTCGAGCATTCCAGCAG-3′) in HPV18 gene sequence 831…853 and the reverse primer (dT)₁₇-p3; 5 µl PCR product from the first round was used as the template. The two primers span bp 814–835 (p1-18) and bp 830–853 (p2-18) in the HPV18 genome. To ensure the specificity of these primers, a negative control (293 cell DNA template) was added to each reaction system.

The HPV18 E7 PCR products for p2-18 were cloned into the pMD-18T vector. Subsequently, Beijing Rui Bo Xing ke Biological Technology sequenced. The results were blasted using the National Center for Biotechnology Information (blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome), HPV18 integration sites in human chromosome were determined for every specimen.

β-actin expression in a total of 189 samples was investigated to evaluate the DNA quality of the tissue specimens. A 290-bp β-actin product was detected in each sample. This result suggested that the DNA from each specimen was of high quality and satisfied the requirements for further experiments. Of the 189 specimens, 168 were positive for HPV DNA, as assessed using MY09/11 primers (Fig. 1A); 33 of these specimens were positive for HPV18 (Fig. 1B), assessed using the HPV18 E6-specific primers.

The sequencing results of samples positive for HPV18 were analyzed with the DNA man software and compared with the HPV18 sequence provided in GenBank (X05015.1). The results of this analysis verified that four of the HPV18 E6-positive samples harbored mutations in the viral DNA: Two samples contained one mutation; the other two contained two mutations (Fig. 2).

HPV18 integration sites were identified in all HPV18 E6-positive specimens. A ~600 bp HPV18 E7 PCR product was identified in three samples using p2-18 primers. The HPV18 E7 PCR product obtained (using primer p2-18) from the three samples was ligated into the pMD-18T vector, and sequence analysis of the HPV18 integration sites was performed. The results of this sequencing analysis indicated that the HPV18 E7 gene PCR product, amplified using the p2-18 primer in these three samples, was part of the HPV18 E7-E1 sequence (Fig. 3A-C). Other sequences from two samples were similar to sequences from human chromosome 5 (Fig. 4A and B) and human chromosome 2 (Fig. 4C).