Human papilloma virus (HPV) infection is controversial as a causative factor in oral tongue cancer. This study aimed to clarify whether HPV directly affects the carcinogenesis and biological behaviour of oral tongue cancer by analyzing HPV prevalence, the physical status of the virus and clinicopathological parameters. Archival tissue was obtained from 36 patients diagnosed with T1 and T2 oral tongue cancer and 25 normal controls. HPV genotyping chip and real-time polymerase chain reaction were used to determine the prevalence, phenotype and physical status of HPV to clarify whether HPV directly affects oncogenesis. The results were also compared with clinicopathological parameters. HPV was detected in 36% (13/36) of oral tongue cancer patients, compared with 4% (1/25) of the control. In the HPV-positive group of oral tongue cancers, HPV-16 was the most common type and its prevalence rate was 85% (11/13). Of the HPV-16 infected oral tongue cancers, the integration rate of HPV-16 was 55% (6/11). The HPV-16 positive group showed shallower stromal invasion than the HPV-16 negative group ( p = 0.045). HPV-16 may be one of the causative factors in early squamous cell oral tongue carcinoma and be associated with its depth of invasion.
Cancers involving the oral cavity account for 2–3% of all malignancies and the tongue is the subsite with the highest incidence of cancer in the oral cavity . Tobacco smoking and alcohol consumption are major causative factors for head and neck squamous cell carcinoma (HNSCC) including oral tongue carcinoma, but viral infection such as high-risk human papilloma virus (HPV) may be an oncogenic factor in HNSCC .
HPV is a double-stranded DNA virus and may play a role in the pathogenesis of HNSCC given the similarities in morphology and susceptibility to HPV exposure between the tissues involved in head and neck cancers and uterine cervical cancer . The tongue may be the first site of exposure to viral microorganisms in the aerodigestive tract and oral tongue cancer could be susceptible to HPV exposure, directly or indirectly. The prevalence of HPV in oral tongue cancer is extremely diverse, ranging from 0% to 100% in the literature, and the prevalence of HPV in HNSCC is not uncommon . The markedly different reports of prevalence of HPV in oral tongue cancer may be due to: mixed samples with the oropharynx; methodological differences for detecting HPV, including less accurate methods; various tumour stages and racial and geographical differences between the studies. Of these, the methodological difference is the most important because results from recent studies using advanced technology for the detection of HPV differ to a great extent compared with the results of previous less accurate studies . The role of HPV in HNSCC is mainly carcinogenesis, leading to the possibility that studies including both early and late stage cancers may be inaccurate.
The prevalence and physical status of HPV purely in oral tongue cancer, excluding cancer of the base of the tongue, especially in the early stages, has not been studied widely. This study aims to clarify whether HPV directly affects the carcinogenesis and biological behaviour of early oral tongue cancer by analyzing the prevalence of HPV, the physical status of the virus and the clinicopathological parameters, using the most up-to-date technology.
Materials and methods
Tissue samples and DNA extraction
Paraffin-embedded tissues were obtained from 36 patients who were diagnosed with T1 or T2 early oral tongue cancer and received surgery as initial treatment between 1995 and 2005. For comparative analysis, 25 pathologically normal oral tongue tissue samples obtained from leukoplakia specimens were included as a control group. The Institutional Review Board of Yonsei University approved the protocol.
Ten micrometer sections were cut from the paraffin blocks and collected in 1.5 ml Eppendorf tubes for DNA extraction. To prevent cross-contamination, each block was cut after thorough cleaning of the microtome blade. Paraffin-embedded samples were placed in xylene for 5 min and centrifuged at 14,000 rpm. DNA extraction was carried out using the QIAamp DNA Mini kit (Qiagen, CA, USA). The quality (ratio of 260 nm/280 nm) and quantity (absorbance at 260 nm) of the isolated DNA were determined by optical density measurement. CasKi and SiHa cells were grown for approximately 5 days in the appropriate medium (CasKi cells and SiHa cells, RPMI 1600 [Gibco-BRL, Grand Island, NY, USA]). DNA isolation was performed with the QIAamp DNA Mini Kit according to the protocol for cultured cells grown in a monolayer.
An HPV genotyping DNA chip (Biocore, Korea, Seoul) arrayed with multiple oligonucleotide probes of L1 sequences from 26 types of HPV was used according to the manufacturer’s protocol. Consensus polymerase chain reaction (PCR) products for L1 were hybridized to the arrayed probes on the HPV chip, and HPV genotypes were identified using a fluorescence scanner (GenPix 4000B, Axon Instruments Inc., CA, USA) with a 532-nm laser for excitation of Cy3. The fluorescence intensity data of the specific probes were then printed out as an Excel spreadsheet.
The copy numbers of the HPV E2 and E6 open reading frames (ORF) were assessed using a TaqMan-based 5′-exonuclease quantitative real-time PCR assay based on the DNA amplification of a 76-bp sequence of the E2 ORF and an 81-bp sequence of the E6 ORF, in the presence of HPV-16 E2- and E6-specific hybridization probes, respectively. The primers and probes for the E2 assay were designed to recognize the E2 hinge region of the E2 ORF, which is most often deleted upon HPV-16 integration in cervical carcinomas. For each specimen, identical amounts of DNA were quantified for the E6 and E2 sequence of HPV-16. Each specimen was assayed three times. PCR amplification was performed in a 25 μl volume containing 1× iQ SuperMix (BioRad, Hercules, CA, USA), 200 nM E2 and E6-specific primers ( Table 1 ), 100 nM dual-labelled (5′Hex and 3′BHQ2) E2 and (5′FAM and 3′BHQ1) E6 fluorogenic hybridization probe, and 200 ng of the genomic DNA template. All experiments were performed using the real-time iCycler™ PCR platform (BioRad, Hercules, CA, USA). In each experiment, two standard curves were included obtained by amplification of a dilution series of the HPV viral copy number using CaSki (American Type Culture Collection, Manassas, VA, USA) cell line genomic DNA, which is known to have 600 copies/genome equivalents (6.6 pg of DNA/genome) . There was a linear relationship between the threshold cycle values plotted against the log of the copy number over the entire range of dilutions. The amplification ramp included two hold programmes of 2 min at 50 °C and 10 min at 95 °C, followed by a two-step PCR cycle with a melting step for 15 s at 95 °C and an annealing step for 1 min at 60 °C for a total of 45 cycles. The ratio of E2 to E6 copy numbers was calculated to determine the physical status of the HPV-16 viral gene. HPV-16 in the pure episomal form is expected to have equivalent copy numbers to those of E2 and E6 genes (i.e. E2/E6 ratio = 1), whereas preferential disruption of E2 upon viral integration should result in fewer E2 gene copies than E6 genes. This means that an E2/E6 ratio of less than 1 would indicate the presence of both the integrated and episomal forms while a ratio of 0 would indicate the presence of only an integrated form. The copy number of the integrated E6 gene was calculated by subtracting the copy number of E2 (episomal) from the total copy number of E6 (episomal and integrated). The ratio of E2 to integrated E6 genes represents the amount of the episomal form in relation to the integrated form. Values less than one indicate the predominance of the integrated form. DNA extracted from the cervical carcinoma cell line SiHa, known to harbour a pure, integrated form of the HPV-16 gene in which the E2 and E4 ORFs are disrupted, was used as the control for E2 (negative) and E6 (positive) amplification . The relative viral load can be estimated by calculating the ratio of copies of E6 in the sample and SiHa cells.
|Name||Sequence||T m (°C)|
Evaluation of clinicopathological parameters
The follow-up period ranged from 13 to 120 months with a mean of 61 months. Surviving patients were followed up for at least 24 months. In order to identify the factors that may be associated with HPV infection, the correlation between clinicopathological factors, such as TNM stage, depth of invasion, recurrence, survival, and HPV prevalence, was analyzed.
The relationship between HPV status and clinicopathological parameters was analyzed using cross-tabulations and Fisher’s exact test with SAS software, version 9.1 (SAS Institute Inc., Cary, NC, USA). The survival rate of the patients was calculated using the Kaplan–Meier method and curves were compared using the log-rank test. A p -value less than 0.05 was considered statistically significant.
In the present study, HPV prevalence in early oral tongue cancer was 36% (13/36). In the HPV-positive tumours, 11 cases (84.8%) were infected with HPV-16 and the others were infected with non-16 high-risk type and low-risk type HPV each and multiple infections were not found in these cases. Corresponding control samples, neighbouring normal tissue of hyperplastic leukoplakia of the tongue, demonstrated rare incidence of HPV infection (1/25; 4%). There was a statistically significant difference in HPV prevalence between early oral tongue cancer and control samples ( p < 0.05) and HPV-16 was the single most common type.
Viral status in head and neck cancers
Both validated assays of the real-time amplification systems for E2 and E6 ORFs using serially-diluted HPV-16 plasmid DNA showed similar amplification efficiencies as reflected by the almost identical slopes of the amplification curves ( Fig. 1 ). The results for the physical states and viral copy numbers are summarized in Table 2 . Integrated E6 was calculated by subtracting the numbers of E2 from E6. The ratio of E2 to integrated E6 represents the amount of the episomal form relative to the integrated form. A value of less than 1.0 indicates a predominance of the integrated form. In the 11 early oral tongue cancer samples with HPV-16 infection, six cases were integrated (55%) ( Table 2 ).
|Case||HPV-16 copies/cell||E2/E6 ratio||E2/integrated E6||Physical status|