The specimens from the 93 patients with malignant squamous tumors, including 64 laryngocarcinoma, 5 oropharyngeal carcinoma, 15 hypopharynx carcinoma and 9 lip carcinoma were included in this study. All cases were hospitalized in the Head and Neck Surgery Department, Peking University Cancer Hospital and Institute, from 2006 to 2011. Among them, 84 were male and 9 were female. The ages of the patients ranged from 27 to 84 years, with the median of 57 years, 87.1% (81/93) and 57.0% (53/93) of the patients had histories of tobacco or alcohol use. The professions of the patients varied largely, and the permanent residences of the patients were distributed widely in mainland of China. All samples were surgically removed and conventionally fixed in 10% formalin and paraffin embedded. Pathological assays verified that all cancers were squamous cancers. The pathological grades of the cancers were assessed by clinical pathologists in the Peking University Cancer Hospital and Institute. The clinical grades of patients with tumors were finally determined by surgeons in the Peking University Cancer Hospital and Institute.

Total DNAs from the tumor tissues were extracted from the paraffin-embedded tissue blocks with a commercial genomic DNA extraction FFPE kit (Qiagen). Briefly, 3–4 formalin-fixed, paraffin-embedded 10 μm sections were soaked in xylene vortex vigorously for at least 1 h. Then pellet was acquired and purified under the kit protocol. Quality of the extracted DNAs was assessed with a settled PCR protocol with a pair of actin-specific primers, which produced a 240-bp long fragment.

The presences of HPVs specific DNA sequences in the extracted DNAs from the tested tumors were screened with a Tellgenplex™ HPV DNA Test kit (Tellgen, China) using Luminex technique, which allowed to detect 26 genotypes of HPVs including 19 high-risk HPV types (HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 82 and 83) and 7 low-risk HPV types (HPV6, 11, 40, 42, 44, 61 and 73). The experiments were performed in Bio-Plex 200 system according to the manufacturer’s instructions. Briefly, 2 μg of the extracted DNA was proceeded the PCR protocol as following: 95°C 30 sec, 58°C 30 sec and 72°C 30 sec for the first 5 circles, and 95°C 30 sec, 55°C 30 sec and 72°C 30 sec for another 35 circles. The PCR products were subjected into a fast hybridization and the data were analyzed with the software supplied by the manufacturer.

Two-rounded PCR techniques for HPV16 and HPV18 were individually established. The primers were P16-1 (5′-GCAAGCAACAG TTACTGCGA-3′) and P16-2 (5′-CAACAAGACATACATCG ACC-3′) for HPV16 E6 sequence and P18-1 (5′-CACTTCACT GCAAGACATAGA-3′) and P18-2 (5′-GTTGTGAAATCGT CGTTTTTCA-3′) for HPV18 E6 sequence. Extracted DNAs (1.0 μg) were mixed with 1 μM of individual primers and 1.25 U Pfu DNA polymerase in a 25 μl volume. PCR was performed with the following conditions: 94°C 1 min, 57°C 1 min and 72°C 1 min, in total 30 cycles, and another 10-min extension. For second round PCR, 5 μl of the first round PCR product was mixed again with 10 μM of individual primers and 1.25 U Pfu DNA polymerase in a 25 μl volume. The PCR condition was 94°C 1 min, 52°C 1 min and 72°C 1 min, totally 40 cycles and another 10 min extension. The targeting fragments of HPV16 E6 and HPV18 E6 were 333 and 322 bp, respectively. All PCR assays were carefully carried out in the PCR laboratory with four separating rooms to avoid of DNA contamination. Actin-specific 240-bp long fragment was produced as control.

The PCR products were analyzed in 2% agarose gel and recovered from gel with QIAquick Gel Extraction kit (Qiagen). Direct sequencing was performed using the same PCR primers and the base sequences were read on ABI PRISMTM 3730XL DNA analyzer.

Paraffin sections (5 μm) were deparaffinized in xylene for 5 min twice and gradually rehydrated routinely. Sections were quenched for endogenous peroxidases in 3% H₂O₂ in methanol for 15 min, pretreated with enzyme digestion antigen retrieval for 1 min. After blocking in 1% normal goat serum, the sections were incubated overnight at 4°C with 1:500-diluted mAb for HPV16/18 E6 (Abcam). The sections were then incubated for 60 min with 1:1000-diluted HRP-conjugated goat anti-mouse secondary antibody (Vector Labs, USA), and visualized by incubation with 3,3-diaminobenzidine tetrahydrochloride (DAB). The slices were dehydrated and mounted in Permount. Photomicrographs were taken with a DP70 digital camera mounted on BX5 microscope (Olympus Optical, Japan).

Statistical analyses were performed using SPSS (Statistical Package for the Social Sciences, Chicago, IL) 11.5 for windows. Prevalence of HPV infection in the head and neck squamous carcinomas of different sites, pathological grades, clinical grades and age groups were analyzed by the χ² test. P<0.05 was considered statistically significant.

Written consent for further investigation and publication was obtained from the patients or the patients’ relatives, respectively. Usage of the stored human samples in this study was approved by the Ethics Committees of Peking University Cancer Hospital and Institute and National Institute for Viral Disease Prevention and Control, China CDC.

The presence of HPV sequences in the fixed tumor tissues was firstly screened by a commercial Luminex technique for HPV genotyping. Out of 93 tumor samples, 7 cases showed positive HPV signals, in which 4 were HPV18-positive, 2 were HPV16-positive and 1 was HPV52-positive. To obtain more detailed evidence, the presence of HPVs sequences in tumor samples were further assayed with the established PCR protocols specific for HPV16, 18 and 52. Among the tested samples, 6 were HPV18-positive, 6 were HPV16-positive and 1 was HPV52-positive. Sequencing assays of the positive PCR products were verified to be E6 sequences of the relative HPVs. Three out of 4 cases showing HPV18-positive in Luminex and the case showing HPV52-positive in Luminex were positive in the PCR assays, whereas 2 cases showing HPV16-positive in Luninex were PCR-negative. Collectively, 7 samples contained HPV18 specific sequences, 8 contained HPV16 sequences and one contained HPV52 sequences.

To assess the HPV-related proteins in tumor tissues, the tissue sections of various cancers were immunohistochemically stained with mAb against HPV16/18 E6 oncoprotein. From 93 cancer cases, 29 samples (31.2%) showed positive signals while none of the five polyps of larynx revealed positive signal. The brown-stained signals were present and restricted to cell nuclei and/or cytoplasm of the cancer cells, without detectable morphological difference among various pathological grades according to cell differentiation (Fig. 1). Additionally, among 16 cancer samples containing HPV16 or 18 sequences, 4 were positive in IHC assay. This highlights a relatively high distribution of HPV associated proteins in the cancer tissues. However, the presence of HPV proteins and HPV gene sequences did not correlate with each other. The total positive rate of HPV genomes and its encoding products in the tested samples was 44.1% (41/93).

Among the 93 tested cancers, 64 were laryngocarcinoma, 5 were oropharyngeal carcinoma, 15 were hypopharynx carcinoma and 9 were lip carcinoma. Table I summarized the results of HPV genomes and proteins in various cancers. The presences of HPV sequences and/or proteins in the cancer tissues seemed to differ largely based on the sites of the malignant tumors, 35.9% (23/64) laryngocarcinoma, 46.7% (7/15) hypopharynx carcinoma, 60.0% (3/5) oropharyngeal carcinoma and 88.9% (8/9) lip carcinoma were HPV-positive either in the assays of viral nucleosides (Luminex and PCR) or in that of viral proteins (IHC). Statistical analysis revealed a significant difference (P=0.022) in the anatomic sites of tumors. It illustrates a clear declining manner of HPV infection in the head and neck tumors from outside (lip) to inside (larynx). Moreover, the positive rates of viral sequences and proteins were different among various tumors. The detection rates of HPV16/18 oncoprotein was significantly higher than that of HPVs DNA in lip cancers (77.8 vs 11.1%, P=0.008) and higher in larynx cancers (28.1 vs 10.9%, P=0.015) having significant difference, while the detection rate of HPV sequences was higher than that of viral protein in hypopharynx cancers (40.0 vs 13.3%, P=0.107) having no significant difference (Table I). Only a few cases (4/93) showed positive both in HPV sequences and proteins.

To address the possible correlation of HPV infection with tumor pathological classification, the prevalence of HPV was sub-grouped according to the tumor pathological grades. Based on the tumor cell differentiation pathologically, the enrolled cancers were divided into the groups of high, moderate and low differentiation. On the whole, the HPV positive rates were higher in the groups of high- (46.6%, 13/28) and moderate-differentiation cancers (46.0%, 23/51) than that in the group of low-differentiation cancers (27.8%, 5/18) (Table II), but statistical analysis of χ² did not reveal difference (P=0.513).

To see the correlation of HPV infection with the clinical progression, all enrolled cancers were classified according to their clinical grades. Generally, the HPV-positive rates in the groups of the clinical phase I, II, III and IV were 51.9 (14/27), 37.1 (13/35), 46.2 (12/26) and 40.0% (2/5), respectively (Table III). Although the HPV-positive rate in the group of phase I was higher than those of the rest, statistic analysis of χ² did not reveal significant difference. Furthermore, the HPV infections in the patients with or without metastasis were analyzed. The HPV-positive rates in the patients with metastasis (53.3%, 8/15) and without metastasis (42.3%, 33/78) were comparable, without statistic difference. It seems that HPV infection in the tumor tissues does not obviously affect the clinical progressions of the oral and throat carcinomas.

Among the enrolled 84 male patients, 40.5% (34/84) cases were HPV-positive, while predominantly more female patients (77.8%, 7/9) showed HPV-positive, revealing an obvious gender-tendency. Among 7 HPV-positive female patients, 4 were lip carcinoma, 2 were larynx carcinoma and 1 was hypopharynx carcinoma. The median age of HPV-positive patients was 58 years, which was slightly older than that of HPV-negative patients (56.5 years old). The HPV-positive data among the different age groups are summarized in Table IV. Most cases were in the age groups of 40–50, 50–60, 60–70 and >70 years and HPV-positive rates in those four groups were 21.4, 48.6, 37.0 and 53.8%, respectively. There were only four patients <40 years, but all were HPV-positive. Statistical analysis did not reveal difference in HPV-positive rates among the age groups of 40–50, 50–60, 60–70 and >70 years. Furthermore, the HPV infections among the patients with or without history of tobacco and alcohol use were analyzed. HPV detection rates in the groups of tobacco and alcohol use were 41.9 (34/81) and 39.6% (21/53), respectively, which were lower than that in the groups without the history of tobacco (58.3%, 7/12) and alcohol (50.0%, 20/40) use, but there is no significant difference between the HPV-positive in the groups using tobacco or alcohol and not using tobacco or alcohol (P=0.289 and 0.321).