| BACKGROUNDHead and neck squamous cell carcinoma (HNSCC) is induced by many risk factors such as genetic inclination, smoking and HPV infections. The accumulating evidence shows that infections with high risk subtypes of HPV, dominantly HPV 16/18, had some tumorigenesis effects on squamous cell cancers, especially for the oropharyngeal carcinomas. The mechanisms of HPV-dependent tumorigenesis were mostly elucidated basing on the molecular evidence in recent researches. Oropharyngeal carcinoma with HPV infection is assumed to be a special kind of cancer considering its characteristics of poorer differentiation but better prognosis. There were some limitations in the earlier researches from China. Some of them did not evaluate accurately the infection state of HPV, or did not investigate the relationship between the expression of Ki-67, p53 and p16, or did not assess the effect of HPV infection on the prognosis of HNSCC.ObjectiveTo detect the infection of human papillomavirus 16/18 (HPV16/18) in the head and neck squamous cell carcinoma by two different methods:real-time fluorescent PCR (RT-PCR) and HPV 16/18 DNA in situ hybridization(ISH). To compare the difference between these two methods. To investigate the relationship between HPV16/18 infection and clinical data of HNSCC, the expression of Ki-67, p53 and p16 protein. To evaluate the effects of high risk factors, such as HPV infection, alcoholism, smoking, lymphoid metastasis, the expressions of Ki-67, p53 and p16, tumor stage on the prognosis of HNSCC.Methods(1) Basical research:The level of HPV 16/18 DNA in the paraffin embedding tumor tissues was measured by RT-PCR and in situ hybridization. The expression of Ki-67, p53 and p16 proteins were measured by immunohistochemistry in tumor tissues(n=78).(2) Clinical study:clinical data, including the sex, age, tumor stage, alcoholism, smoking history, treatment strategies (operation, radiotherapy and chemotherapy) and survive time were collected form their medical records and by telephone follow-up.(3) Statistic methods:Using the Kapp aconsistancy test to evaluate the relation between the RT-PCR and in situ hybridization methods for HPV DNA detection. None-parameter Spearman relationship test and Chi-square test were used to evaluate the relation between HPV infection and the expression of Ki-67, p53, p16 in tumor tissues. COX multiple-factor regression analysis was used to investigate the effects of HPV infection, the expression of Ki-67, p53, p16, tumor stage, lymph nodes metastasis, sex, age, smok history and alcoholism on the patients'survival time.Results(1) The incidence rate of HPV infection was 62.8% in our patients with HNSCC by RT-PCR, while it was 47.4% by in situ hybridization (ISH). One patient (1.28%) was HPV18 DNA and HPV16 DNA positive by ISH. By the way of RT-PCR, the incidence rate of HPV16/18 DNA infection in lip, oral cavity, oropharynx and hypopharynx was 33.3%, 66.67%,70% and 57.14%, respectively. By the way of ISH, the incidence rate of HPV16/18 DNA in lip, oral cavity, oropharynx and hypopharynx was 33.3%,43.8%, 60.0% and 57.1%, respectively. In general, high consistency was found between two methods with Kappa=0.595 (P=0.000). The consistency was highest in lip and hypophrynx (both of them had Kappa=1, P=0.067 and 0.000, respectively). The consistency was lowest in oropharynx carcinomas with kappa=0.348 and P=0.5.(2) Ki-67 protein was expressed in 2-70% cells in each tumor tissues. p53 protein was expressed in 46.15% of the tumor tissues, p16 was expressed in 30.77% of the tumor tissues.(3) HPV infection detected by RT-PCR and ISH had no relation with sex, smoking, alcoholism, clinical tumor stage, tumor site, lyphnoid metastasis(all.P>0.05). HPV infection had a positive relation with pathologic stage (P=0.001 and 0.000, respectively), which mean tumors in higher pathologic stage had a higher incidence rate of HPV infection. In oropharynx and hypopharynx carcinomas subgroup, there was a negative relation between HPV infection and expression of p53(P=0.011 and 0.017 for RT-PCR and ISH methodes, respectively). In the patients with pathology of III grade low differentiation, there was a negative relation between HPV infection and expression of p53(P=0.002 and 0.022 for RT-PCR and ISH methodes, respectively). Positive HPV infection measured by RT-PCR and ISH had no relation with Ki-67(P>0.05), and had a negative relation with expression of pl6(P=0.026 and 0.023 for RT-PCR and ISH methodes, respectively). Furthermore, the expression of p16 had a negative relation with the expression of Ki-67 (P=0.047), and a positive relation with the expression of p53.(4)Survival time and multiple risk factors prognostic analysis in HNSCC:In 78 patients, six of them lost follow-up immediately after operation. The median suvival time of follow up was 21.5 months. The patients were on follow up until his/her death or till September, 2010.①According to the results of RT-PCR, the median survival time in HPV positive group and negative group were 44 months and 25 months respectively,P=0.508.②According to ISH, median survival time in HPV positive group and negative group were 30 months and 28 months, respectively, P=0.63.③The median survival time in the patients with p16 negative was 30 months, and the survival time for the positive did not appeare, because more than half patient were still on live till last follow.④The patients were divided into three subgroups according to their tumor differentiate stages: high differentiation (G1 subgroup), median differentiation (G2 subgroup) and low differentiation (G3 subgroup). The effects of HPV infection on survival time of our patients were evaluated. In G1 group, no median survival time was available. In G2 group, the median survival time was 18 months and 27months in HPV negative and positive respectively, P=0.129. In G3 group, the median survival time was 15 months and 44 months in HPV negative and positive respectively, P=0.098.⑤The patients were divided into lip+oral cavity group and oropharynx+hypopharynx group according to their different tumor sites. In lip+oral cavity group, the median survival time was 28 months and 44 months in HPV negative and positive respectively, P=0.593. In oropharynx+hypopharynx group, the median survival time was 25 months and 28 months in HPV negative and positive respectively, P=0.696.⑥The patients were divided into early stage group (0 stage+I stage+II stage) and late stage group(III stage+IV stage) according to tumor total stages.In early stage group, the median survival time was 28 months in HPV negative patients and the survival time in HPV positive was not available, P=0.553. In late stage group, the median survival time was 18 months and 30 months in HPV negative and positive patients respectively, P= 0.770.⑦The patients were divided into<50y group and≥50y group according to their ages. In the <50y group, the median survival time was 25 months and 35 months in HPV negative and positive patients, respectively, P=0.601. In the≥50y group, the median survival time was 28 months and 44 months in HPV negative and positive patients, respectively, P=0.595.⑧A multivariate Cox regression analysis showed that the two main risk factors for surval time were the expression of Ki-67 (RR 2.341,P=0.012) and lymphonodus metastasis (RR 1.012,P=0.547). The state of HPV infection was not a significant prognostic factor.ConclusionsIn patients with HNCSS, the incidence rate of HPV infection was 62.8% by RT-PCR and 47.7% by in situ hybridation (ISH) methodes. The incidence was varied according to different regions and the highest indicence of HPV infection was in the oropharyngeal carcinomas. Generally speaking, these two methods had high consistency in detection of HPV 16/18 infecton in HNSCC. HPV infection induced HNSCC had a tendency for poorer differentiation. In patients with oropharyx and hypopharyx carcinomas, HPV positive tumor tissues had less expression of p53 and p16 proteine. The expression of p16 had a positive association with p53, and had a negative association with the expression of Ki-67. The results of two methods showed that the median survival time in HPV positive patients was longer than HPV negative patients, although the difference was of no significance. In a multivariate COX regression analysis, the expression of Ki-67 and lymph node metastasis, not HPV infection, were the major risk factors for the survival time of our patients. |
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