ORIGINAL ARTICLE Prognostic Impact of , , Epidermal , and Human Papillomavirus in Oropharyngeal in a Betel Nut–Chewing Area

Jamil N. Al-Swiahb, MD; Chao-Cheng Huang, MD; Fu-Min Fang, MD, PhD; Hui-Ching Chuang, MD; Hsuan-Ying Huang, MD; Sheng-Dean Luo, MD; Chang-Han Chen, PhD; Ching-Mei Chen, MS; Chih-Yen Chien, MD

Objective: To evaluate the prevalence of human Main Outcome Measures: We used the Fisher exact papillomavirus (HPV) and the prognostic significance of test to evaluate the correlation between the clinicopatho- epidermal growth factor receptor (EGFR), p53, and p16 logical variables and the presence of HPV in tumor cells. among patients with oropharyngeal carcinoma. Survival analysis was based on the Kaplan-Meier method.

Design: Retrospective study. Results:We detected HPV in 45 of the 274 patients (16.4%); of these, HPV-16 and -18 were identified in 42 (93.3%) of Setting: Academic Institute of Otolaryngology, the HPV-positive tumors. The HPV-positive oropharyngeal Kaohsiung, Taiwan. were more likely to occur in females, nonsmoking individuals, and those who did not chew betel quid. The Patients: Two hundred seventy-four patients who were HPV-positive tumors significantly expressed p16 and were inversely associated with EGFR and p53 expression (all, diagnosed as having oropharyngeal carcinoma under- Ͻ went testing for the presence of the HPV genome in the P .001). In addition, patients with tumor tissue that was nuclei of their tumor cells from January 1, 1992, through positive for HPV (P=.008) and had negative expression of March 31, 2008. EGFR (P=.01), low expression of p53 (P=.01), and high expression of p16 (P=.04) had a better prognosis. Interventions : The HPV genome was detected by per- Conclusion: Our results suggest that HPV, EGFR, p53, forming polymerase chain reaction–based assays and in and p16 are useful in predicting the clinical situ hybridization on tumor tissue from paraffin blocks. outcomes of oropharyngeal cancer. Immunohistochemistry staining for p16, p53, and EGFR was also performed. Arch Otolaryngol Head Neck Surg. 2010;136(5):502-508

HE HUMAN PAPILLOMAVIRUS eries of HPV in association with oropha- (HPV) is associated with a ryngeal carcinomas became consistent.1 large spectrum of epithelial Currently, we have more molecular evi- lesions. Most of these are be- dence that indicates a relationship between Author Affiliations: nign hyperplasia (warts) that high-risk HPV and the pathogenesis of oro- Departments of Otolaryngology rarelyT progress to cancers. A subgroup of pharyngeal cancers. Expression of HPV e6/ (Drs Al-Swiahb, Chuang, Luo, HPVs, however, are associated with le- e7 are the current criterion stan- Chen, and Chien), Pathology sions that have a tendency to undergo ma- dard for determining a causal role of onco- (Drs C.-C. Huang and lignant transformation.1 Currently, there are genic HPV in human tumors.3 The E6 and H.-Y. Huang), Radiation sufficient molecular and epidemiological E7 produce oncoproteins and are re- Oncology (Dr Fang), and Laboratory Medicine (Ms Chen) data to suggest a pathological significance sponsible for the degradation of p53 and Rb, 3 and Kaohsiung Chang Gung of HPV in head and neck squamous cell car- respectively, whichareimportanteventsthat Head and Neck Oncology cinomas (HNSCCs). The role of HPV in the lead to dysregulation of the and Group (Drs C.-C. Huang, Fang, pathogenesis of HNSCCs was first de- thereby immortalization of the cells. Chuang, H.-Y. Huang, Luo, scribed in 1983,1 when histopathological The silencing of p16 by promoter meth- Chen, and Chien and features consistent with HPV infection were ylation is commonly observed in HNSCC.4 Ms Chen), Chang Gung 2 Memorial Hospital-Kaohsiung identified in oral cancers. Two years later, In contrast, HPV is readily detected in HPV- Medical Center, Chang Gung viral DNA from high-risk HPV-16 was de- positive tumors and is highly correlated with University College of Medicine, tected in an oral carcinoma by Southern blot p16 expression, which is most likely the re- Kaohsiung, Taiwan. hybridization.2 By the early 1990s, discov- sult of transcriptionally active HPV infec-

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©2010 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 tion.5 Epidermal growth factor receptor (EGFR) is a tyro- sine kinase receptor that is expressed in low amounts in Table 1. Primer Sequences for Polymerase Chain Reaction normal squamous cells. Clinically, overexpression of EGFR in HNSCC is correlated with more aggressive tumor be- Product Size, havior and poorer prognosis.6 Therefore, the aims of our Primer Sequence study were to explore the prevalence of HPV and the ex- ␤-Globin PC04 5Ј-CAACTTCATCCACGTTCACC-3Ј 268 Ј Ј pression of p53, p16, and EGFR in oropharyngeal cancer GH20 5 -GAAGAGCCAAGGACAGGTAC-3 HPV L1 MY09 5Ј-CGTCCMARRGGAWACTGATC-3Јa 450 and their relationship to clinicopathological variables in a MY11 5-GCMCAGGGWCATAAYAATGG-3Јa betel nut–chewing area. GP05 5Ј-TTTGTTACTGTGGTAGATAC-3Ј 150 GP06 5Ј-GAAAAATAAACTGTAAATCA-3Ј METHODS a In the primer sequence, M stands for A or C; R, for A or G; W, for A or T; and Y, for T or C. STUDY POPULATION tive test results for ␤-globin DNA by PCR analysis were subse- Our study, which was approved by the Human Research Eth- quently studied using consensus primers MY09 and MY11. The ics Committee of Chang Gung Memorial Hospital, included 274 primer sequences were the same as those previously reported by patients with oropharyngeal carcinoma who underwent diag- Resnick et al,8 flanking the conserved region L1 open reading frame nosis and treatment with surgical resection followed by post- that was found in a broad spectrum of HPV. The PCR analysis operative adjuvant therapy, primary radiotherapy, or concur- was performed by denaturation of the samples at 94°C for 5 min- rent chemoradiotherapy between January 1, 1992, and March utes before the addition of 2.5 U of thermostable Taq polymer- 31, 2008, at the Academic Institute of Otolaryngology, Kaoh- ase, which was then followed by 35 cycles of amplification. A fi- siung, Taiwan. All patients who were treated during this pe- nal extension of 7 minutes completed the last cycle. The PCR riod were enrolled according to the databases of the depart- products were analyzed by electrophoresis and visualized on a ments of Radiation Oncology and Otolaryngology. The patients UV light transilluminator. To further confirm the PCR products underwent clinical staging of their cancer according to the 1997 7 amplified by MY09 and MY11 primers and to overcome the pos- American Joint Committee on Cancer system. Clinicopatho- sible false-negative result for HPV detection potentially caused logical data including age, sex, smoking, betel quid chewing, by DNA degradation in aging archival paraffin-embedded tis- alcohol intake history, nodal status, tumor site, and outcome sues, we also used a second PCR-based strategy with another pair data were obtained retrospectively. of primers, GP05 and GP06, franking a shorter fragment (150 base Group 1, which was used for survival and clinicopathological pairs [bp]) within the L1 open reading frame in supplementary analyses, consisted of 220 patients who received a diagnosis be- analyses for all cases and control specimens (Table 1). tween January 1, 1992, and December 31, 2005, and had a mean Catalyzed reporter deposition in situ hybridization was then age of 51.2 (range, 20-89) years. Group 1 included 111 patients used to determine the type of HPV and to localize the tissue with tonsil cancer, 71 patients with tongue base cancer, and 38 pa- sections of the samples that were positive for HPV L1 by PCR. tients with soft palate cancer. Group 2 consisted of 54 patients who If the type of HPV was beyond the detection of in situ hybrid- were diagnosed as having oropharyngeal squamous cell carcino- ization using the HPV-6, -11, -16, and -18 probes, the HPV type ma from January 1, 2006, through March 31, 2008, with a mean was then determined by results of a linear array HPV genotyp- age of 51.3 (range, 31- 84) years. The group 2 patients were only ing test (Roche Diagnostics GmbH, Mannheim, Germany). used for analyzing the prevalence rate of HPV in oropharyngeal cancer. These patients were not included in the survival analysis because they had only a short-term follow-up for survival. GENERATION OF HPV DNA PROBES Whole genomes of HPV-6, -11, -16, and -18 cloned into a 2743-bp DNA EXTRACTION FOR POLYMERASE pGEM II plasmid (Promega Corporation, Fitchburg, Wiscon- CHAIN REACTION sin) were used as DNA templates. The sequences of the consen- sus primers were identified according to the similarity of DNA To study HPV DNA, the most representative paraffin block of sequences flanking the segments of the noncoding regions de- the tumor for each case was selected by examining the hema- scribed by Huang et al.9 A 50-µL PCR reaction mixture was pre- toxylin-eosin–stained sections. For the cases with small tu- pared using 25 ng of DNA template, 1.5mM magnesium chlo- mor nests on the section, macrodissection was performed to ride, 0.5µM of each primer, 5 U of Taq DNA polymerase, and ensure that the tumor cell purity was greater than 50%. Ten deoxyribonucleotide triphosphates. The concentrations of de- tissue sections, 10 µm thick each, were obtained from the se- oxyadenosine triphosphate, deoxythymidine triphosphate, de- lected tissue block of each specimen and collected in a 1.5-mL oxycytidine triphosphate, and deoxyguanosine triphosphate were Eppendorf tube for DNA extraction. After deparaffinization, the 200µM each in the reaction mixture, but 10% of the deoxythy- genomic DNA was extracted using a tissue (QIAamp; midine triphosphate was substituted with DIG-16-deoxyuridine QIAGEN GmbH, Hilden, Germany) according to the manu- triphosphate (Roche Diagnostics GmbH). The reaction mixture facturer’s recommendation and finally dissolved in 20 µL of dis- was denatured at 94°C for 5 minutes, followed by 35 amplifica- tilled water for later use. tion cycles. The final product was cooled down to 4°C after the reaction, followed by purification using a PCR purification kit DETECTION OF HPV DNA (QIAGEN GmbH), and was diluted to 3.0 ng/µL with DNA in BY POLYMERASE CHAIN REACTION situ hybridization solution (DAKO, Carpinteria, California).

All samples were checked for DNA integrity by amplifying ␤- PREPARATION OF CELL AND TISSUE SECTIONS globin as a housekeeping gene. Primer sequences and the size of polymerase chain reaction (PCR) products for ␤-globin and HPV- Paraffin sections of formalin-fixed CaSki cell line (known to con- DNA analyses are listed in Table 1. The DNA samples with posi- tain 500-600 copies of HPV-16 DNA) were included and detected

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©2010 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 bated with primary anti-DIG–horseradish peroxidase antibody Table 2. Correlation Between the Clinicopathological (DAKO), which was diluted 1:500 in phosphate-buffered saline Features and HPV Status for 30 minutes and finally followed by a TBST wash.

HPV Status, SIGNAL AMPLIFICATION No. of Cases Total No. P AND DEVELOPMENT Variable of Cases Negative Positive Value Sex The GenPoint kit was used for signal amplification. Briefly, the Male 206 182 24 slides were incubated with biotinyl tyramide for 15 minutes and Ͻ.001a Female 14 5 9 then washed and agitated in TBST 3 times for 5 minutes each time. Age, y After the amplification step, streptavidin–horseradish peroxidase Յ49 104 86 18 was applied to the slides, which were then incubated at room tem- .45 Ն50 116 101 15 perature for 15 minutes, followed by three 5-minute washes in Tumor stage TBST. Finally, the signals were developed by diaminobenzidine. T1 and T2 85 71 14 .70 T3 and T4 135 116 19 Nodal status IMMUNOHISTOCHEMICAL STAINING Negative 51 48 3 FOR p16, p53, AND EGFR .04a Positive 169 139 30 TNM stage The representative blocks of the formalin-fixed, paraffin- Stages I and II 22 19 3 embedded biopsy tissue samples were retrieved and sectioned Ͼ.99 Stages III and IV 198 168 30 for the immunohistochemical study. The p16, p53, and EGFR Tobacco use monoclonal antibodies were purchased from Neomarkers Inc Smoking 174 154 20 (Fremont, California), DAKO, and Santa Cruz Biotechnology .009a Nonsmoking 46 33 13 (Santa Cruz, California), respectively. These monoclonal anti- Alcohol use bodies were all diluted 1:100 in phosphate-buffered saline ac- Drinking 74 63 11 Ͼ.99 cording to the manufacturer’s instructions. The sections were de- Nondrinking 146 124 22 paraffinized, incubated for 1 hour at room temperature, and then Betel quid use treated with 3% hydrogen peroxide for 10 minutes to inhibit en- Chewing 160 141 19 .054 dogenous peroxidase activity. They were then microwaved in Nonchewing 60 46 14 10mM citrate buffer (pH, 6.0) to unmask the epitopes. After an- p16 Expression tigen retrieval, the sections were incubated with diluted pri- High 36 5 31 Ͻ.001a mary antibodies for 1 hour, followed by a phosphate-buffered Low 184 182 2 p53 Expression saline wash. Horseradish peroxidase–Fab polymer conjugate High 187 182 5 (PicTure-Plus kit; Zymed Laboratories, South San Francisco, Cali- Ͻ.001a Low 33 5 28 fornia) was then applied to the sections for 30 minutes. After ex- EGFR expression tensive washing, the sections were incubated with peroxidase sub- Positive 195 185 10 strate diaminobenzidine for 5 minutes. Thereafter, the sections Ͻ.001a Negative 25 2 23 were counterstained with Gill hematoxylin and mounted with mounting medium. All the sections were interpreted by a pa- Abbreviations: EGFR, epidermal growth factor receptor; HPV, human thologist (C.-C.H.) who did not know the clinical data. papillomavirus. a Differences were statistically significant. GRADING FOR INTENSITY OF IMMUNOSTAINING in parallel with the sample tissue sections as positive controls. The WITH p16, p53, AND EGFR SiHa cell line (known to contain only 1 to 2 copies of HPV-16) was used to ensure high detection sensitivity for samples with very few Strong nuclear staining as well as strong cytoplasmic staining copies of HPV.9,10 The C33A cell line served as the negative con- was considered positive for p16 expression. Immunostaining trol. The cell blocks used for control samples and the tissue blocks for p16 was regarded as high expression if it was strong and from the specimens that were positive for HPV L1 by PCR were diffuse and if more than 60% of the tumor cells were positive cut into 5-µm-thick sections onto poly-L-lysine–coated slides. for p16.5 When evaluating the intensity of nuclear staining for p53, high expression was defined as expression in greater than 11 IN SITU HYBRIDIZATION FOR HPV DETECTION 50% of the tumor cells. Expression of EGFR was regarded as positive when greater than 50% of cell membranes in the tu- 5 The slides were placed in a 60°C incubator for 30 minutes, followed mor cells were completely stained. by deparaffinization. The target DNA was unmasked by digestion with protease K (DAKO), followed by quenching of the endoge- STATISTICAL ANALYSIS nous peroxidase with 3% hydrogen peroxide at room temperature for 20 minutes. After the DNA probe was added, the sections were We used the Fisher exact test to evaluate the correlation be- denatured by heating at 95°C for 5 minutes and were incubated tween the clinicopathological variables and the status of HPV at 45°C overnight in a humid chamber. After hybridization, the in tumor cells. In addition, the correlation among the expres- slides were rinsed with TBST (TRIS-buffered saline polysorbate 20, sions of EGFR, p16, p53, and HPV was also analyzed. PϽ.05 containing 50mM TRIS hydrochloride [pH, 7.6], 300mM sodium was considered significant in all the statistical analyses. The vari- chloride,and0.1%Tween20,providedintheGenPointkit[DAKO]), ables, including age, sex, T and N stages, TNM stage, the pres- followed by a stringency wash with 0.1ϫ11 saline–sodium citrate ence of HPV in tumor cells, and expression of p16, p53, and and detergent (provided in the GenPoint kit) at 48°C for 20 min- EGFR, were accounted for in survival analysis based on the utes. Slides were then rinsed with TBST for 5 minutes and incu- Kaplan-Meier method. Statistical significance was assessed by

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E F G H

Figure 1. In situ hybridization and immunohistochemical studies in oropharyngeal squamous cell carcinomas. A to D, A human papillomavirus (HPV)–positive tumor. A, The HPV-positive tumor demonstrates nuclear staining for the HPV-16 probe by in situ hybridization. B, Immunohistochemical studies in this tumor show nuclear staining for p16. C, Negative findings are demonstrated for p53. D, Negative findings are also demonstrated for epidermal growth factor receptor (EGFR). E to H, An HPV-negative tumor. E, No staining for the HPV-16 probe is seen by in situ hybridization. F, Immunohistochemical studies are negative for p16. G, Nuclear staining is positive for p53. H, Membranous staining is positive for EGFR (original magnification, ϫ200).

a log-rank test. To determine the distinct prognostic factors for survival, a multivariate analysis was performed according to the Table 3. Cumulative 5-Year Overall Survival Rate According Cox proportional hazards regression model. to the Clinicopathological Features

Variable No. of Cases 5-y Survival Rate, % P Value RESULTS HPV status Positive 33 59.4 .008a There were 206 men and 14 women in group 1 and 54 Negative 187 31.2 men and no women in group 2. Sixteen patients were clas- Age, y sified as having T1 disease; 88, T2; 60, T3; and 110, T4. Յ49 104 33.8 .75 Sixty patients were classified as having N0 disease; 42, Ն50 116 37.2 N1; 129, N2; and 43, N3. There were 4 stage I, 25 stage Sex Male 206 33.3 II, 43 stage III, and 202 stage IV carcinomas. The pres- .008a ence of HPV in the tumor nuclei was more likely to be Female 14 71.4 found among females (PϽ.001), nonsmoking patients Tumor stage T1 and T2 85 59.0 Ͻ.001a (P=.009), patients with positive nodal metastasis (P=.04), T3 and T4 135 20.1 and those who did not chew betel nuts (P =.054) Nodal stage (Table 2). The HPV-positive tumors were significantly Negative 51 34.8 .74 more likely to express p16 and were inversely associ- Positive 169 36.2 ated with EGFR and p53 expression (all, P Ͻ .001) TNM stage Stages I and II 22 55.7 (Figure 1). In addition, patients with the presence of .045a HPV (P=.008), negative expression of EGFR (P=.01), low Stages III and IV 198 33.9 p16 Expression expression of p53 (P=.01), and high expression of p16 High 36 54.7 .04a (P=.04) in tumor cell nuclei had better prognoses of cu- Low 184 32.2 mulative 5-year overall survival (Table 3 and Figure 2). p53 Expression However, the Cox proportional hazards regression analy- High 187 31.4 .01a sis demonstrated that HPV status (P=.02) and T stage Low 33 62.3 (PϽ.001) were the only independent factors for cumu- EGFR expression Positive 195 32.7 lative 5-year overall survival in this cohort (Table 4). .01a In total, 45 of the 274 patients (16.4%) were identi- Negative 25 60.8 fied with HPV within the nuclei of their tumor cells in Abbreviations: EGFR, epidermal growth factor receptor; HPV, human both groups. Thirty-five patients (77.8%) had positive papillomavirus. findings for HPV-16; 4 (8.9%), for HPV-18; 3 (6.7%), for a Differences were statistically significant.

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0.8

0.7 HPV-positive tumors 0.6 High p16–expression tumors

0.5 Cumulative Overall Survival 0.4 HPV-negative tumors Low p16–expression tumors 0.3

0.2

C D 1.0 P = .01 P = .01 0.9

0.8

0.7 Low p53–expression tumors EGFR–negative tumors 0.6

0.5 Cumulative Overall Survival 0.4 High p53–expression tumors EGFR–positive tumors 0.3

0.2 040206080 100 120 140 160 040206080 100 120 140 160 Survival Time, mo Survival Time, mo

Figure 2. The cumulative overall survival by Kaplan-Meier analysis for patients with oropharyngeal cancer. A, The patients with a human papillomavirus (HPV)–positive tumor have significantly better survival than those with an HPV-negative tumor. B, The patients with a tumor showing high p16 expression have significantly better survival than those with low p16 expression. C, The patients with a tumor showing low p53 expression have significantly better survival than those with high p53 expression. D, The patients with an epidermal growth factor receptor (EGFR)–negative tumor have significantly better survival than those with an EGFR-positive tumor.

an increasing trend to 22.2%. In group 2, the prevalence Table 4. Risk Factors Affecting Cumulative 5-Year Overall rates of HPV in tonsil cancer, tongue base cancer, and Survival Rate Determined by Cox Proportional Hazards soft palate cancer were 17.9%, 25.0%, and 27.3%, Regression Analysis respectively.

Variable RR (95% CI) P Value HPV status, negative vs positive 2.04 (1.12-3.69) .02a COMMENT Tumor stage, T3 and T4 vs T1 and T2 2.85 (1.91-4.26) Ͻ.001a

Abbreviations: CI, confidence interval; HPV, human papillomavirus; Because the patients were enrolled in a wide time frame RR, relative risk (1992-2008), there were great variations in the thera- a Differences were statistically significant. peutic strategy. The survival might be affected by differ- ent methods of treatment, especially for the patients with HPV-16 and -18; 1 (2.2%), for HPV-31; 1 (2.2%), for stages III and IV cancer. Accordingly, we also compared HPV-16 and -31; and 1 (2.2%), for HPV-6 and -11. In the survivals of the patients with stages III and IV can- group 1, the prevalence rates of HPV in tonsil cancer, cer who underwent treatment before and after 1999 in tongue base cancer, and soft palate cancer were 12.6%, this study cohort based on the Kaplan-Meier method. The 18.3%, and 15.8%, respectively. The prevalence rate of patients treated after 1999 had better survival rates com- HPV in group 1 patients who were enrolled from 1992 pared with those treated before (P=.007). This finding to 2005 was 15%. The prevalence rate of HPV in group might be attributed to the evolution of surgical and ra- 2 patients who were enrolled from 2006 to 2008 showed diation techniques, a better imaging study in the selec-

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©2010 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 tion of patients, and, most important, the intervention females, nonsmoking individuals, and those who did of multidisciplinary team work for the treatment of pa- not chew betel nuts among patients with oropharyngeal tients in recent years. cancer. The HPV-positive tumors showed a rising trend It is widely accepted that the most common risk fac- among these patients in the last 2 years of the study. tors for HNSCC are tobacco use, heavy alcohol consump- This might have been caused by a change in lifestyle, tion, betel quid chewing, and high-risk HPV infection. particularly in sexual behavior. To our knowledge, this High-risk HPVs are currently well accepted as causative study is the first combining the promising predictive agents for most cervical cancers and have recently been markers, including HPV DNA and p16, p53, and EGFR associated with a subset of head and neck cancers.12-14 expression in a large series of oropharyngeal cancer in Oropharyngeal cancer has emerged as another mucosal an area of betel nut chewing such as Taiwan. For clini- cancer that is associated with HPV infection.13 Klussmann cians, a combined immunohistochemical study for p16, et al15 reported HPV DNA to be present in 20% of all p53, and EGFR expression and detection of HPV DNA HNSSCs and in nearly 60% of tonsillar cancers. In con- are easily applicable in a routine pathology laboratory, trast, our study showed that only 12.6% of tonsillar can- and it can provide invaluable prognostic information cers were HPV positive in a betel nut–chewing area. for individual patients with more relevance than staging Patients with HPV tend to be younger than those with- alone. out the virus.16 This finding may be the result of high- risk sexual behaviors that are more common among younger patients. However, our study was inconsistent Submitted for Publication: May 26, 2009; final revision in this observation because our patients showed a higher received October 23, 2009; accepted November 25, 2009. percentage of betel nut chewing and represented a mean Correspondence: Chih-Yen Chien, MD, Department of age of approximately 50 years. In addition, the most com- Otolaryngology, Chang Gung Memorial Hospital- monly detected HPV in HNSCC is HPV-16, which rep- Kaohsiung Medical Center, 123 Ta-Pei Road, Niao-Song resents 90% to 95% of all HPV-positive HNSCC cases.17,18 Hsiang, Kaohsiung County, 833 Taiwan (cychien3965 In the present series, the prevalence rate of HPV was low @adm.cgmh.org.tw). in oropharyngeal cancer, but HPV-16 was still shown to Author Contributions: Drs Al-Swiahb and C.-C. Huang be the dominant type. In addition, the presence of HPV contributed equally to this study. Drs Al-Swiahb, C.-C. in the tumor nuclei was more likely to be found among Huang, and Chien had full access to all the data in the patients with metastasis to the neck in this study. The study and take responsibility for the integrity of the data reason is unclear, but more studies are necessary to ex- and the accuracy of the data analysis. Study concept and plore this answer. design: Al-Swiahb, C.-C. Huang, and Chien. Acquisition Patients with HPV-positive oropharyngeal cancer re- of data: Al-Swiahb, C.-C. Huang, Fang, Chuang, and Luo. sponded well to treatment and showed good prog- Analysis and interpretation of data: C.-C. Huang, Chuang, noses.18,19 Absence of p16 mutation and high expression H.-Y. Huang, Luo, C.-H. Chen, and C.-M. Chen. Draft- of p16 are also distinguishing molecular markers of HPV- ing of the manuscript: Al-Swiahb and Chien. Critical re- positive HNSCC.20 Therefore, it has been suggested that vision of the manuscript for important intellectual content: high expression of p16 may be used as a surrogate marker C.-C. Huang, Fang, Chuang, H.-Y. Huang, Luo, C.-H. to identify HPV-positive HNSCC. In this study, strong Chen, C.-M. Chen, and Chien. Statistical analysis: Fang. expression of p16 was significantly associated with HPV- Administrative, technical, and material support: Al- positive tumors and with better clinical outcomes. These Swiahb, C.-C. Huang, Chuang, H.-Y. Huang, Luo, C.-H. findings are consistent with other studies.5 In HNSCC, Chen, C.-M. Chen, and Chien. Study supervision: Chien. p53 is frequently mutated,21 overexpressed,22 and asso- Financial Disclosure: None reported. ciated with poorer prognosis.23 In contrast, in the present Funding/Support: This study was supported by grant series, low expression of p53 and the presence of HPV CMRPG860512 from the Chang Gung Memorial Hos- in tumors were associated with better prognosis. Gen- pital. erally, p53 is inactivated by HPV E6 in an HPV- Previous Presentation: This study was presented at the positive tumor, which may be the reason for low expres- Asia-Pacific Congress on Oral Cavity Cancer in Con- sion of p53 in the tumor cells. junction with the 12th Annual Meeting of the Taiwan Co- Growth receptors that are normally expressed at lower operative Oncology Group; December 6, 2008; Taipei, rates in most epithelial cells, in particular EGFR, have Taiwan. attracted attention owing to their inherent ability to stimu- late the proliferation of epithelial cells.24 In HNSCC, EGFR REFERENCES is usually overexpressed and is related to poor progno- sis.5,25 Our data showed results consistent with this. In 1. Ha PK, Califano JA. The role of human papillomavirus in oral carcinogenesis. the present series, the molecular explanation for the in- Crit Rev Oral Biol Med. 2004;15(4):188-196. 2. Gillison ML. 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Call for Papers

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