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Human papillomavirus in tonsillectomy specimen from China and Pakistan — Prevalence and genotype distribution
Accepted Manuscript Title: Human papillomavirus in tonsillectomy specimen from China and Pakistan — Prevalence and genotype distribution Authors: Hasan Mujtaba, Ying Wang, Yixin Duan, Meng Cao, Nana Zhang, Iffat Batool, Ali Murtaza, Xiaoli Chen, Yili Wang PII: DOI: Reference:
S0344-0338(18)30835-5 https://doi.org/10.1016/j.prp.2018.08.032 PRP 52175
To appear in: Received date: Revised date: Accepted date:
5-7-2018 17-8-2018 28-8-2018
Please cite this article as: Mujtaba H, Wang Y, Duan Y, Cao M, Zhang N, Batool I, Murtaza A, Chen X, Wang Y, Human papillomavirus in tonsillectomy specimen from China and Pakistan — Prevalence and genotype distribution, Pathology - Research and Practice (2018), https://doi.org/10.1016/j.prp.2018.08.032 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Human papillomavirus in tonsillectomy specimen from China and Pakistan – Prevalence and genotype distribution.
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Ridhima Charles Data Administrator Elsevier. Subject : List of Authors, their affiliation for the article PRP 52175.
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You can find the list of authors and affiliation for the said article in the attached world file and also herein the mail. Secondly, kindly make sure that we have two corresponding authors for our article which is marked by asterisk (*) and also stated under the heading “Corresponding authors”.
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Our reference: PRP 52175 Article reference: PRP_2018_755 Article title: Human papillomavirus in tonsillectomy specimen from China and PakistanPrevalence, genotype characterization and geographical variation To be published in: Pathology - Research and Practice
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“Human papillomavirus in tonsillectomy specimen from China and Pakistan – Prevalence, genotype characterization and geographical variation”.
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Hasan Mujtaba a, Ying Wang a, Yixin Duan a , Meng Cao a , Nana Zhang a, Iffat Batool b, Ali Murtaza c , *Xiaoli Chen b , *Yili Wang a Institute for Cancer Research, School of Basic Medical Science, Xi’an Jiaotong University, 710061 Xi’an, Shaanxi, China
c
Consultant Pathologist, Ali Laboratory, Rawalpindi, Pakistan Department of Pathology, Second Affiliated Hospital, Xi’an Jiaotong University, Xian, China
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b
Department of Pediatrics, Fauji Foundation Hospital, Rawalpindi, Pakistan
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b
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a
email of authors in order: [email protected] [email protected] [email protected] [email protected] 1
[email protected] [email protected] [email protected]
Corresponding authors (*)
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Xiaoli Chen Department of Pathology, Second Affiliated Hospital, Xi’an Jiaotong University, Xian, China [email protected] Yili Wang [email protected]
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Institute for Cancer Research, School of Basic Medical Science, Xi’an Jiaotong University, 710061 Xi’an, Shaanxi, China
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Regards
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Wang Yili.
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Abstract
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Background: The expected corresponding increase in tonsillar human papillomavirus (HPV) infection associated with increasing incidence of oropharyngeal squamous cell carcinoma
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(OPSCC) substantiate the evaluation of normal tonsillar tissue in different population. The
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epidemiology of HPV in tonsillar tissue varies geographically . This study evaluated samples from two countries to determine the prevalence in respective samples.
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Objective: To characterize HPV infection in non-malignant tonsillar tissue from Shaanxi, China (herein after referred to as China) and Punjab, Pakistan (herein after referred to as Pakistan). Methodology: The DNA extracted from formalin-fixed, paraffin-embedded tumor free tonsillar tissue was evaluated using polymerase chain reaction (PCR). A total of 367 cases from China
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and 139 cases from Pakistan were screened for HPV DNA using GP5+/GP6+ consensus primer. Genotype of the positive cases was determined for common HPV types (6, 11, 16, 18, 52, 58) simultaneously by type-specific PCR.
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Results: The mean age of cohorts in China was 13.42 (Median age 7, Range 2-72 years) while in Pakistan it was 10.77 (Median age 8, Range 3-42 years) the gender distribution was 61.6% male in China and in Pakistan they were 56.8%, rest were females. The overall prevalence of HPV in China was 2.45% and 2.16% in Pakistan. High risk human papillomavirus was 1.63% in China with 5 cases positive for HPV 16 and HPV 58 in 1 case. In Pakistan, 2 cases (1.43%) of HPV 16
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were detected. Low-risk types include HPV 11 present in 2 cases from China, while HPV 6 was
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detected in 1 case each from both the countries.
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Conclusion: A low prevalence of HPV was found in China and Pakistan; high-risk and low-risk HPV were detectable in tonsillar tissue from both countries. Age stratification (< 5 years, 5-14
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years, 15-25 years, > 25 years) suggest that sexual and non-sexual transmission of the virus can
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occur. The difference in the genotype distribution geographically within China and with Pakistan
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was observed in the case of HPV 58. The most common type in both the countries was HPV 16. Keywords: Human papillomavirus, tonsils, polymerase chain reaction, oropharyngeal squamous
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cell carcinoma, China, Pakistan
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Introduction
The 225% incidence increase of human papillomavirus (HPV) positive oropharyngeal squamous cell carcinoma (OPSSC) in the past two decades from developed countries is disconcerting [1,2]. It is estimated that after 2020, the incidence will surpass cases of viral-mediated cervical cancer
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in the United States [1]. Almost 70% of OPSCC patients are HPV-positive, amongst them HPV 16 is involved in 90% of the cases [3]. HPV are highly transmissible and primarily transferred in sexually active individuals [4]. Rising
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incidence of HPV-positive OPSCC indicates that the virus has a favorable tropism for this site. It is due to tonsillar reticulated crypts having porous basement membranes and exposed basal cells, hence a susceptible site for primary infection [5–8]. Once these cells are infected, the virus attains its productive life cycle and is dependent on the epithelial maturation [4]. While the viral carcinogenicity is the activity of high-risk type E6 and E7 genes which deactivate cellular p53
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and pRB tumor suppressor genes [4,9].
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Generally, a natural immune response clears HPV infection in 90% of cases in a period of 2
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years. Those who are unable to clear it are at risk of developing cancer [10]. Persistent infection for a period of 10-20 years can ultimately lead to cancer [9]. In OPSCC patients, detection of
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antibodies against E6 appears 10 years before the diagnosis of cancer, suggest that a long
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duration of infection is necessary for malignant transformation [11]. However, in the cervical region where HPV is frequently detectable and the precancerous lesions develop, tonsillar tissue
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acts otherwise with a low rate of detectable HPV, ill-defined natural history and no precancerous
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lesion [4]. It is also reported that oral lesion rarely present HPV [12]. Indicating that much is still unknown about the natural history of this virus [13].
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Incidence of HPV-driven OPSCC indicates that similar patterns of increased infection from the virus in normal tonsils should be detectable. Sexual behavior including oral sex is considered a significant contributor of HPV transmission in the oral cavity [14,15]. Though vertical, horizontal and auto-inoculation of HPV are also possible but not considered a major contributor
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[16-18]. Other risk factors associated with oral HPV infection are the male gender, the number of sexual partners, sexual orientation and tobacco [15]. In tonsils limited studies struggle to provide consistent results of HPV prevalence. The
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discrepancy in the prevalence different countries are primarily attributed to geographical variation, social context and the method of HPV detection. We collected the tonsillectomy specimen from two countries and determined the status in respective cohort. The objectives of the study were (ⅰ) to detect HPV prevalence in Shaanxi province China (hereinafter referred to as China) and Punjab, Pakistan (hereinafter referred to as Pakistan), countries with different
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geographical and anthropological background. (ⅱ) to determine the associated genotype with
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type specific polymerase chain reaction (PCR).
Specimen Collection and study design
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Materials and Methods
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After approval from the Institute Review Board, the study was conducted at Xi’an Jiaotong University School of Medicine in China. The Institutional Research Ethics committee, China and
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National Bioethical Committee, Pakistan granted the formal approval of the study. Formalin-fixed paraffin-embedded (FFPE) tonsillar tissues were collected from the archive of
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the laboratory at the Second Affiliated Hospital, Xi’an, China and the Ali Laboratory,
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Rawalpindi, Pakistan. Patients from both the countries had undergone tonsillectomy procedure between January 2013 and February 2018. Inclusion criteria were routine tonsillectomy of patients due to hypertrophy, chronic tonsillitis or sleep-disordered breathing. Exclusion criteria include the patients presented with/or clinical
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suspected case of oropharyngeal malignancy and immunosuppression. Patients were deidentified and only data pertaining to age and gender were collected from clinical histories in each case. DNA Extraction, PCR and Electrophoresis
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A total of 399 samples from China and 153 from Pakistan were included in the study. FFPE tissue was subjected to microtomy. To prevent cross-contamination the blades of the microtome were changed between each paraffin block. Fifteen sections of 5 µm thickness were subjected to deparaffinization with xylene. Rehydration of the tissue with graded ethanol was carried out.
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Genomic DNA extraction was performed by following the standard instructions in the TIANamp Genomic DNA kit, Tiangen. The quality of extracted DNA and its ability to amplify was verified
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by PCR amplification and DNA electrophoresis of the human β-actin housekeeping gene.
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HPV screening was done by Gp5+/GP6+ consensus primers under standard conditions [19]. Briefly, the PCR reaction was performed in a final of 25µl reaction containing 4µl of genomic
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DNA (~50 ng), 2µl of each primer, 12.5 µl 2x Taq PCR MasterMix Tiangen, Beijing, China
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[(0.1 U/µl) Taq polymerase, 0.5 mM each dNTP, 20mM Tris-HCl [pH 8.3], 100 mM KCl, 3mM
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MgCl2] and 4.5 µl of water. Positive (Caski DNA) and negative (DEPC water) controls were run alongside the samples. Using the described thermocycler setting for the consensus primer,
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reactions were carried out in a total of 35 cycles [19]. HPV genotyping was performed by typespecific primers, sequence generated by retrieving DNA files for HPV type 6, 11, 16, 18, 52 and
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58 from Genbank (https://www.ncbi.nlm.nih.gov/genbank/). DNA from Hela cells was used as a positive control for HPV 18 while purified plasmid DNA of HPV (6, 11, 52, 58) were used as a positive control for respective genotype. Primers for each HPV type were designed and specificity confirmed by BLAST analysis. The sequence of the primers is shown in Table 1.
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After amplification, the samples were subjected to electrophoresis on 2% agarose gel stained with ethidium bromide. Band sizes were compared against a 100bp molecular marker. Photographic records were taken from a UV light transilluminator.
HPV 11 E7 HPV 16 E7 HPV 18 E7 HPV 52 E7
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HPV 58 E7
Primer Position 1083/1084 1247/1248 467/468 696/697 362/363 531/532 136/137 261/262 695/696 780/781 690/691 776/777 671/672 761/762
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HPV 6 E7
Primer sequence (5’) FP- TGGCACCCAGCACAATGAA RP-AGTCATAGTCCGCCTAGAAG FP-GACCTGCAACCTCCAGACCCTG RP-TCGGTGCGCAGATGGGACACAC FP-GGGAAAGGCACGCTTCATAAA RP-TGTCCACCTTGTCCACCTCATC FP-AACCGGACAGAGCCCATTAC RP-AGATGGGGCACACAATTCCT FP- AAGAAAACGATGAAATAGATGGA RP- GGCTTCACACTTACAACACA FP- GCAGAACAAGCCACAAGCAA RP- TAGAGTACGAAGGTCCGTCG FP-CGAGGATGAAATAGGCTTGG RP-ACACAAACGAACCGTGGTGC
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Primers β-actin
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Table 1: Primer sequences for HPV genotyping
AT(°C) 56
Product length (bp) 183
54
250
56
190
56
144
56
104
54
105
54
109
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AT-Annealing temperature FP-Forward Primer RP-Reverse Primer
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Statistical Analysis
Statistical analysis was performed using SPSS software version 18. Descriptive analysis of the
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data was carried out and determination of means, standard deviation and p-value were performed. χ2 test was used to determine the association of HPV positivity with age, gender and
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nationality. Results
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The β-actin housekeeping gene was successfully amplified in 367 (92%) cases from China and 139 (90%) cases from Pakistan. Overall the male subjects in China were 61.6% and in Pakistan 56.8%, rest were females. We found both high-risk HPV (HR-HPV) and low-risk HPV (LRHPV) in the study cohort of respective countries with an overall prevalence of 2.45% in China and 2.16% in Pakistan. Amongst the positive cases detected by consensus primer no sample was 7
presented with unknown or multiple HPV types. The positive cases were simultaneously screened for the HPV genotypes which include HPV (6, 11, 16, 18, 52, 58).The prevalence of HR-HPV in China was 1.63% (6 cases), which include 5 cases positive for HPV 16 and 1 case
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positive for HPV 58. The age-group distribution of positive HR-HPV cases were, 1 case of HPV 16 in less than 5 years age group, 1 case (HPV 16) in pre-puberty (5-14 years), 1 case of HPV 16 in the 15-25 years age group while in greater than 25 years 3 cases were positive (2 cases of HPV 16 and 1 cases of HPV 58). LR-HPV was detected in a total of 3 cases, 1 case each of HPV 6 and HPV 11 in 5-14 years age group and 1 cases of HPV 11 in greater than 25 years age group.
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In Pakistan, HPV 16 was the only detectable high-risk type with 2 (1.43%) cases, 1 in pre-
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puberty and 1 in 15-25 years age group. HPV 6 was the low-risk type detected in 5-14 years age
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group.
Overall prevalence of HPV in both the countries seem to increase in aging groups: 1.10% (< 5
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years), 1.68% (5-14 years), 2.56% (15-25 years), 6.33% (> 25 years).
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HPV-positive status was not associated with age, gender and nationality of the subjects in our
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study cohort, determined by using χ2 test and with statistical significance limit set at P < 0.05. Characteristics of the study subjects along with HPV status are given in Table 2, while the
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stratification of HPV genotypes with age groups and country are given in Table 3. Table 4
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represent the characteristics of positive cases.
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Table 2: Characteristics of study subjects.
Number of Patients
China n (%)
Pakistan n (%)
Total N (%)
367 (72.5)
139 (27.5)
506 (100)
226 (61.6) 141 (38.4)
79 (56.8) 60 (43.2)
305 (60.3) 201 (39.7)
80 (21.8) 194 (52.9) 25 (6.8) 68 (18.5)
11 (7.9) 103 (74.1) 14 (10.1) 11 (7.9)
91 (17.9) 296 (58.5) 41 (8.1) 78 (15.4)
3 (1.3) 3 (2.1) 3 (1.3) 0 (0)
1 (1.2) 1 (1.6) 1 (1.2) 0 (0)
4 (1.3) 4 (1.9) 4 (1.3) 0 (0)
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Parameter
Male Female Age distribution (years) < 5 Preschool 5-14 School going 15-25 Young adults >25 Midlife and above
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HPV status
HR-HPV in Male HR-HPV in Female LR-HPV in Male LR-HPV in Female
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Gender
Age Group
Less than 5
Country
China
Total Country
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Country
Country
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25
Total
Total
1
1
79
80
0
0
11
11
1
1
90
91
1
3
191
194
Pakistan
1
0
1
2
101
103
2
1
2
5
292
297
China
1
1
24
25
Pakistan
0
0
14
14
1
1
38
39
China
1
2
1
4
64
68
Pakistan
0
1
0
1
10
11
1
3
1
5
74
79
Total Country
Total
1
Total
Greater than
Overall HPV Positive Negative
1
Total
15-25
HPV 58
China
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5-14
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Pakistan
HPV Genotype HPV 11 HPV 16
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HPV 6
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Table 3: Stratification of HPV Genotypes with Age groups and Country.
China
1
2
5
1
9
358
367
Pakistan
1
0
2
0
3
136
139
2
2
7
1
12
494
506
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Age Group
Gender
Country
Genotype
1
3
1
F
China
HR-HPV 16
2
9
2
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China
LR-HPV 11
3
13
2
M
China
LR-HPV 6
4
14
2
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China
HR-HPV 16
5
24
3
F
China
HR-HPV 16
6
27
4
F
China
HR-HPV 16
7
29
4
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China
HR-HPV 16
8
33
4
M
China
HR-HPV 58
9
41
4
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China
LR-HPV 11
10
7
2
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Pakistan
HR-HPV 16
11
12
2
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Pakistan
LR-HPV 6
12
31
4
F
Pakistan
HR-HPV 16
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Age
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N
S. No
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Table 4: Characteristics of Positive cases
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Discussion
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OPSCC is multifactorial disease and HPV positive cancer are now being considered as distinct
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subset which are etiologically driven by the virus. In head and neck squamous cell carcinoma oropharyngeal cancers depict the highest percentage of HPV positivity and genotype detected
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commonly is HPV16 [3]. Increasing incidence of HPV positive OPSCC from the developed
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countries [2,20], imperatively demands the status of virus to be determined in normal tonsils among different population. Thus, we collected tonsillectomy sample which are appropriate to
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detect HPV.
In the tonsils from both countries the most common type was HR-HPV 16, this genotype was present across all the age groups in China. Another high-risk genotype detected in the samples from China was HPV58 present in adult male, this genotype is prevalent in cervical lesions within Shaanxi, China [21]. The presence of HPV 58 in benign tonsils has only been reported 10
from Korea previously [8]. Though individual sexual behavior was not considered in our study, non-sexual or sexual transmission could be involved. Vertical transmission of HPV has been reported in new born [16-18], conclusive evidence for horizontal transmission are still lacking
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[22], while oral sex is the major factor for transmission in the adults [14,15]. In Chinese pediatric tonsillectomy specimen HPV status have been reported by two studies. A study from Sun et al. in Zhejiang detected 2 (1%) LR-HPV in 177 cases while the study from Xue et al. in the autonomous region of Ningxia detected no HPV in 146 subjects [23,24]. In Shaanxi, we were able to detect both high-risk (HPV 16) and low-risk (HPV 6 and HPV 11)
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viruses in the pediatric group.
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In Pakistan, HPV 16 was the only detectable high-risk type with 1 case each in the pediatric and
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adult populations. One case of LR-HPV 6 was presented in 5-14 years age group. Significant variability in the HPV prevalence have been observed across Ningxia (having significant muslim
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population from China), reporting 0% [23] and Turkey 6.6% [25]. Despite religious similarities,
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variable HPV prevalence is observed in Pakistan, Ningxia and Turkey.
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In discordant literature, the prevalence of HPV in tonsillar tissue ranged from 0-12.5% with a total of 91 (1.42%) detectable cases from 6400 individuals (for detailed analysis see Table 5).
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Though the exact parameters in these studies are not comparable because of inherent differences in the studies as some studies only detected high-risk type others screened pediatric or adult
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subjects only. Our findings (2.45% in China and 2.16% in Pakistan) are close to the pooled prevalence of HPV (1.42%) from previous studies. HPV infection detected in the mouth through oral rinse and gargle was 4.5% in a systemic review of 4,600 individual and it ranged from 0.2-20.7% [26]. Our combined findings from
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China and Pakistan for the adult group (> 15 years) show 6 (5.08%) positive cases from a total of 118 subjects, hence in accordance with the rinse and gargle technique [26]. Another technique to determine HPV is through oral swabs and cyto-brush which is reported to provide insufficient
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DNA in the tonsils [27], but in the oral cavity efficient demonstration of HPV DNA was reported as 3.12% [28].
To screen HPV, multiple assay choices are available which has resulted in controversy [29]. Considering the paraffin-embedded tissue with fragmented DNA, we simply selected GP5+/GP6+ consensus primers for the study. It should be able to amplify the smallest region of
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150bp in the L1 region of the HPV genome even in the fragmented case.
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HPV infection may be transient or persistent, infectivity also depends on genotype, site of
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involvement and gender. It is observed that HPV 16 is slowest to be cleared in men [30]. While, the infection in the genital skin of male is weaker than the epithelium of cervix and vagina [31].
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It seems that susceptible tonsillar epithelium is strongly protected by the immune response
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underneath and virus itself in the tonsils are suppressed to a minimum level and mostly cleared
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within a period of one year [32,33].
Other factors responsible for the varied HPV prevalence reported in studies include difference in
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population studied, choice of assay, sampling technique and evolving sexual orientation. Our study has one of the biggest sample size and we evaluated total of 506 samples from two
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different regions. The limitations of study were that we could not differentiate between nonsexual or sexual-transmission, duration of infection and between transient or persistent infection. Secondly, majority of individuals in our study are children (<14 years): China 75%: Pakistan 82% thus relevance of the data to sexually active adults is reduced.
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The U.S Food and Drug Administration (FDA) has approved PCR consensus and type specific primers for virus detection but all the HPV detection assay have some pros and cons. It is recommended that quantitative DNA test with transcription detection are patho-physiologically
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more relevant [34] Additional studies with improved design using recently developed assay such as RT-qPCR or ddPCR should be conducted. These assays can demonstrate the presence of viral transcripts and viral loads quantification and could be helpful in unfolding the unknowns of HPV. Lastly in China and Pakistan there is no country wide vaccination program against HPV, immunization
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against HPV is evoking and the public acceptance for vaccination is increasing though majority
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is still unvaccinated [35,36]. Vaccination against HPV is also believed to reduce the incidence of
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OPSCC. As there is no validate technique to screen HPV mediated cancers in the oral cavity,
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vaccination seems to be the best method for prevention.
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Conclusion
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The prevalence of HPV in tonsillar tissue is low in both countries but a noteworthy proportion of
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individuals have oncogenic and non-oncogenic HPV. The vague natural history of HPV causing OPSCC without a detectable precancerous lesion in the tonsils emphasize the importance of
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vaccination against HPV as the most efficient way to deal with the transmission chains and reduce endemicity. Further studies are necessary to address the increasing incidence of HPV-
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mediated carcinogenesis to clarify the role of viral, host and behavioral variables.
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Table 5 : Comparison of studies examining HPV in tonsils.
Tissue type
Assay
HPV-positive / Sample size (%)
Type Identified
Strome et al. 2002 [37] United States
Adults
FFPE
Southern Blot
3/48 (6.25)
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Syrjänen 2004 [38] literature review*
Children and adults
Varies
PCR, ISH, Southern Blot
17/200 (8.5)
6,11,16
Chen et al. 2005 [39] Finland
Children and adults
Fresh frozen
Nested PCR (MY09/11 and GP5+/GP6+)
13/206 (6.3)
16
Ribeiro et al. 2006 [40] Brazil
2-13 years
Fresh frozen
PCR MY09/11
0/100 (0)
None
Sisk et al. 2006 [41] United States
2-18 years
Fresh frozen
MY 09/11
2/50 (4)
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Mammas et al. 2006 [42] Greece
2-14 years
FFPE
PCR GP5+/GP6+
9/106 (8.5)
16
N/S
FFPE
Consensus Primer (N/S)
8/69 (11.6)
16, 58
5-21 years
FFPE
PCR MY/GP
16/252 (6.3)
6, 11, 16, 31
Ernster et al. 2009 [43] United States
Greater than 21 years
FFPE
Type specific PCR for HPV 16 & 18)
0/226 (0)
None
Duray et al. 2011[44] Belgium
Children and adults
FFPE
PCR GP5+/GP6+
10/80 (12.5)
16, 18, 31
Sun et al. 2012 [24] China
Children
FFPE
PCR (N/S)
2/177 (1)
6,11
Palmer et al. 2014 [45] United Kingdom
Children and adults
FFPE & Fresh homogenized
PCR GP5+/GP6+
0/3377 (0) & 0/511 (0)
None
Xue et al. 2014 [22] China
3-12 years
FFPE
qPCR MY09/11
0/146 (0)
None
Rusan et al. 2015 [46] Denmark
8-30 years
Fresh frozen
Nested PCR (MY 09/11 and GP5+/GP6+)
1/80 (1.25)
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Cockerill et al. 2016 [5] United States
Children and adults
Fresh Unfrozen and swab
Holgic Aptima test & Roche Cobas test
0/193 (0)
None
Ilmarinen et al. 2017 [47] Finland
Children and adults
Fresh frozen
Luminex xMAP
5/477 (1.05)
16, 52, 66, 68
Rieth et al. 2018 [48] United States
Adults
FFPE
Nested PCR (MY09/11 and GP5+/GP6+)
5/102(4.9)
16,18
A
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Baloglu et al. 2010 [25] Turkey
ED
Kim et al. 2007 [8] South Korea
HPV Human papillomavirus
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Age Range
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Author / City
* Compiled small studies
N/S Not specified
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Abbreviations HPV: human papillomavirus; OPSCC: oropharyngeal squamous cell carcinoma; PCR: polymerase chain reaction
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Conflict of Interest The authors declare no conflict of interest Acknowledgements
This study was funded by the Shaanxi Provincial Innovation Foundation (No. 2011KTCL03-18).
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We are grateful to Ding Haiyan and Mr. Guo Shangwen for the tissue sections and assistance
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throughout the research work.
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S0344-0338(18)30835-5 https://doi.org/10.1016/j.prp.2018.08.032 PRP 52175
To appear in: Received date: Revised date: Accepted date:
5-7-2018 17-8-2018 28-8-2018
Please cite this article as: Mujtaba H, Wang Y, Duan Y, Cao M, Zhang N, Batool I, Murtaza A, Chen X, Wang Y, Human papillomavirus in tonsillectomy specimen from China and Pakistan — Prevalence and genotype distribution, Pathology - Research and Practice (2018), https://doi.org/10.1016/j.prp.2018.08.032 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Human papillomavirus in tonsillectomy specimen from China and Pakistan – Prevalence and genotype distribution.
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Ridhima Charles Data Administrator Elsevier. Subject : List of Authors, their affiliation for the article PRP 52175.
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You can find the list of authors and affiliation for the said article in the attached world file and also herein the mail. Secondly, kindly make sure that we have two corresponding authors for our article which is marked by asterisk (*) and also stated under the heading “Corresponding authors”.
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Our reference: PRP 52175 Article reference: PRP_2018_755 Article title: Human papillomavirus in tonsillectomy specimen from China and PakistanPrevalence, genotype characterization and geographical variation To be published in: Pathology - Research and Practice
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“Human papillomavirus in tonsillectomy specimen from China and Pakistan – Prevalence, genotype characterization and geographical variation”.
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Hasan Mujtaba a, Ying Wang a, Yixin Duan a , Meng Cao a , Nana Zhang a, Iffat Batool b, Ali Murtaza c , *Xiaoli Chen b , *Yili Wang a Institute for Cancer Research, School of Basic Medical Science, Xi’an Jiaotong University, 710061 Xi’an, Shaanxi, China
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Consultant Pathologist, Ali Laboratory, Rawalpindi, Pakistan Department of Pathology, Second Affiliated Hospital, Xi’an Jiaotong University, Xian, China
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Department of Pediatrics, Fauji Foundation Hospital, Rawalpindi, Pakistan
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b
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email of authors in order: [email protected] [email protected] [email protected] [email protected] 1
[email protected] [email protected] [email protected]
Corresponding authors (*)
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Xiaoli Chen Department of Pathology, Second Affiliated Hospital, Xi’an Jiaotong University, Xian, China [email protected] Yili Wang [email protected]
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Institute for Cancer Research, School of Basic Medical Science, Xi’an Jiaotong University, 710061 Xi’an, Shaanxi, China
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Regards
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Wang Yili.
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Abstract
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Background: The expected corresponding increase in tonsillar human papillomavirus (HPV) infection associated with increasing incidence of oropharyngeal squamous cell carcinoma
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(OPSCC) substantiate the evaluation of normal tonsillar tissue in different population. The
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epidemiology of HPV in tonsillar tissue varies geographically . This study evaluated samples from two countries to determine the prevalence in respective samples.
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Objective: To characterize HPV infection in non-malignant tonsillar tissue from Shaanxi, China (herein after referred to as China) and Punjab, Pakistan (herein after referred to as Pakistan). Methodology: The DNA extracted from formalin-fixed, paraffin-embedded tumor free tonsillar tissue was evaluated using polymerase chain reaction (PCR). A total of 367 cases from China
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and 139 cases from Pakistan were screened for HPV DNA using GP5+/GP6+ consensus primer. Genotype of the positive cases was determined for common HPV types (6, 11, 16, 18, 52, 58) simultaneously by type-specific PCR.
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Results: The mean age of cohorts in China was 13.42 (Median age 7, Range 2-72 years) while in Pakistan it was 10.77 (Median age 8, Range 3-42 years) the gender distribution was 61.6% male in China and in Pakistan they were 56.8%, rest were females. The overall prevalence of HPV in China was 2.45% and 2.16% in Pakistan. High risk human papillomavirus was 1.63% in China with 5 cases positive for HPV 16 and HPV 58 in 1 case. In Pakistan, 2 cases (1.43%) of HPV 16
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were detected. Low-risk types include HPV 11 present in 2 cases from China, while HPV 6 was
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detected in 1 case each from both the countries.
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Conclusion: A low prevalence of HPV was found in China and Pakistan; high-risk and low-risk HPV were detectable in tonsillar tissue from both countries. Age stratification (< 5 years, 5-14
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years, 15-25 years, > 25 years) suggest that sexual and non-sexual transmission of the virus can
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occur. The difference in the genotype distribution geographically within China and with Pakistan
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was observed in the case of HPV 58. The most common type in both the countries was HPV 16. Keywords: Human papillomavirus, tonsils, polymerase chain reaction, oropharyngeal squamous
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cell carcinoma, China, Pakistan
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Introduction
The 225% incidence increase of human papillomavirus (HPV) positive oropharyngeal squamous cell carcinoma (OPSSC) in the past two decades from developed countries is disconcerting [1,2]. It is estimated that after 2020, the incidence will surpass cases of viral-mediated cervical cancer
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in the United States [1]. Almost 70% of OPSCC patients are HPV-positive, amongst them HPV 16 is involved in 90% of the cases [3]. HPV are highly transmissible and primarily transferred in sexually active individuals [4]. Rising
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incidence of HPV-positive OPSCC indicates that the virus has a favorable tropism for this site. It is due to tonsillar reticulated crypts having porous basement membranes and exposed basal cells, hence a susceptible site for primary infection [5–8]. Once these cells are infected, the virus attains its productive life cycle and is dependent on the epithelial maturation [4]. While the viral carcinogenicity is the activity of high-risk type E6 and E7 genes which deactivate cellular p53
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and pRB tumor suppressor genes [4,9].
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Generally, a natural immune response clears HPV infection in 90% of cases in a period of 2
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years. Those who are unable to clear it are at risk of developing cancer [10]. Persistent infection for a period of 10-20 years can ultimately lead to cancer [9]. In OPSCC patients, detection of
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antibodies against E6 appears 10 years before the diagnosis of cancer, suggest that a long
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duration of infection is necessary for malignant transformation [11]. However, in the cervical region where HPV is frequently detectable and the precancerous lesions develop, tonsillar tissue
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acts otherwise with a low rate of detectable HPV, ill-defined natural history and no precancerous
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lesion [4]. It is also reported that oral lesion rarely present HPV [12]. Indicating that much is still unknown about the natural history of this virus [13].
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Incidence of HPV-driven OPSCC indicates that similar patterns of increased infection from the virus in normal tonsils should be detectable. Sexual behavior including oral sex is considered a significant contributor of HPV transmission in the oral cavity [14,15]. Though vertical, horizontal and auto-inoculation of HPV are also possible but not considered a major contributor
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[16-18]. Other risk factors associated with oral HPV infection are the male gender, the number of sexual partners, sexual orientation and tobacco [15]. In tonsils limited studies struggle to provide consistent results of HPV prevalence. The
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discrepancy in the prevalence different countries are primarily attributed to geographical variation, social context and the method of HPV detection. We collected the tonsillectomy specimen from two countries and determined the status in respective cohort. The objectives of the study were (ⅰ) to detect HPV prevalence in Shaanxi province China (hereinafter referred to as China) and Punjab, Pakistan (hereinafter referred to as Pakistan), countries with different
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geographical and anthropological background. (ⅱ) to determine the associated genotype with
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type specific polymerase chain reaction (PCR).
Specimen Collection and study design
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Materials and Methods
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After approval from the Institute Review Board, the study was conducted at Xi’an Jiaotong University School of Medicine in China. The Institutional Research Ethics committee, China and
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National Bioethical Committee, Pakistan granted the formal approval of the study. Formalin-fixed paraffin-embedded (FFPE) tonsillar tissues were collected from the archive of
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the laboratory at the Second Affiliated Hospital, Xi’an, China and the Ali Laboratory,
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Rawalpindi, Pakistan. Patients from both the countries had undergone tonsillectomy procedure between January 2013 and February 2018. Inclusion criteria were routine tonsillectomy of patients due to hypertrophy, chronic tonsillitis or sleep-disordered breathing. Exclusion criteria include the patients presented with/or clinical
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suspected case of oropharyngeal malignancy and immunosuppression. Patients were deidentified and only data pertaining to age and gender were collected from clinical histories in each case. DNA Extraction, PCR and Electrophoresis
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A total of 399 samples from China and 153 from Pakistan were included in the study. FFPE tissue was subjected to microtomy. To prevent cross-contamination the blades of the microtome were changed between each paraffin block. Fifteen sections of 5 µm thickness were subjected to deparaffinization with xylene. Rehydration of the tissue with graded ethanol was carried out.
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Genomic DNA extraction was performed by following the standard instructions in the TIANamp Genomic DNA kit, Tiangen. The quality of extracted DNA and its ability to amplify was verified
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by PCR amplification and DNA electrophoresis of the human β-actin housekeeping gene.
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HPV screening was done by Gp5+/GP6+ consensus primers under standard conditions [19]. Briefly, the PCR reaction was performed in a final of 25µl reaction containing 4µl of genomic
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DNA (~50 ng), 2µl of each primer, 12.5 µl 2x Taq PCR MasterMix Tiangen, Beijing, China
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[(0.1 U/µl) Taq polymerase, 0.5 mM each dNTP, 20mM Tris-HCl [pH 8.3], 100 mM KCl, 3mM
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MgCl2] and 4.5 µl of water. Positive (Caski DNA) and negative (DEPC water) controls were run alongside the samples. Using the described thermocycler setting for the consensus primer,
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reactions were carried out in a total of 35 cycles [19]. HPV genotyping was performed by typespecific primers, sequence generated by retrieving DNA files for HPV type 6, 11, 16, 18, 52 and
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58 from Genbank (https://www.ncbi.nlm.nih.gov/genbank/). DNA from Hela cells was used as a positive control for HPV 18 while purified plasmid DNA of HPV (6, 11, 52, 58) were used as a positive control for respective genotype. Primers for each HPV type were designed and specificity confirmed by BLAST analysis. The sequence of the primers is shown in Table 1.
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After amplification, the samples were subjected to electrophoresis on 2% agarose gel stained with ethidium bromide. Band sizes were compared against a 100bp molecular marker. Photographic records were taken from a UV light transilluminator.
HPV 11 E7 HPV 16 E7 HPV 18 E7 HPV 52 E7
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HPV 58 E7
Primer Position 1083/1084 1247/1248 467/468 696/697 362/363 531/532 136/137 261/262 695/696 780/781 690/691 776/777 671/672 761/762
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HPV 6 E7
Primer sequence (5’) FP- TGGCACCCAGCACAATGAA RP-AGTCATAGTCCGCCTAGAAG FP-GACCTGCAACCTCCAGACCCTG RP-TCGGTGCGCAGATGGGACACAC FP-GGGAAAGGCACGCTTCATAAA RP-TGTCCACCTTGTCCACCTCATC FP-AACCGGACAGAGCCCATTAC RP-AGATGGGGCACACAATTCCT FP- AAGAAAACGATGAAATAGATGGA RP- GGCTTCACACTTACAACACA FP- GCAGAACAAGCCACAAGCAA RP- TAGAGTACGAAGGTCCGTCG FP-CGAGGATGAAATAGGCTTGG RP-ACACAAACGAACCGTGGTGC
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Primers β-actin
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Table 1: Primer sequences for HPV genotyping
AT(°C) 56
Product length (bp) 183
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250
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190
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144
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104
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105
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AT-Annealing temperature FP-Forward Primer RP-Reverse Primer
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Statistical Analysis
Statistical analysis was performed using SPSS software version 18. Descriptive analysis of the
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data was carried out and determination of means, standard deviation and p-value were performed. χ2 test was used to determine the association of HPV positivity with age, gender and
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nationality. Results
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The β-actin housekeeping gene was successfully amplified in 367 (92%) cases from China and 139 (90%) cases from Pakistan. Overall the male subjects in China were 61.6% and in Pakistan 56.8%, rest were females. We found both high-risk HPV (HR-HPV) and low-risk HPV (LRHPV) in the study cohort of respective countries with an overall prevalence of 2.45% in China and 2.16% in Pakistan. Amongst the positive cases detected by consensus primer no sample was 7
presented with unknown or multiple HPV types. The positive cases were simultaneously screened for the HPV genotypes which include HPV (6, 11, 16, 18, 52, 58).The prevalence of HR-HPV in China was 1.63% (6 cases), which include 5 cases positive for HPV 16 and 1 case
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positive for HPV 58. The age-group distribution of positive HR-HPV cases were, 1 case of HPV 16 in less than 5 years age group, 1 case (HPV 16) in pre-puberty (5-14 years), 1 case of HPV 16 in the 15-25 years age group while in greater than 25 years 3 cases were positive (2 cases of HPV 16 and 1 cases of HPV 58). LR-HPV was detected in a total of 3 cases, 1 case each of HPV 6 and HPV 11 in 5-14 years age group and 1 cases of HPV 11 in greater than 25 years age group.
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In Pakistan, HPV 16 was the only detectable high-risk type with 2 (1.43%) cases, 1 in pre-
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puberty and 1 in 15-25 years age group. HPV 6 was the low-risk type detected in 5-14 years age
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group.
Overall prevalence of HPV in both the countries seem to increase in aging groups: 1.10% (< 5
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years), 1.68% (5-14 years), 2.56% (15-25 years), 6.33% (> 25 years).
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HPV-positive status was not associated with age, gender and nationality of the subjects in our
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study cohort, determined by using χ2 test and with statistical significance limit set at P < 0.05. Characteristics of the study subjects along with HPV status are given in Table 2, while the
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stratification of HPV genotypes with age groups and country are given in Table 3. Table 4
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represent the characteristics of positive cases.
8
Table 2: Characteristics of study subjects.
Number of Patients
China n (%)
Pakistan n (%)
Total N (%)
367 (72.5)
139 (27.5)
506 (100)
226 (61.6) 141 (38.4)
79 (56.8) 60 (43.2)
305 (60.3) 201 (39.7)
80 (21.8) 194 (52.9) 25 (6.8) 68 (18.5)
11 (7.9) 103 (74.1) 14 (10.1) 11 (7.9)
91 (17.9) 296 (58.5) 41 (8.1) 78 (15.4)
3 (1.3) 3 (2.1) 3 (1.3) 0 (0)
1 (1.2) 1 (1.6) 1 (1.2) 0 (0)
4 (1.3) 4 (1.9) 4 (1.3) 0 (0)
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Parameter
Male Female Age distribution (years) < 5 Preschool 5-14 School going 15-25 Young adults >25 Midlife and above
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N
HPV status
HR-HPV in Male HR-HPV in Female LR-HPV in Male LR-HPV in Female
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Gender
Age Group
Less than 5
Country
China
Total Country
CC
Country
Country
A
25
Total
Total
1
1
79
80
0
0
11
11
1
1
90
91
1
3
191
194
Pakistan
1
0
1
2
101
103
2
1
2
5
292
297
China
1
1
24
25
Pakistan
0
0
14
14
1
1
38
39
China
1
2
1
4
64
68
Pakistan
0
1
0
1
10
11
1
3
1
5
74
79
Total Country
Total
1
Total
Greater than
Overall HPV Positive Negative
1
Total
15-25
HPV 58
China
EP
5-14
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Pakistan
HPV Genotype HPV 11 HPV 16
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HPV 6
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Table 3: Stratification of HPV Genotypes with Age groups and Country.
China
1
2
5
1
9
358
367
Pakistan
1
0
2
0
3
136
139
2
2
7
1
12
494
506
9
Age Group
Gender
Country
Genotype
1
3
1
F
China
HR-HPV 16
2
9
2
M
China
LR-HPV 11
3
13
2
M
China
LR-HPV 6
4
14
2
M
China
HR-HPV 16
5
24
3
F
China
HR-HPV 16
6
27
4
F
China
HR-HPV 16
7
29
4
M
China
HR-HPV 16
8
33
4
M
China
HR-HPV 58
9
41
4
M
China
LR-HPV 11
10
7
2
M
Pakistan
HR-HPV 16
11
12
2
M
Pakistan
LR-HPV 6
12
31
4
F
Pakistan
HR-HPV 16
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Age
A
N
S. No
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Table 4: Characteristics of Positive cases
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Discussion
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OPSCC is multifactorial disease and HPV positive cancer are now being considered as distinct
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subset which are etiologically driven by the virus. In head and neck squamous cell carcinoma oropharyngeal cancers depict the highest percentage of HPV positivity and genotype detected
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commonly is HPV16 [3]. Increasing incidence of HPV positive OPSCC from the developed
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countries [2,20], imperatively demands the status of virus to be determined in normal tonsils among different population. Thus, we collected tonsillectomy sample which are appropriate to
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detect HPV.
In the tonsils from both countries the most common type was HR-HPV 16, this genotype was present across all the age groups in China. Another high-risk genotype detected in the samples from China was HPV58 present in adult male, this genotype is prevalent in cervical lesions within Shaanxi, China [21]. The presence of HPV 58 in benign tonsils has only been reported 10
from Korea previously [8]. Though individual sexual behavior was not considered in our study, non-sexual or sexual transmission could be involved. Vertical transmission of HPV has been reported in new born [16-18], conclusive evidence for horizontal transmission are still lacking
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[22], while oral sex is the major factor for transmission in the adults [14,15]. In Chinese pediatric tonsillectomy specimen HPV status have been reported by two studies. A study from Sun et al. in Zhejiang detected 2 (1%) LR-HPV in 177 cases while the study from Xue et al. in the autonomous region of Ningxia detected no HPV in 146 subjects [23,24]. In Shaanxi, we were able to detect both high-risk (HPV 16) and low-risk (HPV 6 and HPV 11)
N
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viruses in the pediatric group.
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In Pakistan, HPV 16 was the only detectable high-risk type with 1 case each in the pediatric and
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adult populations. One case of LR-HPV 6 was presented in 5-14 years age group. Significant variability in the HPV prevalence have been observed across Ningxia (having significant muslim
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population from China), reporting 0% [23] and Turkey 6.6% [25]. Despite religious similarities,
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variable HPV prevalence is observed in Pakistan, Ningxia and Turkey.
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In discordant literature, the prevalence of HPV in tonsillar tissue ranged from 0-12.5% with a total of 91 (1.42%) detectable cases from 6400 individuals (for detailed analysis see Table 5).
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Though the exact parameters in these studies are not comparable because of inherent differences in the studies as some studies only detected high-risk type others screened pediatric or adult
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subjects only. Our findings (2.45% in China and 2.16% in Pakistan) are close to the pooled prevalence of HPV (1.42%) from previous studies. HPV infection detected in the mouth through oral rinse and gargle was 4.5% in a systemic review of 4,600 individual and it ranged from 0.2-20.7% [26]. Our combined findings from
11
China and Pakistan for the adult group (> 15 years) show 6 (5.08%) positive cases from a total of 118 subjects, hence in accordance with the rinse and gargle technique [26]. Another technique to determine HPV is through oral swabs and cyto-brush which is reported to provide insufficient
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DNA in the tonsils [27], but in the oral cavity efficient demonstration of HPV DNA was reported as 3.12% [28].
To screen HPV, multiple assay choices are available which has resulted in controversy [29]. Considering the paraffin-embedded tissue with fragmented DNA, we simply selected GP5+/GP6+ consensus primers for the study. It should be able to amplify the smallest region of
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150bp in the L1 region of the HPV genome even in the fragmented case.
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HPV infection may be transient or persistent, infectivity also depends on genotype, site of
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involvement and gender. It is observed that HPV 16 is slowest to be cleared in men [30]. While, the infection in the genital skin of male is weaker than the epithelium of cervix and vagina [31].
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It seems that susceptible tonsillar epithelium is strongly protected by the immune response
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underneath and virus itself in the tonsils are suppressed to a minimum level and mostly cleared
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within a period of one year [32,33].
Other factors responsible for the varied HPV prevalence reported in studies include difference in
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population studied, choice of assay, sampling technique and evolving sexual orientation. Our study has one of the biggest sample size and we evaluated total of 506 samples from two
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different regions. The limitations of study were that we could not differentiate between nonsexual or sexual-transmission, duration of infection and between transient or persistent infection. Secondly, majority of individuals in our study are children (<14 years): China 75%: Pakistan 82% thus relevance of the data to sexually active adults is reduced.
12
The U.S Food and Drug Administration (FDA) has approved PCR consensus and type specific primers for virus detection but all the HPV detection assay have some pros and cons. It is recommended that quantitative DNA test with transcription detection are patho-physiologically
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more relevant [34] Additional studies with improved design using recently developed assay such as RT-qPCR or ddPCR should be conducted. These assays can demonstrate the presence of viral transcripts and viral loads quantification and could be helpful in unfolding the unknowns of HPV. Lastly in China and Pakistan there is no country wide vaccination program against HPV, immunization
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against HPV is evoking and the public acceptance for vaccination is increasing though majority
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is still unvaccinated [35,36]. Vaccination against HPV is also believed to reduce the incidence of
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OPSCC. As there is no validate technique to screen HPV mediated cancers in the oral cavity,
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vaccination seems to be the best method for prevention.
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Conclusion
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The prevalence of HPV in tonsillar tissue is low in both countries but a noteworthy proportion of
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individuals have oncogenic and non-oncogenic HPV. The vague natural history of HPV causing OPSCC without a detectable precancerous lesion in the tonsils emphasize the importance of
CC
vaccination against HPV as the most efficient way to deal with the transmission chains and reduce endemicity. Further studies are necessary to address the increasing incidence of HPV-
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mediated carcinogenesis to clarify the role of viral, host and behavioral variables.
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N U SC RI PT
Table 5 : Comparison of studies examining HPV in tonsils.
Tissue type
Assay
HPV-positive / Sample size (%)
Type Identified
Strome et al. 2002 [37] United States
Adults
FFPE
Southern Blot
3/48 (6.25)
16
Syrjänen 2004 [38] literature review*
Children and adults
Varies
PCR, ISH, Southern Blot
17/200 (8.5)
6,11,16
Chen et al. 2005 [39] Finland
Children and adults
Fresh frozen
Nested PCR (MY09/11 and GP5+/GP6+)
13/206 (6.3)
16
Ribeiro et al. 2006 [40] Brazil
2-13 years
Fresh frozen
PCR MY09/11
0/100 (0)
None
Sisk et al. 2006 [41] United States
2-18 years
Fresh frozen
MY 09/11
2/50 (4)
11
Mammas et al. 2006 [42] Greece
2-14 years
FFPE
PCR GP5+/GP6+
9/106 (8.5)
16
N/S
FFPE
Consensus Primer (N/S)
8/69 (11.6)
16, 58
5-21 years
FFPE
PCR MY/GP
16/252 (6.3)
6, 11, 16, 31
Ernster et al. 2009 [43] United States
Greater than 21 years
FFPE
Type specific PCR for HPV 16 & 18)
0/226 (0)
None
Duray et al. 2011[44] Belgium
Children and adults
FFPE
PCR GP5+/GP6+
10/80 (12.5)
16, 18, 31
Sun et al. 2012 [24] China
Children
FFPE
PCR (N/S)
2/177 (1)
6,11
Palmer et al. 2014 [45] United Kingdom
Children and adults
FFPE & Fresh homogenized
PCR GP5+/GP6+
0/3377 (0) & 0/511 (0)
None
Xue et al. 2014 [22] China
3-12 years
FFPE
qPCR MY09/11
0/146 (0)
None
Rusan et al. 2015 [46] Denmark
8-30 years
Fresh frozen
Nested PCR (MY 09/11 and GP5+/GP6+)
1/80 (1.25)
6
Cockerill et al. 2016 [5] United States
Children and adults
Fresh Unfrozen and swab
Holgic Aptima test & Roche Cobas test
0/193 (0)
None
Ilmarinen et al. 2017 [47] Finland
Children and adults
Fresh frozen
Luminex xMAP
5/477 (1.05)
16, 52, 66, 68
Rieth et al. 2018 [48] United States
Adults
FFPE
Nested PCR (MY09/11 and GP5+/GP6+)
5/102(4.9)
16,18
A
CC E
PT
Baloglu et al. 2010 [25] Turkey
ED
Kim et al. 2007 [8] South Korea
HPV Human papillomavirus
A
Age Range
M
Author / City
* Compiled small studies
N/S Not specified
14
Abbreviations HPV: human papillomavirus; OPSCC: oropharyngeal squamous cell carcinoma; PCR: polymerase chain reaction
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Conflict of Interest The authors declare no conflict of interest Acknowledgements
This study was funded by the Shaanxi Provincial Innovation Foundation (No. 2011KTCL03-18).
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We are grateful to Ding Haiyan and Mr. Guo Shangwen for the tissue sections and assistance
D
M
A
N
throughout the research work.
A.K. Chaturvedi, E.A. Engels, R.M. Pfeiffer, B.Y. Hernandez, W. Xiao, E. Kim, B. Jiang, M.T. Goodman, M. Sibug-Saber, W. Cozen, L. Liu, C.F. Lynch, N. Wentzensen, R.C. Jordan, S. Altekruse, W.F. Anderson, P.S. Rosenberg, M.L. Gillison, Human papillomavirus and rising oropharyngeal cancer incidence in the United States, J. Clin. Oncol. 29 (2011) 4294–4301. doi:10.1200/JCO.2011.36.4596.
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