ORIGINAL RESEARCH


https://doi.org/10.5005/jp-journals-10015-2261
World Journal of Dentistry
Volume 14 | Issue 7 | Year 2023

Human Papillomavirus Prevalence and p16INK4a Expression in Formalin-fixed, Paraffin-embedded Tissues of Head and Neck Squamous Cell Carcinomas: A Retrospective Study


Siribang-on P Khovidhunkit1, Nuha A Guddoos2, Sopee Poomsawat3, Sudaporn Kengkarn4, Puangwan Lapthanasupkul5, Phaibul Punyarit6, Pimsupak Boonmongkolraksa7, Sirima Sanguansin8

1Department of Advanced General Dentistry, Faculty of Dentistry, Mahidol University, Bangkok, Thailand

2Master Dental Care Centre, Male City, Maldives

3,5Department of Oral and Maxillofacial Pathology, Faculty of Dentistry, Mahidol University, Bangkok, Thailand

4Faculty of Medical Technology, Rangsit University, Pathum Thani, Thailand

6Department of Clinical Pathology, Army Institute of Pathology, Phramongkutklao Medical Center and Pathology Division, Bangkok, Thailand; Department of Surgery, Bumrungrad International Hospital, Bangkok, Thailand

7Department of General Dentistry, Faculty of Dentistry, Srinakharinwirot University, Bangkok, Thailand

8Department of Oral Biology, Faculty of Dentistry, Mahidol University, Bangkok, Thailand

Corresponding Author: Sirima Sanguansin, Department of Oral Biology, Faculty of Dentistry, Mahidol University, Bangkok, Thailand, Phone: +6622007849, e-mail: sirima.san@mahidol.ac.th

Received on: 02 June 2023; Accepted on: 04 July 2023; Published on: 31 August 2023

ABSTRACT

Aim: To investigate the prevalence of human papillomavirus (HPV) in formalin-fixed, paraffin-embedded (FFPE) head and neck squamous cell carcinoma (HNSCC) tissues.

Materials and methods: A total of 176 FFPE tissues of HNSCC were retrospectively collected. The integrity of DNA extracts was evaluated by polymerase chain reaction (PCR) analysis using primers specific for the β-globin gene and 54 DNA extracts were positive for further evaluation. Nested PCR analysis using consensus primers specific to the L1 region of HPV was subsequently performed. HPV typing was investigated using DNA sequencing analysis. The p16INK4a expression was examined in HPV-positive samples using immunohistochemical staining with p16INK4a antibody.

Results: Out of 54 samples, only one sample (1.85%) was positive for HPV-DNA. DNA sequencing analysis confirmed the presence of HPV type 16. The HPV-positive sample came from a married male patient with a history of smoking and alcohol consumption. The tumor was located on the laryngeal area with the diagnosis of squamous cell carcinoma. Strongly positive p16INK4a was detected in the HPV-positive sample. The majority of HPV-negative patients had a history of smoking, alcohol consumption, and betel nut chewing habit.

Conclusion: Very low prevalence of HPV suggested that HPV might not play an important role in the etiopathogenesis of HNSCC in this group of patients. However, strong p16INK4a expression was exhibited in the sample with the presence of a high-risk type of HPV.

Clinical significance: Smoking, alcohol consumption, and betel nut chewing habit might play a more important role than HPV infection in the carcinogenesis of HNSCC in the present study. The p16INK4a overexpression could be used to predict the presence of high-risk HPV.

How to cite this article: Khovidhunkit SP, Guddoos NA, Poomsawat S, et al. Human Papillomavirus Prevalence and p16INK4a Expression in Formalin-fixed, Paraffin-embedded Tissues of Head and Neck Squamous Cell Carcinomas: A Retrospective Study. World J Dent 2023;14(7):569–575.

Keywords: Formalin-fixed paraffin-embedded, Head and neck cancer, Human papillomavirus, Polymerase chain reaction, p16INK4a expression

INTRODUCTION

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide.1 This group of cancers represents a cohort of neoplasms emerging from the oral cavity, oropharynx, hypopharynx, nasopharynx, and larynx.1

The cause of HNSCC is multifactorial, and well-established risk factors of HNSCC are smoking, alcohol abuse, and betel quid chewing habit. However, in some cases of HNSCC, no smoking or drinking history has been reported, and recent studies have shown a strong association between human papillomavirus (HPV) infections and HNSCC.2,3

Human papillomavirus (HPV) is a virus with a circular, double-stranded DNA genome and a tropism for basal cells of the epithelium.4 HPVs can be divided into low-risk and high-risk groups depending on their association with malignancies. A meta-analysis study reported that the predominant high-risk types of HPV associated with HNSCC are HPVs 16 and 18.3 Although many studies indicated a high frequency of HPV in HNSCC specimens, the detection rate of HPV varied considerably depending on the environmental risk factors, the status of the disease, detection techniques, and tissue origin.3,5 A systematic review by Kreimer et al. reported that among 5,046 cases from 60 eligible studies in 26 countries, 26% of all HNSCC biopsy specimens were HPV positive.6 This study also suggested that HPV appeared to play an etiological role in a considerable fraction of oropharyngeal cancers but in a small subgroup of cancers of the oral cavity and larynx.6

The prevalence rates of HPV in HNSCC and oral squamous cell carcinoma (OSCC) vary in the same country. Two studies performed in Thailand reported different prevalence rates. A study conducted by Khovidhunkit et al. using polymerase chain reaction (PCR) analysis of DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues showed that out of 32 OSCC, 17 leukoplakia, and 16 oral lichen planus specimens, only one OSCC specimen was positive for HPV.7 This is in contrast with Swangphon and colleagues’ study which performed an in situ hybridization of HPV E6/E7 messenger RNA (mRNA) in FFPE tissue samples and found HPV infection in 41 and 50% of the oral cavity and oropharynx cancers, respectively.8

The p16INK4a, a tumor suppressor, plays a crucial role in controlling carcinogenesis.9 It inhibits cell cycle progression by restricting retinoblastoma tumor suppressor protein phosphorylation during G1 to S phase of the cell cycle. Aberration of p16INK4a has been found in oral potentially malignant disorders and OSCC in the Thai population.5,10 In many cases, a lack of expression of p16INK4a has been found. In those cases infected with a high-risk type of HPV, overexpression of p16 has been demonstrated and suggested as a marker for such infection in a systematic review and meta-analysis study.11 Using p16 for predicting the presence of HPV 16/18 has a sensitivity of 40% and specificity of 79.3% in OSCC in the Thai population.5

The objective of the present study was to investigate the prevalence of HPV in more existing FFPE tissues of HNSCC. In addition, the expression of p16INK4a was also examined in HNSCC with HPV-positive specimens.

MATERIALS AND METHODS

The present study was conducted during 2013–2015 in the Department of Oral and Maxillofacial Pathology and the Research Unit, Faculty of Dentistry, Mahidol University, Bangkok, Thailand, after obtaining approval from the Institutional Ethical Committee (MU-DT/PY-IRB 2013/015.1203 and IRB/RTA 1511/2555). The ethical guidelines of the Declaration of Helsinki were followed in the present study. The data of all patients were kept confidential.

Specimen Selection Criteria

Histopathological diagnosis as HNSCC cases with clinical data and information related to lifestyles from two institutes in the central part of Thailand; the Pathology Division, Army Institute of Pathology, and the Department of Oral and Maxillofacial Pathology, Faculty of Dentistry, Mahidol University, Bangkok, Thailand, between 2005 and 2013 were included. Histopathological examinations from both institutes were performed according to World Health Organization-Tumor Node Metastasis classification to identify the staging and grading of HNSCC.12

Study Specimen

A total of 176 FFPE specimens of HNSCC were screened and only 102 FFPE tissue blocks had sufficient tissues for DNA extraction. Around 77 of these specimens were retrieved from the Pathology Division, Army Institute of Pathology, and 25 specimens from the Department of Oral and Maxillofacial Pathology, Faculty of Dentistry, Mahidol University, Bangkok, Thailand.

DNA Extraction

The DNA was extracted from 102 FFPE HNSCC specimens using QIAamp® DNA FFPE tissue kit (QIAGEN® GmbH, Hilden, Germany). The extraction procedure was performed according to the manufacturer’s protocol, except that the 1-hour time period of incubation at 90°C was changed to 5 minutes because longer incubation time at high temperatures resulted in more DNA fragmentation. The concentration and purity of the extracted DNA were spectrophotometrically measured at 260 and 280 nm (Epoch™ Gen5™ BioTek®, Vermont, United States of America). DNA concentration of all the samples was adjusted to 100 ng/μL using deionized water.

β-globin PCR Analysis

The amplification of the β-globin housekeeping gene was performed for DNA quality control in all 102 specimens. KM29–KM138 primers were utilized for the amplification of a 262 bp product. The primer sequences, PCR condition, and thermocycling of the PCR reaction had previously been described.13 In each reaction, 400 ng of DNA was used and sterile distilled water was added to the total volume of 20 μL. All β-globin-positive samples were subjected to PCR for HPV-DNA investigation.

Polymerase Chain Reaction (PCR) for HPV Detection

Nested PCR analysis using MY09–MY11 primers as outer primers and HPV1003–HPV1004 primers as inner primers were conducted in 54 β-globin-positive specimens as previously described.13 In each reaction, 400 ng of DNA was used. PCR products from the first PCR analysis were employed as templates for the second amplification. Around 1 μL of the MY09–MY11 PCR product was diluted in the ratio of 1:8. Around 2 μL of the diluted PCR products were then used as a template for the nested PCR amplification with HPV1003–HPV1004 primers.

Around 5 μL of the amplified PCR products were subjected to 1.5% agarose gel electrophoresis. The gel was stained with ethidium bromide and analyzed under ultraviolet transillumination (GelDoc, Bio-Rad®, California, United States of America). Two positive controls were DNA extracted from HPV 18-containing HeLa cells and DNA extracted from FFPE cervical cancer specimen positive for HPV type 18. The negative control was deionized sterile water. Each sample was performed in duplicate.

Immunohistochemistry of p16INK4a

Immunohistochemical staining for p16 was performed in a positive HPV-PCR specimen, as previously described by Sihavong et al.14 The FFPE sections were first deparaffinized in xylene and then rehydrated. Antigen retrieval was performed in a target retrieval solution (S1699, Dako, Carpinteria, California, United States of America) using a microwave, and the sections were immersed in 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase activity. The slides were then incubated with a mouse monoclonal antihuman p16INK4a antibody at a dilution of 1:60 (#Z2117, Zeta Corporation, Arcadia, California, United States of America) at 4°C in a humidified chamber overnight. Subsequently, the sections were incubated with a biotinylated secondary antibody and diaminobenzidine (EnVision Detection Systems Peroxidase/DAB, Dako, Gilstrap, Denmark) according to the manufacturer’s instructions. The sections were finally counterstained with hematoxylin. Negative control sections were obtained by omitting the primary antibody. Samples of cervical cancer were used as positive controls on each run.

Polymerase Chain Reaction (PCR) and Immunohistochemical Analysis

For DNA quality control using β-globin PCR, a positive sample showed a band at 262 bp on an agarose gel. For HPV detection, subsequent to the second nested PCR using HPV1003–HPV1004 primers, a band at 150 bp were considered to be HPV positive. For immunohistochemical analysis of p16INK4a expression, a positively stained specimen represented yellow-brown staining in the nucleus and cytoplasm of >70% of epithelial tumor cells.

Statistical Analysis

Statistical Package for the Social Sciences (SPSS) version 16.0 (IBM, Armonk, New York, United States of America) was utilized for statistical analysis. The relationship between the presence of HPV and clinicopathological characteristics was examined using a Chi-square test. A p-value < 0.05 was considered statistically significant.

RESULTS

Patient Characteristics and Evaluation of DNA Integrity using β-globin PCR

After reviewing 176 FFPE specimens of patients with HNSCC diagnosed between 2005 and 2013, only 102 had sufficient tissues. Among those, 54 specimens (53%) indicated good quality by giving positive results for the β-globin gene (Fig. 1) and were included for further HPV analysis. Patient’s demographic characteristics and risk factors are presented in Table 1. The majority of these 54 patients were male. The most frequent site of the tumor was the oral cavity (67%, 36/54), followed by the larynx (24%, 3/54) and pharynx (9%, 5/54), and the majority had stage IV HNSCC (50%, 27/54).

Fig. 1: PCR analysis of β-globin gene. Lane M, molecular marker (100 base pairs); lane 1, negative control; lane 2, positive control (HeLa cells); lane 3, positive control (cervical cancer sample); lane 4, negative β-globin gene sample; lanes 5–7, positive β-globin gene samples

Table 1: Clinicopathologic data and statistical analysis (N = 54)
Characteristics Study samples HPV-positive HPV-negative p-value
Total number 54 (100%) 1 (1.85%) 53 (98.15%)
Gender 0.550
 Male 41 (76%) 1 (2%) 40 (98%)
 Female 13 (24%) 0 (0%) 13 (100%)
Age (years) 0.059
 Below and = 50 13 (24%) 1 (7.7%) 12 (92.3%)
 Above 50 41 (76%) 0 (0%) 41 (100%)
Habits
 Smoking 29 (54%) 1 (3%) 28 (97%) 0.259
 Alcohol consumption 22 (41%) 1 (5%) 21 (95%) 0.223
 Betel nut chewing 5 (9%) 0 (0%) 5 (100%) 0.721
Anatomical site 0.475
 Oral cavity 36 (67%) 0 (0%) 36 (100%)
 Not oral cavity 18 (33%) 1 (5.6%) 17 (94.4%)
• Larynx 13 (24%) 1 (7.7%) 12 (92.3%)
• Oropharynx 5 (9%) 0 (0%) 5 (100%)
Clinical staging 0.475
 Stages I and II 20 (37%) 0 (0%) 20 (100%)
• Stage I 13 (24%) 0 (0%) 13 (100%)
• Stage II 7 (13%) 0 (0%) 7 (100%)
 Stages III and IV 34 (63%) 1 (3%) 33 (97%)
• Stage III 7 (13%) 0 (0%) 7 (100%)
• Stage IV 27 (50%) 1 (3.7%) 26 (96.3%)
Histologic grade *0.033
 Well-differentiated 12 (22%) 0 (0%) 12 (100%)
 Moderately differentiated 35 (65%) 0 (0%) 35 (100%)
 Poorly differentiated 7 (13%) 1(14%) 6 (86%)

*Statistically significant as p-value < 0.05

Evaluation of the Presence of HPV-DNA using Nested PCR Analysis

The presence of HPV-DNA was investigated by nested PCR analysis using two primer sets, including MY09–MY011 (Fig. 2A) and HPV1003–HPV1004 (Fig. 2B). Both primer sets recognized the L1 region of HPV. After PCR analysis of HPV using MY09–MY011 primers, only one positive band was observed in one positive control (Fig. 2A). After the second PCR amplification using HPV1003–HPV1004 primers, two positive controls, and one HNSCC DNA sample showed positive bands (Fig. 2B).

Figs 2A and B: (A) First nested PCR analysis of HPV using MY09–MY011 primers; (B) Second nested PCR analysis of HPV using HPV1003–HPV1004 primers. Lane M, molecular marker (100 bp); lane 1, negative control; lane 2, positive control (HeLa cells); lane 3, positive control (cervical cancer sample); lane 4, positive HPV sample; lanes 5–8, negative HPV samples

According to DNA sequencing analysis, the only one HPV-positive sample was HPV type 16. Medical history indicated that this was a specimen from laryngeal carcinoma of a married male patient aged 50 years with a history of heavy smoking (two packs a day) and heavy alcohol consumption (approximately 180 mL of whisky a day). The tumor was poorly differentiated squamous cell carcinoma with clinical stage IV (Table 1). Statistical analysis showed that only one factor, poorly differentiated histological grade, had a significant effect on HPV positivity as p-value = 0.033.

Expression p16INK4a of using Immunohistochemistry

The investigation of p16 expression using immunohistochemistry in the positive HPV-DNA case revealed strong cytoplasmic staining in the tumor cells (Fig. 3).

Figs 3A to F: Immunohistochemical analysis of p16INK4a in HNSCC specimen with positive high-risk HPV. Strong cytoplasmic staining of p16INK4a in the tumor nests of cells. (A) 40× magnification; (B) 100× magnification; (C) 200× magnification; (D) 400× magnification; (E) negative control cervical sample (100× magnification); (F) positive control cervical sample (100× magnification)

DISCUSSION

The role of HPV in the pathogenesis of HNSCC is well established and a wide range of detection rates has been reported,6,15 which may be attributable to ethnogeographical differences, the sensitivity of the applied diagnostic technique, method of sample storage, specimen type, HPV detection procedure, number of samples analyzed, and the risk factors for HPV infection of the subjects.

In Thailand, the prevalence of HPV in OSCC has previously been evaluated. In the central part, especially in Bangkok, the prevalence of HPV in cancers of the hypopharynx and larynx was low.16 Only one out of 80 DNA samples was positive for the HPV-62 serotype. The positive specimen was from a 72-year-old male patient with no risk factors for HPV infection but had habitual smoking and drinking behaviors.16 On the contrary, a high prevalence of HPV in OSCC has been reported in the northeastern part of Thailand. Using in situ hybridization of HPV E6/E7 mRNA in FFPE tissue samples, HPV infection was found in 41 and 50% of cancers of the oral cavity and oropharynx, respectively.8 The present study conducted in the central part of Thailand indicated a low prevalence of HPV. These results implied that divergent HPV prevalences might be attributed to the different geographical locations.

The method to identify the presence of HPV is also critical for the estimation of HPV prevalence. Due to weak viral productivity, a highly sensitive system of HPV-DNA detection is required. A PCR-based method could detect 40% of HPV in OSCCs compared to 30% using an in situ hybridization-based method.17 The use of MY09–MY11 and either GP5/GP6 or GP5+/GP6+ primers in a nested PCR assay has been shown to increase the overall sensitivity compared to that of each primer pair alone.18 In the present study, nested PCR using two sets of consensus primers was performed. In every PCR reaction, HPV-DNA could be amplified from the two positive controls suggesting that the nested PCR reaction was sensitive enough to detect HPV-DNA, but the samples of DNA extracts might not contain HPV-DNA.

The site of cancer is one of the factors that may determine the prevalence of HPV. In a meta-analysis (n = 2,739) and large case series (n = 1,042) of laryngeal squamous cell carcinoma (LSCC), it was shown that the true rate of HPV-driven LSCC was 8.6% and <5%, respectively.19 It was suggested that HPVs were more associated with oropharyngeal cancers than the oral cavity and laryngeal cancers. Since the majority of the tumors in the present study were from the oral cavity, it is not surprising that the prevalence of HPV might be low. In addition, the positive sample came from squamous cell carcinoma of the larynx; therefore, HPV might not play a significant role in this group of OSCC.

The only HPV-positive sample in the current study came from a male patient and the majority of the patients were male. Several previous studies have reported male predominance.5,20 A study conducted in the northern part of Thailand showed that all OSCC-DNA samples with positive high-risk HPV-DNA were from male patients.5 A retrospective study in the United States of America by Settle et al. reported that out of 68 HPV-positive samples, 54 (79%) were from males, and 14 (21%) were from females.21 These variations might be attributable to different lifestyles in different gender groups.

The majority of patients in the current study, including the only HPV-positive patient, had a history of smoking and alcohol drinking. Smoking has been shown to increase the risk of high-risk HPV infection and HPV-associated HNSCC.22,23 This suggested an additive effect of the two risk factors on carcinogenesis—smoking and HPV. One recent study has also shown that alcohol intake was associated with a significantly increased risk for HPV infection in men.24

The p16 expression has been proposed as a marker for malignant transformation, especially in cervical cancer.25 A study also suggested using a combination of p16 immunohistochemistry and the detection of high-risk HPV in identifying a person who is at risk of cervical cancer.26 Although p16INK4a is postulated to be a surrogate marker for high-risk HPV infection in cervical cancer, the discordance of the association of high-risk HPV and p16 overexpression in head and neck cancer exists.27 A study in Thailand by Swangphon et al. found that expression of p16INK4a was significantly more frequent in HPV-negative (78.9%) than in HPV-positive HNSCC and about half of HPV-positive samples did not express p16.8 In a large cohort study of 504 patients in Thailand, Nopmaneepaisarn et al. observed a low prevalence of p16-positive HPV-related head and neck cancer.27 In the current study, the high-risk HPV-positive case showed strong cytoplasmic expression of p16INK4a throughout the tumor cells. Because of the discordance of the use of p16INK4a as a surrogate marker for high-risk HPV infection in HNSCC, it is, therefore, recommended that for HNSCC, p16 immunohistochemistry should be used in conjunction with other molecular methods for the detection of high-risk HPV infection.28

There are some limitations in the current study. First, the sample size was small. Although p16INK4a is considered to be an indirect marker of HPV-induced HNSCC, only one sample was positive for both HPV and p16INK4a in this present study. This might render a difficulty in drawing any conclusion on whether p16 could be used as a marker for HPV-positive samples. Second, good quality DNA extracted from FFPE tissues for PCR analysis in the current study was approximately half (54 out of 102 samples or 53%). It is known that the fixation process, including time, temperature, formaldehyde concentration, as well as pH plays an important role in the quality of DNA,29 although successful DNA extraction from FFPE tissues for HPV detection using PCR analysis has been reported by others.8,20,21,26 Therefore, the limitation in using FFPE tissue for molecular biological study must be taken into account.

CONCLUSION

A low prevalence of HPV in HNSCC was observed, suggesting that HPV may not play a significant role in the etiopathogenesis of HNSCC in this group of patients. Strong staining of p16INK4a expression was detected in an HPV-positive HNSCC specimen.

Funding

This research was supported by a grant from the Faculty of Dentistry, Mahidol University, Bangkok, Thailand (2014).

Manufacturer Name:

  • QIAamp® DNA FFPE tissue kit (QIAGEN® GmbH, Hilden, Germany).

  • Spectrophotometer (Epoch™ Gen5™ BioTek®, Vermont, United States of America).

  • UV transillumination (GelDoc, Bio-Rad®, California, United States of America).

  • Target Retrieval Solution (S1699, Dako, Carpinteria, California, United States of America).

  • Mouse monoclonal anti-human p16INK4A (#Z2117, Zeta Corporation, Arcadia, California, United States of America).

  • Biotinylated secondary antibody and diaminobenzidine (EnVision Detection Systems Peroxidase/DAB, Dako, Gilstrap, Denmark).

  • The SPSS version 16.0 (IBM, Armonk, New York, United States of America).

Acknowledgment

The authors would like to thank all the staff in the Research Unit of the Faculty of Dentistry, Mahidol University, Bangkok, Thailand for their support.

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