velopment of some human malignancies. One such virus is human papillomavirus (HPV), which is an important factor in the development of squamous cell carcinoma of the uterine cervix and orophar­

INTRODUCTION

It is now known that viruses are involved in the de­

velopment of some human malignancies. One such virus is human papillomavirus (HPV), which is an important factor in the development of squamous cell carcinoma of the uterine cervix and orophar­

ynx.

HPV is a DNA­containing papovavirus with over 100 subtypes that are classified by oncogenic risk level into high­risk types (including HPV 16, 18, 31, 33, 35, 39, 45, 51, 52 and 58) and low­risk types (including HPV 6, 11, 42, 43 and 44).

HPV is detected in over 90% of patients with cervical can­

cer. Either HPV 16 or 18 is detected in about 70%

of patients, whereas other high­risk types are de­

tected in less than 5%.

HPV is also detected in about 34% of patients with oropharyngeal cancer. In them, HPV 16 is

found in about 91% of the patients.

Reported rates of HPV infection are 9% to 67% in patients with squamous cell carcinoma, with figures varying widely among studies.

p53 controls the cell cycle by regulating cyclin­dependent kinase (CDK) at the G1 checkpoint. When p53 binds to the E6 protein in HPV 16 or 18, p53 is ubiquitinated and de­

graded. Inactivation of p53 suppresses expression of p21, a CDK inhibitor induced by p53, which re­

sults in immortalization of the cell.

Rb is another tumor suppressor gene similar to p53, which works with cyclin D and p16 to regulate the cell cycle at the G1 checkpoint in response to genetic damage from stress or other causes.

In normal cells, Rb, which is phosphorylated by cyclin D, regulates the cell cycle by releasing E2F.

However, the HPV E7 protein binds directly to Rb without involvement of cyclin D, which inhibits the

Investigation of human papillomavirus infections and the expression of related genes in epithelial dysplasias and squamous cell carcinomas of the oral mucosa

Yukihiro Uehara, Kaname Tsuji, Tomio Iseki and Shosuke Morita

First Department of Oral and Maxillofacial Surgery, Osaka Dental University, 5-17 Otemae 1-chome, Chuo-ku, Osaka-shi, Osaka 540-0008, Japan

We studied the prevalence of human papillomavirus (HPV) infections in patients with epi­

thelial dysplasia and squamous cell carcinoma of the oral mucosa, as well as the expres­

sion of p53, p53 (mutant type), Rb, p16, p21 and cyclin D1. The subjects were patients with irritation fibroma (IF), mild dysplasia (MiD), moderate dysplasia (MoD), severe dys­

plasia (SD), carcinoma in situ (CIS), well differentiated squamous cell carcinoma (WSCC) and poorly differentiated squamous cell carcinoma (PSCC) diagnosed at Osaka Dental University Hospital. Six patients, each with one of these conditions were examined. HPV was detected by in situ hybridization (ISH). Expression of p53, p53 (mutant type), Rb, p16, p21 and cyclin D1 was assessed by immunohistochemical staining (IHC).

All cases were negative for HPV by ISH. For IHC, marked expression of p53 and mutant type p53 was seen in WSCC and PSCC. We could not detect expression of p21 in CIS, WSCC or PSCC. Marked expression of Rb and cyclin D1 was seen in CIS, WSCC and PSCC. We could not detect expression of p16 in WSCC or PSCC. The results suggested that HPV infections have low correlation with oral cancer. We found that p16 is a useful indicator of HPV infection. (J Osaka Dent Univ 2017 ; 51 : 137­144)

Key words : Human papillomavirus ; Squamous cell carcinoma ; Oral mucosa ; p16 ;

p53 ; Rb

function of Rb and accelerates the cell cycle.

Overexpression of Rb has been reported in carci­

noma in situ and squamous cell carcinoma of the oral mucosa.

Although the CDK4 inhibitor p16 attenuates inactivation of Rb by binding to the cy­

clin D/CDK complex, the E7 protein of HPV 16 binds directly to Rb, causing the cell cycle to ad­

vance due to loss of normal Rb function. Rb is itself inactivated by E7, which causes loss of normal p16 function.

Although previous studies have shown the involvement of HPV in oral cancer, they have been few in number and their results have been in­

consistent.

We examined the prevalence of HPV infection in patients with epithelial dysplasia and squamous cell carcinoma of the oral mucosa by in situ hybridiza­

tion (ISH). We also assessed expression of the tu­

mor suppressor genes p53 and Rb as well as re­

lated factors p21, p16 and cyclin D1 by immunohis­

tochemical staining to determine how each relates to HPV infection.

MATERIALS AND METHODS Specimens

We used six samples each of irritation fibroma (IF), mild dysplasia (MiD), moderate dysplasia (MoD), severe dysplasia (SD), carcinoma in situ (CIS), well differentiated squamous cell carcinoma (WSCC) and poorly differentiated squamous cell carcinoma (PSCC) of the oral mucosa obtained from Osaka Dental University Hospital. The specimens were fixed in 10% formalin solution, dehydrated in a graded ethanol series, and embedded in paraffin.

The age, gender and site distribution are shown in Tables 1 and 2. This research was approved by the Ethics Committee at Osaka Dental University (Ap­

proval number 110813).

In situ hybridization (ISH)

Sections 4 μm thick were deparaffinised in Hemo­D and rehydrated through a graded ethanol series.

The sections were immersed in Target Retrieval Solution (DakoCytomation, Glostrup, Denmark) for 40 min at 95° C for nucleic acid retrieval. After treat­

ing the sections with proteinase K for 10 min, endo­

genous peroxidase was blocked by 0.3% hydrogen peroxidase. After rehydration of the sections with ethanol, an HPV wide spectrum probe (DakoCyto­

mation), was applied to each section, which was then covered with glass. The sections were dena­

Table 1 Subjects

Lesion Age (yrs) Gender

Average Range M F

IF MiD MoD SD CIS WSCC PSCC

52 60 69 72 58 68 61

30­73 46­65 54­80 68­79 36­77 33­95 49­83

3 3 3 4 5 4 5

3 3 3 2 1 2 1

Total 63 30­95 27 15

IF : Irritation fibroma, MiD : Mild dysplasia, MoD : Moderate dysplasia, SD : Severe dysplasia, CIS : Carcinoma in situ, WSCC : Well differentiated squamous cell carcinoma, PSCC : Poorly differentiated squamous cell carcinoma.

Table 2 Site of lesion

Site Cases Lesion

IF MiD MoD SD CIS WSCC PSCC

Tongue Maxillary gingiva Mandibular gingiva Buccal mucosa Floor of mouth Hard palate

18 7 9 3 3 2

2 1 2 1 0 0

1 2 1 0 0 2

5 0 0 1 0 0

3 1 1 1 0 0

3 1 1 0 1 0

3 1 1 0 1 0

1 1 3 0 1 0

Total 42 6 6 6 6 6 6 6

tured for five min at 90° C and hybridized for 18 h at 37° C using a Hybridizer (DakoCytomation).

After hybridization, the sections were washed in Stringent Wash Concentrate (DakoCytomation) for 45 min at 55° C. In this process, Gene Point System (DakoCytomation) was used according to the manu­

facturer’s instructions : First, the sections were incubated in primary steptavidin­HRP at a dilution of 1 : 100 for 15 min. After washing, they were incu­

bated in biotin tyramide solution for 10 min, followed by secondary steptavidin­HRP for 15 min.

The sections were then visualized by 3,3’­diamin­

obenzidine­tetrahydrochloride (DAB ; DakoCytoma­

tion, Carpinteria, CA, USA) and counter stained with hematoxylin.

Immunohistochemistry (IHC)

Sections 4 μm thick were deparaffinized in Hemo­

D

(Falma, Tokyo, Japan) and rehydrated through a graded ethanol series. Antigen retrieval to anti­

human retinoblastoma mouse monoclonal antibody (Rb), anti­human p21 mouse monoclonal antibody (p21) and anti­human cyclin D1 mouse monoclonal antibody (cyclin D1) was carried out by autoclaving at 121° C for 15 min in 0.01 M citrate buffer at pH 6.0 for Rb and p21, and at pH 7.0 for cyclin D1.

Antigen retrieval to anti­human p16 mouse mono­

clonal antibody (p16), anti­human p53 mouse mon­

oclonal antibody (p53) and anti­human p53 mouse monoclonal antibody mutation (p53 (mutant type)) was carried out by autoclaving at 121° C for 15 min in 0.01 M tris buffer containing EDTA at pH 9.0.

Endogenous peroxidase was blocked by 0.3%

hydrogen peroxidase. Rb, p21, cyclin D1, p53, p53 (mutant type) and p16 were diluted with Antibody Diluent solution (DakoCytomation, Glostrup, Den­

mark), and each of the sections was reacted for 60 min at room temperature. The sections were then incubated with peroxidase dextran polymer (Envi­

sion＋ ; DakoCytomation, Carpinteria, CA, USA) for 30 min at room temperature. The sections were visualized using 3,3’­diaminobenzidine­tetrahydro­

chloride (DAB, DakoCytomation) and counter­stained with hematoxylin. The antigens used, in terms of original source, dilution and manufacturer, are sum­

marized in Table 3.

Evaluation of ISH and IHC

Expression of ISH and IHC were evaluated accord­

ing to Nishioka et al.

: ( − ) less than 10% positive cells, (1 ＋ ) 10­50% positive cells, (2 ＋ ) 50­75% posi­

tive cells and (3 ＋ ) more than 75% positive cells.

How each disorder relates to the expression of Rb, p21, cyclinD1, p53, p53 (mutant type) and p16 was examined by Fisher’s exact test. A value of 0.05 or less was considered significant.

RESULTS ISH

All cases were negative for HPV mRNA.

IHC (Figs. 1 and 2)

p16

p16 immunoreactivies were indicated by nuclear and cytoplasm staining. Five cases of IF and MoD, and four of MiD were (1 ＋ ). All cases of WSCC and PSCC, five of CIS and three of SD were ( − ) (p

＜0.05).

p53

p53 immunoreactivies were revealed by nuclear staining. All cases of IF and MiD, four of MoD and three of SD were (1 ＋ ). For CIS and WSCC, one case each was ( − ), one (1 ＋ ) and two each (2 ＋ ) and (3 ＋ ). For PSCC, one case was ( − ), two (2 ＋ ) and three (3 ＋ ) (p＜0.05).

p53 (mutant type)

p53 (mutant type) immunoreactivies were revealed by nuclear staining. All cases of MiD, five of IF and

Table 3 Sources and dilutions of the primary antibodies

Antibody Source Dilution

p16 p53

p53 (mutant type) p21

Rb Cyclin D1

DAKO, Denmark DAKO, Denmark DAKO, Denmark DAKO, Denmark NOVO, United Kingdom DAKO, Denmark

1 : 1

1 : 100

1 : 100

1 : 50

1 : 50

1 : 200

All of the antibodies were monoclonal mouse antibodies.

four each of MoD and SD were ( − ). In CIS and WSCC, two cases were ( − ). In PSCC, one case was ( − ) and three cases were (3 ＋ ).

p21

p21 immunoreactivies were indicated by nuclear staining. All cases of IF, and five of MiD were (1 ＋ ).

In MoD, SD, CIS, WSCC and PSCC, between three and five cases were ( − ).

Rb

Rb immunoreactivies were indicated by nuclear

staining. All cases of IF and MiD were (1 ＋ ). In MoD, two cases each were ( − ), (1 ＋ ) and (2 ＋ ). In SD, two cases were (1 ＋ ), three (2 ＋ ) and one (3 ＋ ).

In WSCC, four cases were (2 ＋ ) and two (3 ＋ ). In CIS, four cases were (2 ＋ ) and one (3 ＋ ). In PSCC, one case was (2 ＋ ) and four (3 ＋ ) (p＜0.01).

cyclin D1

cyclin D1 immunoreactivies were indicated by nu- clear staining. In each of IF, MiD and MoD, one case was ( − ) and five (1 ＋ ). In SD, five cases were (1 ＋ ) and one (2 ＋ ). In CIS, two cases were (1 ＋ ),

Fig. 1 Immunohistochemical results.

and four (2 ＋ ). In WSCC and PSCC, three cases each were (1 ＋ ).

Relation between p53 and p21 expression (Ta- ble 4)

All cases of IF and five of MiD were p53 positive/

p21 positive. One case each of CIS, WSCC and

Fig. 2 Immunohistochemical staining. (A-D : Original ×100, E and F : Orginal ×40).

A : Overexpression of cyclin D1 in PSCC, B : Overexpression of Rb in WSCC, C : Non-expression of p21 in PSCC, D : Expression of p21 in IF, E : Overexpression of p53 (mutant type) in CIS, F : Overexpression of p53 in CIS.

Table 4 Relation between p53 and p21 expression

Expression of p53/p21 IF MiD MoD SD CIS WSCC PSCC

p53 positive/p21 positive p53 positive/p21 negative p53 negative/p21 positive p53 negative/p21 negative

6 0 0 0

5 1 0 0

2 4 0 0

3 3 0 0

1 4 0 1

2 3 0 1

1 4 0 1

*p＜0.05

*

PSCC was p53 negative/p21 negative (p＜0.05).

Relation between Rb and p16 expression (Table 5)

Five cases of IF and MiD, four of MoD and three of SD were Rb positive/p16 positive. In CIS, WSCC and PSCC, almost all cases were Rb positive/p16 negative (p＜0.05).

DISCUSSION

There are two ways of HPV detection, PCR and ISH. Wang et al. studied the infection rate of HPV in oropharyngeal cancer. They said that there was no significant difference in detection rate between PCR and ISH. We used ISH to detect HPV.

Many papers have studied the infection rate of HPV in oral carcinoma. Recently, Dong et al. reported that the infection rate of HPV in oral carcinoma is 9%

and that of oropharyngeal cancer is 79%. The in- fection rate of HPV is significantly higher in oropha- ryngeal cancer.

Smitha et al. used meta analysis to examine the association between the HPV infec- tion rate and cancers of the oropharynx and oral cavity. They reported that although oropharyngeal cancer has a strong correlation with HPV infection, oral cancer does not because of its low infection rate.

We were unable to detect HPV in CIS, WSCC or PSCC.

Our results were similar to those of other reports.

However, it has been suggested that there are not enough cases to investigate the infection rate of HPV. We feel it necessary to do research using more cases in the future. p53 is a tumor suppres- sor gene whose role includes regulation of the cell cycle and induction of apoptosis ; abnormal p53 expression has been reported in epithelial dysplasia

and squamous cell carcinoma of the oral mucosa.

Field et al.

and Ogden et al.

detected overex- pression of p53 in epithelial dysplasia and squ- amous cell carcinoma. p53 is also expressed in healthy cells, and when p53 becomes overex- pressed due to squamous cell carcinoma, p21 ex- pression decreases. In the present study, we found that this relationship was significant.

Hossain et al. found that a large percentage of patients with epithelial dysplasia and squamous cell carcinoma are p21 positive, which suggests that p21 negative patients have a mutation in p53. This in turn suggests that overexpression of p21 is only induced by wild type p53.

The p53 monoclonal an- tibodies (clone DO-7) we used for immunohisto- chemical staining react with both the wild type and mutants. In addition to DO-7, we also used p53 monoclonal antibodies that react to the mutant type of p53 (clone PAb 240). This is the first study in which both antibodies were used.

Patients with CIS, WSCC, or PSCC who stained positive for p53 with DO-7 also stained positive for mutant p53. Some reports have noted that there was overexpression of the mutant type of p53 with HPV negative oropharyngeal cancer. This is consis- tent with our study.

Rb is another tumor suppres- sor gene which, like p53, works with cyclin D and p16 to regulate the cell cycle in response to genetic damage from stress or other causes. Cyclin D has subtypes cyclin D1, D2 and D3. In this study, we examined cyclin D1, which is most strongly associ- ated with Rb in the G1 phase. Both p16 and cyclin D1 regulate Rb. Nishioka et al. found that Rb over- expression is associated with decreased p16 ex- pression.

We found that, as reported in previous studies, expression of the Rb related factor p16 is

Table 5 Relation between Rb and p16 expression

Expression of Rb/p16 IF MiD MoD SD CIS WSCC PSCC

Rb positive/p16 positive Rb positive/p16 negative Rb negative/p16 positive Rb negative/p16 negative

5 1 0 0

5 1 0 0

4 0 2 0

3 3 0 0

0 5 1 0

0 6 0 0

0 5 0 1

*p＜0.05

*

decreased in patients with Rb overexpression, and that this decrease is significant.

We recently found that p16 is as indicator of HPV infections in the oropharynx.

We were unable to detect p16 or HPV infection in CIS, WSCC or PSCC. We found that the same was true with HPV negative oropharyngeal cancer.

This suggests that p16 can also be an indicator of HPV infection. We detected overexpression of the other Rb related factor, cyclin D1, in patients with CIS, WSCC or PSCC. Overexpression of cyclin D1 in epithelial dysplasia and squamous cell carcinoma of the oral mucosa is likely due to the loss of the inhibitory effect of cyclin D1 on CDK, resulting from loss of p16 expression.

Nishikawa et al. proposed that cyclin D1 expres- sion is an indicator of cellular proliferation in epithe- lial dysplasia and squamous cell carcinoma of the oral mucosa, and noted that overexpression may indicate hidden potential for malignancy.

It is said that lesions with an has overexpression of cyclin D1 or p53 have a poor prognosis.

From these reports, we believe that cyclin D1 and p53 may also be an indicator of the prognosis and an aid in treatment planning.

Our results suggest that oral cancer has a low correlation with HPV infections and should not be considered a risk factor. They also suggest that p16 is a useful indicator of HPV infection. From this study, there was a correlation between negative HPV infection, non-expression of p21 and overex- pression of mutant type p53. Further research with more cases is needed into the correlation between HPV infections and oral cancer, and into the inter- relationship of p16, p21 and other factors. This re- search will be useful for formulating prognosis and treatment planning.

We would like to thank members of the First Department of Oral and Maxillofacial Surgery, Osaka Dental University, for their kind advice and help. We would also like to thank members of the Department of Oral Pathology, Osaka Dental University, for their advice concerning histopathol- ogy.

REFERENCES

1. Scheffner M, Werness BA, Huibregtse JM, Levine AJ, Howley M. The E6 oncoprotein encoded by human papillo- mavirus types 16 and 18 promotes the degradation of p53.

Cell 1990 ; 63 : 1129-1136.

2. Munoz N, Bosch FX, Sanjose S, Herrero R, Castellsague X, Shah KV, Snijders PJF, Meijer CJLM. Epidemiologic classi- fication of human papillomavirus types associated with cer- vical cancer. N Engl J Med 2003 ; 348 : 518-527.

3. Kawana K, Yasugi T, Taketani Y. Human papillomavirus vaccine : current issues & future. Indian J Med Res 2003 ; 130 : 341-347.

4. Thomas JT, Laimins LA, Ruesch MN. Perturbation of cell cycle control by E6 and E7 oncoproteins of human papillo- maviruses. Papillomavirus Report 1998 ; 9 : 59-64.

5. Maruyama H, Yasui T, Ishikawa-Fujiwara T, Morii E, Yamamoto Y, Yoshii T, Takenaka Y, Nakahara S, Todo T, Hongyo T, Inohara H. Human papillomavirus and p53 muta- tions in head and neck squamous cell carcinoma among Japanese population. Cancer Sci 2014 ; 105 : 409-417.

6. Castro TPG, Filbo IB. Prevalence of human papillomavirus (HPV) in oral cavity and oropharynx. Braz J Otorhinolaryn- gol 2006 ; 72 : 272-282.

7. Sosorbaram K, Sugita Y, Suzumura Y, Sato E, Maeda H, Kameyama Y. Human papillomavirus infection in the normal oral cavity of young people in Mongolia. Oral Med Pathol 2006 ; 11 : 105-112.

8. Sugiyama M, Bhawal UK, Dohmen T, Ono S, Miyauchi M, Ishikawa T. Detection of human papillomavirus-16 and HPV -18 DNA in normal, dysplastic, and malignant oral epithe- lium. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003 ; 95 : 594-600.

9. Summersgill KF, Smith EM, Levy BT, Allen JM, Haugen TH, Turek LP. Human papillomavirus in the oral cavities of chil- dren and adolescents. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001 ; 91 : 62-69.

10. Terai M, Sata T, Matsukura T, Takagi M. Human papilloma- virus (HPV) in papilloma, leukoplakia and squamous cell carcinoma of the oral cavity. Oral Med Pathol 1999 ; 4 : 17 -23.

11. Hartwell LH, Weihert TA. Checkpoints : controls that ensure the order of cell cycle events. Science 1989 ; 246 : 629- 634.

12. Sherr CJ, Roberts JM. CDK inhibitors : positive and nega- tive regulators of G1-phase progression. Genes Dev 1999 ; 13 : 1501-1512.

13. Nishioka M, Win SS, Myint B, Takagi M. Detection of p53, Bax, Rb, p16 and human papillomavirus DNA in betel quid associated oral squamous cell carcinoma in Myanmar. Oral Med Pathol 2003 ; 8 : 7-11.

14. Fregonesi PAG, Teresa DB, Duarte RA, Neto CB, Oliveira MRB, Soares CP. p16 INK4A immunohistochemical overex- pression in premalignant and malignant oral lesions with hu- man papillomavirus. J Histochem Cytochem 2003 ; 51 : 1291-1297.

15. Cunningham LL, Pagano GM, Li M, Tandon R, Holm SW, White DK, Lele SM. Overexpression of p16 INK4 is a reli- able marker of human papillomavirus-induced oral high- grade squamous dysplasia. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006 ; 102 : 77-81.

16. Wang Z, Xia RH, Ye DX, Li J. Human papillomavirus 16 in-

fection and TP53 mutation : Two distinct pathogeneses for oropharyngeal squamous cell carcinoma in an eastern Chi- nese population. PLoS One 2016 ; 17 : 11 : e01644912016.

17. Dong J, Feng Q. HPV16 infection in oral cavity and oropha- ryngeal cancer patients. J Oral Sci 2016 ; 58 : 265-269.

18. Smitha T, Mohan CV, Hemavathy S. Prevalence of human papillomavirus 16 DNA and p16 INK4a protein in oral squamous cell carcinoma : A systemic review and meta- analysis. J Oral Maxillofac Pathol 2017 ; 21 : 76-81.

19. Field JK, Spandidos DA, Malliri A, Gosney JR, Yiagnisis M, Stell PM. Elevated p53 expression correlates with a history of heavy smoking in squamous cell carcinoma of the head and neck. Br J Cancer 1991 ; 64 : 573-577.

20. Ogden GR, Kiddie RA, Lunny DP, Lane DP. Assessment of p53 protein in normal, benign, and malignant oral mucosa. J Pathol 1992 ; 166 : 389-394.

21. Hossain MMI, Uzzaman MA, Nishioka M, Sakamoto K, Es-

guerra RL, Yamaguchi A, Takagi M. Expression of p53, mdm 2 and p21 proteins in betel quid and tobacco associ- ated oral squamous cell carcinoma in Bangladeshi popula- tion. Oral Med Pathol 2005 ; 10 : 23-31.

22. Pande P, Mathur M, Shukla NK, Ralhan R. pRb and p16 protein alterations in human oral tumorgenesis. Oral Oncol 1998 ; 34 : 396-403.

23. Sartor M, Steingrimsdottir H, Elamin F, Gaken J, Warnaku- lasuriya S, Partridge M, Thakker N, Johnson NW, Tavassoli M. Role of p16/MTS1, cyclin D1 and RB in primary oral cancer and oral cancer cell lines. Br J Cancer 1999 ; 80 : 79-86.

24. Nishikawa T, Hara R, Ito Y, Masuno K, Tominaga K, Wato

M, Watanabe Y, Mori M, Tanaka A. Aberrant expression

patterns of histone H3 mRNA, p53 cylin D1, and cyclin B1

in oral neoplastic epithelial lesions. Acta Histochem Cyto-

chem 2005 ; 38 : 53-59.