Context: p16 is an important tumor suppressor gene and responsible for regulating the cell cycle. Diffuse positivity with p16 in the cervix and head/neck carcinomas can be regarded as a surrogate marker of the presence of high-risk human papillomavirus (HPV). Aim: The aim of our study was to search the existence of p16 expression in pterygium. We also analyzed the association of p16 expression with epithelial dysplasia and HPV expression. Subjects and Methods: The study enrolled 75 cases of pterygium. The conjunctival tissues of 10 patients excised by the strabismus surgery were used as control group. All of the slides were stained with p16 via the immunohistochemical method. Results: 49 (65%) of pterygiums showed low-grade epithelial dysplasia. None of the control groups showed dysplasia. Positive expression of p16 in patient group was significantly higher (P < 0.001). Staining percentage (SP) of p16 was between 0 and 26% in pterygium; mean SP was 5.1%. There was no staining in the control group. A total of 59 (72%) pterygium cases were positive with p16. Appoximately 42 of 49 (85%) cases with dysplasia showed p16 staining. There was a significant relation between dysplasia and positive expression of p16 (P < 0.001). Conclusions: P16 is significantly expressed in pterygium and correlated with epithelial dysplasia. Furthermore, the existence of p16 expression suggests that HPV is a possible ethiological factor in pterygium. We think that examination of p16 expression and analysis of HPV DNA in p16 positive cases can help us to understand the etiopathogenesis of the disease better.
Keywords: Dysplasia, HPV, ımmunohistochemistry, p16, pterygium
|How to cite this article:
Süren E, Nergiz D, Süren D, Alikanoğlu AS, Yıldırım HT, Altun ZA. Expression of p16 in pterygium and its relation with epithelial dysplasia and possible etiologic role of HPV. Indian J Pathol Microbiol 2022;65:258-61
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Süren E, Nergiz D, Süren D, Alikanoğlu AS, Yıldırım HT, Altun ZA. Expression of p16 in pterygium and its relation with epithelial dysplasia and possible etiologic role of HPV. Indian J Pathol Microbiol [serial online] 2022 [cited 2022 May 4];65:258-61. Available from: https://www.ijpmonline.org/text.asp?2022/65/2/258/343171
Pterygium is a common sporadic disease. The etiopathogenesis of pterygium is still unknown. Scattering of light can induce pterygium. This thought was put newly in etio-pathogenesis of pterygium. UV is known as the most important risk factor in the formation and the progression of pterygium., UV causes genetic, biochemical and morphologic changes in the limbal stem cells. The UV rays absorbed by the cornea and conjunctiva causes cell injury and reconstruction of extracellular matrix by the help of this fibrovascular proliferation starts and pterygium forms.,,, Histopathologically, pterygium are characterized by hyperplastic limbal epithelial cells accompanied by Bowman’s layer dissolution, epithelial-mesenchymal transition, and an activated fibroblastic stroma with inflammation, neovascularization, and matrix remodeling. [Figure 1]a.
|Figure 1: (a) Typical histopathological features of pterygium (H&E X100), (b) Low-grade epithelial dysplasia in a pterygium (H&E X400)|
p16(INK4a) is one of the major cell cycle tumor suppressor genes that can be used as immunohistochemical markers in cancer diagnosis, treatment, and prognosis, both inactivation and overexpression during cancer development.
In recent years, immunohistochemistry with p16 antibodies has been used as a diagnostic marker in gynecopathology. Data show that strong nuclear and cytoplasmic p16(INK4A) overexpression is a reliable indicator for high-risk human papillomavirus (HPV) subtypes such as type 16 and 18 in oropharyngeal squamous cell carcinomas and dysplasias.
There are few reports about p16 expression in pterygium in literature. In this article, we examined the expression of p16 via the immunohistochemistry method in pterygium samples and examined its relation with epithelial dysplasia, and also discussed its relation with possible HPV infection.
|Materials and Methods|
Our retrospective study initially included 85 cases of pterygium which were diagnosed histopathologically between 2014 and 2017, and the conjunctival tissues of 10 patients excised by the strabismus surgery were used as the control group. Ten cases were excluded from the study, because of the suboptimal sampling or non-appropriated orientation of the samples. The rest 75 cases included the study as the patient group. The clinical data were obtained from the patient files. Informed consent was obtained from the patient files taken before the operation. The research followed the tenets of the Declaration of Helsinki. The ethical approval was taken from the ethics committee of Health Sciences University, Antalya Training and Research Hospital. (Approval number: 6/2, Date: 18 -03/2018).
Tissue preparation and immunohistochemical staining
Materials obtained by surgical resection were put in 10% formaldehyde for fixation for 24 h. Afterward routine tissue processing was applied and the samples were embedded in paraffin. After evaluating hematoxylin and eosin stained sections, cross-sections of 4-μm thickness were obtained for immunohistochemical staining. All slides were stained with primary antibody p16 (mouse monoclonal, dilution 1: 4000, ABM, Richmond, Canada). For positive control, cervical high-grade squamous intraepithelial lesion (HSIL) secondary to HPV infection was used. The primary antibody was omitted for negative control.
Histopathological examination and evaluation of immunohistochemically stained sections
All cases are re-evaluated by 2 pathologist and diagnosis was confirmed. Also the hematoxylin and eosin stained sections were histopathologically examined for the presence of epithelial dysplasia [Figure 1]b. Expression rates of p16 in the epithelium of pterygium and normal conjunctiva were evaluated by 2 pathologists blinded to clinical and histopathological features. Nuclear staining was considered positive for p16. For immunohistochemical evaluation, first the staining was determined as negative or positive. Then if there was a staining, the hot zone (the area containing more positive cells) was determined and staining percentage (SP) was added by counting positive cells/100 epithelial cells [Figure 2].
|Figure 2: (a) p16 negativity in a pterygium (b) Diffuse p16 positivity in a pterygium (a-b, p16 IHC, mouse monoclonal, dilution 1: 4000, ABM, Richmond-Canada, X200)|
Statistical analyses were performed by using SPSS software version 24.0 for Windows (SPSS, Inc., Chicago, IL, USA). Descriptive analyses were presented as mean ± standard deviation. Cross-tables were made and the mean percentages of p16 expressions in control group and pterygium patients were identified. Differences between groups were analyzed using the Chi-square test. A value of P < 0.05 was considered to indicate statistically significant difference.
The study included a total of 85 cases, of which 75 (88%) are pterygium and 10 (12%) are control cases. 39 (52%) of the pterygium cases were female and 36 (48%) male. 5 (50%) of the control cases were female and 5 (50%) male. The age interval of the patient group was 28-82 years with the median age 55.06 (Standard deviation: 14.16).
65% of pterygium cases showed low-grade epithelial dysplasia, dysplasia was negative in 35% of cases. None of the control group cases showed dysplasia. For dysplastic pterygium samples, dysplasia seen at the head part of the pterygium. Positive expression of p16 was significantly higher at patient group than the control group (P < 0.001).
Staining percentage (SP) of p16 was between 0 and 26% in the patient group, mean SP was 5.1%. There was no staining in the control group (0%). When any staining over 0% was accepted as positive, 54 (72%) pterygium cases were positive, whereas 21 (28%) cases were negative. The 42 of 49 (85%) cases with epithelial dysplasia showed p16 staining. In the patient group, there was a significant relation between dysplasia and positive expression of p16 (P < 0.001).
Many genes and proteins have been discovered in the etiopathogenesis of pterygium and are thought to have different mechanisms in the occurrence of the disease. Nevertheless, the mechanism of the disease development is not fully understood and is still controversial. In addition to many studies advocating that pterygium is a cellular proliferative disease, there are also studies suggesting that there is a defect in the regulation of apoptosis in the cell cycle characterized by the development of the disease.,,,,,,
P16 (INK4a) is one of the most important tumor suppressor genes and responsible for regulating the cell cycle. It is codified by a gene localized on chromosome 9p21 within the INK4a/ARF locus. It is a member of the INK4 family of CDK (cyclin-dependent kinase) inhibitors. The classic role of p16 (INK4a) is to check the cell cycle in early G1 phase and inhibit further transition of the cell cycle from G1 to S phase as a component of a multiprotein regulatory complex. p16 has been reported to play an important role in cell differentiation, cell quiescence, and cell senescence, which makes it not just a tumor suppressor protein but a cell regulatory protein that plays a critical role in regulating terminal differentiation and the aging process. As a result; p16 (INK4a) is one of the major cell cycle tumor suppressor genes that can be used as immunohistochemical markers in cancer diagnosis, treatment, and prognosis, both inactivation and overexpression during cancer development.
In recent years, immunohistochemistry with p16 antibodies has been used as a diagnostic aid in various scenarios in gynecologic pathology. Diffuse (as opposed to focal) positivity with p16 in the cervix can be regarded as a surrogate marker of the presence of high-risk HPV (types 16, 18, 31, 33, 51, 52, 58, 59, 68). In cervical squamous lesions, p16 is positive in most high-grade cervical intraepithelial neoplasia (CIN) and in some cases of low-grade CIN, usually those associated with high-risk HPV.
HPV is also a known risk factor for the development of benign and malignant mucosal head and neck lesions. Data show that strong nuclear and cytoplasmic p16 (INK4A) overexpression is a reliable surrogate indicator for HPV in oropharyngeal squamous cell carcinomas and dysplasias. But, p16 should not be considered a globally specific marker of HPV infection; indeed, this tumor suppressor protein can be overexpressed in a variety of non-HPV-related malignancies such as myometrial leiomyosarcoma, oropharyngeal squamous cell carcinoma, prostate carcinoma, endometrial carcinoma, basal cell carcinoma of the skin.,,,,
There are only two studies in the literature that searched the immunohistochemical p16 expression in pterygium. Chen et al. searched the status of p16 methylation in pterygium and normal conjunctival by methylation–specific polymerase chain reaction and immunohistochemical method.
p16 gene promoter hypermetylation was detected in 21 (16.3%) of 129 pterygial specimens. Among them, 46 (35.7%) were positive for p16 protein expression, and 83 (64.3%) were negative. Staining for p16 was limited to the nuclei of the epithelial layer. They observed a significant reverse correlation between hypermethylation of the p16 promoter and the expression of p16 protein (P = 0.006). Their data provided molecular evidence that p16 gene promoter methylation occurs in pterygia and that it may play a role in the their development.
Ramalho et al. found an increased p16 expression in all of the recurrent and primary pterygium cases in their study. They obtained a cytoplasmic p16 expression in primary cases and cytoplasmic/nuclear expression in recurrent cases. Their claim is that mainly the recurrent form may demonstrate some similarity to aggressive squamous lesions, which can explain the high index of recurrence. In our study, all cases showed a strong nuclear and weak cytoplasmic p16 expression.
P16 positivity was found as 72% in the pterygium cases in our study. This rate is not as low as 35.7% rate found in the study of Chen et al. and not high as 100% rate found in the study of Ramelho et al. In three of the studies different clones of p16 were used and Chen et al. accepted the cut-off value as 10%. These two factors may explain the different results of these studies. However, in case of the 10% cut-off margin used in the study of Ramalho et al., the positivity rate found as 100% in their study is partly incompatible with the results of our study. This result may be due to the different patient populations and the different clones of the immunohistochemical markers.
According to our knowledge, there is no report which investigated the relationship between the epithelial dysplasia in pterygium and p16 expression. In our study, we found 42/49 (85%) cases with epithelial dysplasia showed p16 staining. These findings let us to think p16 expression plays an important role of pterygium development and have a potential for determining tumor progression and would let us to select the cases for close follow-up. There are several limitations to be mentioned concerning the present study. The data regarding the prognosis and follow-up of the patients after the diagnosis could not be evaluated because high-grade dysplasia was found in none of the cases in our study. We considered that the data in this study can contribute to the literature but there is a need for further retrospective and prospective randomized studies to be performed in larger series including cases with both low and high-grade dysplasia to correctly evaluate data regarding the prognosis and follow up.
Also we wanted to search if there is a relation between p16 expression and HPV positivity as found in cervical lesions. So, we analyzed the existence of HPV types 6, 11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 56, 58, 59, 68 in 4 cases in which p16 expression was over 20% and the DNA isolation was sufficient, by hybrid capture method using Diagene hc2 HPV DNA test kit. HPV DNA was not found in 2 cases and was found positive in the other 2 cases. The positive cases were positive for HPV type 68 (the p16 expression was 22% and 26% in these two cases respectively). Although these primitive results are not useful for a significant conclusion, they may be correlated with the role of HPV in the etiyopathogenesis of pterygium by wide study series. As well known, HPV infection is one of the most common sexually transmitted diseases and it may contaminate the ocular surface by otoinoculation with contaminated fingers.
In conclusion, p16 is significantly expressed in pterygium cases and it is correlated with epithelial dysplasia. In some pterygium cases, the existence of p16 expression suggests the probable etiologic role of HPV DNA. We suggest that the analysis of p16 expression and analysis of HPV DNA and subtypes of HPV especially in p16 positive cases may help us to understand the etiopathogenesis of pterygium better.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
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Conflicts of interest
There are no conflicts of interest.
Mooren JJ, Gültekin SE, Straetmans JM, Haesevoets A, Peutz-Kootstra CJ, Huebbers CU, et al. P16(INK4A) immunostaining is a strong indicator for high-risk-HPV-associated oropharyngeal carcinomas and dysplasias, but is unreliable to predict low-risk-HPV-infection in head and neck papillomas and laryngeal dysplasias. Int J Cancer 2014;134:2108-17.
Source of Support: None, Conflict of Interest: None