INTRODUCTION
Penile cancer is a type of cancer that is relatively rare with an incidence of 0.3 cases per 100,000 men per year in western countries. However, in developing countries, such as in Southeast Asia, Latin America, and Africa, the incidence of penile cancer significantly increased by 2.8–6.8 per 100,000 men [1]. The
anatomical pathology registration data in Indonesia shows that the highest number of penile cancers is found in Bali compared to other provinces in Indonesia [2]. Age-specific incidence penile can be found in 2.1 per 100,000 men in Bali [3]. Around 95% of malignancies in the penis are diagnosed with squamous cell carcinoma, and the rest are other types of rare cancer such as basal cell carcinoma and sarcoma [4].
The Proportion of p16 Expression in Penile Squamous Cell Carcinoma Based on Immunohistochemistry Examination in the Balinese Population
Komang Ariningrum Dwita Lestari Ardhaputri
1*, Desak Putu Oki Lestari
2, Ni Wayan Armerinayanti
2, Ni Luh Putu Eka Kartika Sari
3, Luh Yeni Laksmini
4, I Made Jati Luhur
4, I Nyoman Upadana
5, Kadek Pramesti Dewi
51 Faculty of Medicine and Health Sciences, Warmadewa University, Denpasar, Bali, Indonesia
2 Department of Anatomical Pathology, Faculty of Medicine and Health Sciences, Warmadewa University, Bali, Indonesia
3 Department of Biomolecular, Faculty of Medicine and Health Sciences, Warmadewa University, Bali, Indonesia
4 Department of Anatomical Pathology, Singaraja General Hospital, Bali, Indonesia
5 Department of Anatomical Pathology, Sanjiwani General Hospital, Bali, Indonesia
A R T I C L E I N F O Received : 24 August 2021 Reviewed : 18 October 2021 Accepted : 18 January 2022 Keywords:
HPV, penile squamous cells carcinoma, p16 overexpression
A B S T R A C T
Background: Penile cancer is a rare type of cancer in men and was the highest in Bali compared to other regions in Indonesia. Around 95% of malignancies in the penis are squamous cell carcinoma (SCC) type. One of the risk factors associated with penile squamous cell carcinoma is an infection caused by Human Papillomavirus (HPV). Human Papillomavirus E6 and E7 oncoproteins play a significant role in penile squamous cell carcinoma carcinogenesis.
Overexpression of p16 protein can be used as a marker of HPV infection. This study aims to determine the expression of p16 protein in penile squamous cell carcinoma.
Methods: This study was conducted to determine the prevalence of p16 protein expression was assessed by immunohistochemical examination from paraffin block tumors of penile cancer patients. Expression of p16 was observed to be positive when stained with a strong brown color in the nucleus and cytoplasm of tumor cells. The analysis method used in this study is a descriptive statistic to provide a general description of the characteristics of each research variable. The data were processed using SPSS to determine the frequency and median of the age of patients with penile squamous cell carcinoma and the prevalence of p16 protein expression in cell penile squamous cell carcinoma. The data shown in this study are the age range and prevalence of p16 protein expression in penile squamous cell carcinoma using the prevalence table.
Results: This study used 142 penile squamous cell carcinoma samples derived from biopsy, partial, and total penectomy. The results of the IHC examination showed that the expression of p16 protein was positive in penile cancer tumor cells in 68 cases (47.9%), with the highest age distribution in the 50–59-year age group (28.2%).
Conclusions: Further research is needed to confirm the correlation between HPV infection and p16 protein overexpression in penile squamous cell carcinoma, including HPV genotyping.
*Corresponding author:
Komang Ariningrum Dwita Lestari Ardhaputri
Faculty of Medicine and Health Science, Warmadewa University, Denpasar, Bali, Indonesia [email protected]
One of the risk factors associated with penile squamous cell carcinoma is an infection caused by Human Papillomavirus (HPV) [1,5,6]. The integration of high risk types-HPV (hr-HPV) in host cell DNA is associated with malignancy, resulting in increased expression of the genes produced by HPV, namely oncoproteins E6 and E7, which play a significant role in the carcinogenesis of penile squamous cell carcinoma [7–9].
Initially, when the transcription factor E2F binds to the pRb protein, it remains inactive. Overexpression of the hr-HPV E7 oncoprotein will disrupt the E2F-pRB complex by replacing the transcription factor E2F attached to pRb [7,10,11]. With this release, E2F becomes active and causes a cell phase transition from G1 to S. In the cell cycle, the G1 to S phase is controlled by the interaction of p16 protein with pRb protein. The main function of pRb is to bind and inhibit E2F, which causes a decrease in products involved in DNA formation and chromosomal replication. This disruption allows CDK-Inhibitor p16 to accumulate in the cell nucleus and inhibit CDK4 and CDK6 in G1 and leads to the inactivation of the tumor suppressor protein Rb [6,7,11,12].
Inactivation of pRb protein by oncoprotein E7 causes upregulation of p16 [7,10]. The expression of p16 then triggers negative feedback control of pRb and blocks the release of the transcription factor E2F. However, p16 could not neutralize the E2F transcription factor- mediated release of the HPV oncoprotein E7, resulting in an overaccumulation of the p16 protein in cells [7,10,12]. Disruption of the regulation of the pRb-p16 cell cycle pathway results in uncontrolled cell proliferation that ultimately contributes to malignant transformation.
Overexpression of the E6 oncoprotein acts by a different mechanism. Oncoprotein E6 inhibits the tumor suppressor protein p53 through ubiquitination so that the function of p53 is lost and causes uncontrolled cell activity and inhibits apoptosis [9].
In normal cells, p16 protein expression is only found in the parabasal epithelial cell nucleus, whereas in penile squamous cell carcinoma, p16 protein expression was found in all layers and not only in the cell nucleus but also in the cytoplasm of tumor cells [11]. Therefore, in tumors caused by hr-HPV, p16 protein overexpression may serve as an immunohistochemical marker and could be a target for antigen-specific immunotherapy [7,13,14].
This study aims to determine the expression of p16 protein in penile squamous cell carcinoma.
METHODS
This study is a one-time observational study involving 142 samples taken from paraffin blocks of penile cancer tumor tissues in 2015–2020 diagnosed with squamous cell carcinoma from 3 anatomic pathology laboratory centers in Bali. The research took place at the biomedical laboratory at the Faculty of Medicine and Health
Sciences, Warmadewa University, and the Anatomic Pathology Laboratory at hospitals throughout Bali. The total sampling method was used in this study. Initially, there were 150 samples. The enrolled samples met the following inclusion criteria: (1) paraffin block containing tumor mass; (2) paraffin block with good quality. Thus, after implementing the inclusion criteria, only 142 samples were used.
Paraffin tumor blocks were cut with a thickness of 2 microns and placed on a glass slide coated with polylysine. Deparaffination of the preparations was carried out using xylene and rehydration with a graded concentration of alcohol in running water. The rehydrated practices were then heated in citrate buffer solution for 35 minutes at a temperature of 95°C in a Biogear RG-1 type cooker; then, the preparations were cooled for 20 minutes. The samples were then washed with phosphate-buffered saline (PBS) twice for five minutes each for antigen retrieval purposes. The blocking of endogenous peroxidase with drops of Excell Block allowed it to stand for 5 minutes. The slides were dripped with p16 Cell Marque monoclonal primary antibody clone number:42 with a dilution of 1:100, incubated at room temperature for 60 minutes in a humidity chamber. The preparations were rewashed with PBS twice for five minutes each. The trials were then dripped with secondary antibody (Excell Link) and allowed to stand for 15 minutes, followed by washing back into PBS twice for 5 minutes each. The preparations were then dripped with Excell HRP, allowed to stand for 20 minutes, followed by washing using PBS twice for five minutes each. Chromogen 3,3’-diaminobenzidine (DAB) then leaked onto the preparation and was allowed to stand for a maximum of 1 minute. The samples were washed in running water after the administration of DAB and then administered with a counterstain using Mayer’s hematoxylin for 1 minute. The samples were washed with running water for 5 minutes, followed by drops of lithium carbonate, and rest for 30 seconds. The samples were then rewashed with running water for 5 minutes, followed by a dehydration process using graded alcohol and clearing with xylol. The following preparation was mounted and covered with a cover glass.
The expression of p16 was assessed by immunohistochemical examination and categorized into positive and negative. It was positive if intense brown stains appeared diffusely in the nucleus and cytoplasm in most tumor cells and negative if no stains in the nucleus and cytoplasm of tumor cells. Observations were made using an Olympus microscope with an objective lens magnification of 100–400x. The analysis method used in this study is a descriptive statistic to provide a general description of the characteristics of each research variable. The data shown in this study are the age range and prevalence of p16 protein expression in penile squamous cell carcinoma using prevalence tables.
This study was held after approval from the Ethics Committee, Faculty of Medicine, Udayana University/
RSUP Sanglah (Study number 2020.01.2.1082).
RESULT
Sample characteristics of the study population A total of 142 cases of penile squamous cell carcinoma that met the inclusion showed a varied age range of patients, namely from 33–91 years old, with a mean age of 60.42 ± 1.07 and the median age of 59.50. The distribution of research subjects based on age was categorized into seven groups. There were 6 samples aged between 30–39 years (4.2%), 25 samples aged between 40–49 years (17.6%), 40 samples aged between 50–59 (28.2%), 30 samples aged between 60–69 (21.1%), 32 samples aged between 70–79 (22.5%), 8 samples aged between 80–89 (5.6%), and 1 sample aged between 90–99 (0.7%). The highest number of samples was in the age range of 50–59 years (28.2%) (as shown in Table 1).
There were 68 cases with positive p16 protein overexpression and 74 cases with negative expression in this study. The mean age of penile squamous cell carcinoma patients with positive overexpression of p16 protein was 59 years with the youngest age being 33 years and the oldest being 86 years. Meanwhile, the mean age of penile squamous cell carcinoma patients with p16 protein that is negatively expressed is 62 years with the youngest age being 36 years and the oldest being 91 years.
Prevalence of p16 protein expression in penile squamous cell carcinoma
Based on the protein immunohistochemical detection method (p16-IHC), p16 protein expression was divided into two groups, namely positive and negative expressions. In this study, we found 68 cases with positive p16 overexpression (47.9%) and 74 cases (52.1%) with negative expression (as shown in Table 2).
Table 1. Sample characteristics of the study population
Age Frequency
(n) Percentage
(%)
30–39 years 6 4.2
40–49 years 25 17.6
50–59 years 40 28.2
60–69 years 30 21.1
70–79 years 32 22.5
80–89 years 8 5.6
90–99 years 1 0.7
Total 142 100
Table 2. Prevalence of p16 protein Expression in Penile Squamous Cell Carcinoma
Age Frequency
(n) Percentage
(%)
Positive 68 47.9
Negative 74 52.1
Total 142 100
DISCUSSION
The age range of penile squamous cell carcinoma samples varied from 33 to 91 years. The highest number of patients was between 50 and 59 years (28.2%). The results of this study are from several studies which found that the average age of diagnosis was around 60 years of age and rarely found under 55 years of age [1,14–16]. Age has a vital role in the risk of developing malignancy. The risk of penile squamous cell carcinoma will increase with age, with the age group most susceptible to penile squamous cell carcinoma between the 5th to 7th decade [1,15]. Many risk factors contribute to the development of tumors, such as environmental influences and genetic background, lack of circumcision, phimosis, multiple sex partners, HPV infection, early and high sexual activity [2,17].
Figure 1. (A&B) Positive p16 overexpression staining (100x magnification, 400x magnification respectively);
(C) Negative p16 staining (100x magnification)
The carcinogenesis of penile cancer is not yet fully known. In general, the carcinogenesis of penile squamous cell carcinoma comes from a chronic inflammatory process due to HPV or non-HPV infection that can trigger malignant transformation. The carcinogenesis mechanism of non-HPV was not fully understood [18]. The chronic inflammatory process results in reactive oxygen species (ROS) and reactive nitrogen intermediates that induce DNA damage and genomic instability [19]. The inflammatory mediator cyclooxygenase-2 (COX-2) is associated with tumor progression in the SCC penis [20].
HPV infection in invasive penile cancer varies from 30 to 70%, depending on the detection method [21,22].
Increasing cases of penile squamous cell carcinoma and increasing age, an intrinsic biological factor, can be associated with the risk of acquiring HPV infection in the oncogenesis pathway. This can occur due to the lack of an adaptive immune response that increases the chances of HPV infection, which then develops into malignancy [20–22]. In addition to the risk of HPV infection, age was also associated with the risk of viral persistence. The increase in age also increases the chance of hr-HPV infection. This can happen because of the impaired function of the immune system or the virulence of HPV [1,23].
The p16 protein is a tumor suppressor protein that is important in regulating the normal cell cycle. Infections caused by HPV will disrupt cell regulation and cause the accumulation of p16 protein in the nucleus. This accumulation will lead to uncontrolled cell proliferation, which will significantly contribute to the transformation towards malignancy [7]. hr-HPV infection types 16 and 18 were found in 88.6% of penile squamous cell carcinoma [24]. The data showed that 77.7% of cases indicated that the predominant oncogenic type was hr- HPV type 16 [24]. According to the World Health Organization and the International Society of Urological Pathology, it was decided that penile squamous cell carcinoma is a neoplasm associated with HPV infection, so it is recommended to use p16 immunohistochemical staining as a marker in HPV infection to determine histological classification [4,14].
Immunohistochemical staining of p16 protein was used to identify the presence of p16 protein overexpression in penile cancer based on antigen and antibody reactions [25]. Immunohistochemical staining of p16 protein was carried out according to the protocol specified by Vantagebio using a monoclonal mouse antibody with clone number: 42. In the sample used in this study, tissue preservation was carried out with formalin-fixed paraffin-embedded (FPPE), which was processed into colorless slides. The use of heat-induced epitope retrieval (HIER) in immunohistochemical staining procedures aims to remove the formalin blockage and free the targeted epitope. HIER in this study was carried out for 30 minutes at a temperature of 95°C. After
HIER. The 1:100 diluted p16 Biocare primary antibody was then dripped and attached to the antigen epitope in the target tissue. Then, chromagen dye was given to visualize the antigen-antibody reaction, and the last step was hematoxylin-eosin staining. The expression of p16 protein was assessed by immunohistochemical staining and categorized into two groups: positively expressed if stained with solid brown in the nucleus and cytoplasm in most tumor cells and negatively expressed if not stained in the nucleus and cytoplasm of tumor cells.
According to a study done in 2018, the examination of p16 protein by immunohistochemistry showed a 100%
sensitivity, 87 specificities, a negative predictive value of 72%, and a positive predictive value of 100% [26].
This test is superior to other methods of detecting hr- HPV because the p16 protein can describe gene expression. Pre-cancerous and cancerous lesions caused by hr-HPV infection will show solid and diffuse nuclear and cytoplasmic features. In this study, it was found that there were more negative expressions than positive expressions. The expression of p16 protein in penile squamous cell carcinoma is closely related to the presence of hr-HPV so the negative expression of p16 protein indicates the presence of low-risk HPV (lr-HPV) infection or the absence of HPV infection. This may be due to the possibility of non-HPV oncogenesis associated with the pRb pathway or the ability of some lr-HPV genotypes to induce malignant transformation in epithelial cells such as hr-HPV-induced malignancies [26–28]. A study conducted by Martinez-Bailon et al.
[29] in 2019, which linked HPV genotyping with overexpression of the p16 protein, stated that although p16 protein can be used as a surrogate marker in identifying malignancies caused by hr-HPV infection, there are still around 10% of cases with the hr-HPV infection expressed as p16 negative. This occurs because of other infections caused by HPV, the mechanism of inactivation of the gene encoding the protein p16 CDNK2A, and loss of heterozygosity [27]. Detection of p16 protein through immunohistochemical staining can be used as a morphological indicator in penile squamous cell carcinoma and an accurate determining indicator in diagnosing HPV infection when combined with HPV genotyping using PCR [30].
The limitation of this study is that it only shows the proportion of p16 expression. Further research is needed to determine the relationship of p16 with clinical and prognostic variables such as tumor size, grade, and stage.
CONCLUSIONS
This study found 68 cases with positive p16 overexpression (47.9%) and 74 cases (52.1%) with negative expressions in penile squamous cell carcinoma. Further research is needed to confirm the correlation between
HPV infection and p16 protein overexpression in penile squamous cell carcinoma, including HPV genotyping.
DECLARATIONS
Ethics ApprovalThis study was held after approval from the Ethics Committee, Faculty of Medicine, Udayana University/
RSUP Sanglah (Study number 2020.01.2.1082).
Competing of Interest
The authors declare no competing interest in this study.
Acknowledgment
We are thankful to all the laboratory staff for helping with the research.
REFERENCES
1. Douglawi A, Masterson TA. Penile cancer epidemiology and risk factors: A contemporary review. Curr Opin Urol. 2019;29(2):145–9.
2. Lestari DPO, Armerinayanti NW. Increasing trend of penile cancer cases in Bali compared to other regions in Indonesia. IOP Conf Ser Mater Sci Eng.
2018;434(1):0–5.
3. Lestari DP, Riasa IN, Niramayah PK, et al. The Burden of Penile Cancer in Bali Compared to Other Provinces in Indonesia: Penile Cancer. Open Access Macedonian Journal of Medical Sciences. 2021;9(E):1273-8.
4. Moch H, Cubilla AL, Humphrey PA, et al. The 2016 WHO Classification of Tumours of the Urinary System and Male Genital Organs—Part A: Renal, Penile, and Testicular Tumours. Eur Urol.
2016;70(1):93–105.
5. Pereira-Lourenço M, Vieira e Brito D, Peralta JP, et al. Prognostic value of p16INK4a overexpression in penile cancer. Arch Ital di Urol e Androl.
2020;92(1):11–6.
6. De Andrade Martins V, Pinho JD, Júnior AALT, et al. P16INK4a expression in patients with penile cancer. PLoS One. 2018;13(10).
7. Wai KC, Strohl MP, van Zante A, Ha PK. Molecular Diagnostics in Human Papillomavirus-Related Head and Neck Squamous Cell Carcinoma. Cells.
2020;9(2):500.
8. Chipollini J, Chaing S, Azizi M, et al. Advances in understanding of penile carcinogenesis: The search for actionable targets. Int J Mol Sci. 2017;18(8).
9. Rampias T, Sasaki C, Psyrri A. Molecular mechanisms of HPV induced carcinogenesis in head and neck.
Oral Oncol. 2014;50(5):356–63.
10. Laksmini LY, Widiana GR, Sriwidyani NP, Mulyadi K.
Ekspresi P16INK4a pada squamous cell carcinoma serviks teri. Maj Patol. 2014;23(3):24–31.
11. Ciesielska U, Zatonski T, Nowinska K, et al. Expression of cell cycle-related proteins p16, p27 and Ki-67 proliferating marker in laryngeal squamous cell carcinomas and in laryngeal papillomas. Anticancer Res. 2017;37(5):2407–15.
12. Kidd LC, Chaing S, Chipollini J, et al. Relationship between human papillomavirus and penile cancer- implications for prevention and treatment. Transl Androl Urol. 2017;6(5):791–802.
13. Sand FL, Rasmussen CL, Frederiksen MH, et al.
Prognostic significance of HPV and p16 status in men diagnosed with penile cancer: A systematic review and meta-analysis. Cancer Epidemiol Biomarkers Prev. 2018;27(10):1123–32.
14. Eich ML, del Carmen Rodriguez Pena M, Schwartz L, et al. Morphology, p16, HPV, and outcomes in squamous cell carcinoma of the penis: a multi- institutional study. Hum Pathol. 2020;96:79–86.
15. Hansen BT, Orumaa M, Lie AK, et al. Trends in incidence, mortality and survival of penile squamous cell carcinoma in Norway 1956–2015. Int J Cancer.
2018;142(8):1586–93.
16. Vieira CB, Feitoza L, Pinho J, et al. Profile of patients with penile cancer in the region with the highest worldwide incidence. Sci Rep. 2020;10(1):1–7.
17. Bruno L. Risk factors associated with the development of penile neoplasms. J Chem Inf Model.
2019;53(9):1689–99.
18. Thomas A, Necchi A, Muneer A, et al. Penile cancer.
Nat Rev Dis Prim. 2021;7(1):1–24.
19. Khansari N, Shakiba Y, Mahmoudi M. Chronic Inflammation and Oxidative Stress as a Major Cause of Age- Related Diseases and Cancer. Recent Pat Inflamm Allergy Drug Discov. 2009;3(1):73–80.
20. Lestari DPO, Mulyadi K, Moestikaningsih. Hubungan Positif Ukuran Tumor, Grade, Invasi Uretra, Invasi Intra Vasa, Invasi Perineural Dan Overekspresi Cox- 2 Pada Karsinoma Sel Skuamosa Penis Dengan Terjadinya Metastasis Ke Kelenjar Getah Bening Inguinal. Majalah Patologi. 2014;24(3):34-40.
21. White MC, Holman DM, Boehm JE, et al. Age and cancer risk: A potentially modifiable relationship.
Am J Prev Med. 2014;46(3 SUPPL. 1):S7.
22. Montes Cardona CE, García-Perdomo HA. Incidence of penile cancer worldwide: systematic review and meta-analysis. Rev Panam Salud Pública. 12;41:1–10.
23. Yu YB, Wang YH, Yang XC, et al. The relationship between human papillomavirus and penile cancer over the past decade: A systematic review and meta-analysis. Asian J Androl. 2019;21(4):375–80.
24. Slovin SF, Augusto Ornellas A, Ye D, et al. Importance of HPV in Chinese Penile Cancer: A Contemporary Multicenter Study. Front Oncol. 2020;10:1521.
25. Ozawa H. Principles and basics of immunohistochemistry. Folia Pharmacol Jpn.
2019;154(4):156–64.
29. Martínez-Bailón C, Mantilla-Morales A, Méndez- Matías G, et al. Human papillomavirus genotypes and P16INK4A expression in squamous penile carcinoma in Mexican patients. BMC Infect Dis.
2019;19(1):1–8.
30. Zito Marino F, Ronchi A, Stilo M, et al. Multiplex HPV RNA in situ hybridization/p16 immunohistochemistry: A novel approach to detect papillomavirus in HPV-related cancers. A novel multiplex ISH/IHC assay to detect HPV. Infect Agent Cancer. 2020;15(1):4–11.
26. Arista A, Murtiastutik D, Setyaningrum T, et al. Profil Ekspresi p16 ink4a dan Tipe Human papillomavirus (HPV) pada Pasien Kondilomata Akuminata Wanita (p16 ink4a Expression Profile and Human papillomavirus (HPV) Type in Female Condyloma Akuminata Patients). Period Dermatology Venereol.
2018;30(2):2–8.
27. De Andrade Martins V, Pinho JD, Júnior AALT, et al. P16INK4a expression in patients with penile cancer. Tornesello ML, editor. PLoS One. 2018;13(10):
e0205350.
28. Lohneis P, Boral S, Kaufmann AM, et al. Human papilloma virus status of penile squamous cell carcinoma is associated with differences in tumour- infiltrating T lymphocytes. Virchows Arch.
2015;466(3):323–31.
