11Archivio Italiano di Urologia e Andrologia 2020; 92, 1 ORIGINAL PAPER Prognostic value of p16INK4a overexpression in penile cancer Mário Pereira-Lourenço 1, Duarte Vieira e Brito 1, Miguel Eliseu 2, Noémia Castelo-Branco 3, João Pedro Peralta 1, Ricardo Godinho 1, Paulo Conceição 1, Mário Reis 1, Carlos Rabaça 1, Amílcar Sismeiro 1 1 Urology department Portuguese Institute of Oncology Coimbra, Coimbra, Portugal; 2 Urology and Kidney transplant department Coimbra Hospital University Centre, Coimbra, Portugal; 3 Pathology department Portuguese Institute of Oncology Coimbra, Coimbra, Portugal. Introduction: Penile cancer is rare, account- ing for less than 1% of all male cancers in industrialized countries. It is most common in areas of high prevalence of HPV, being a third of cases attributed to the car- cinogenic effect of HPV. Tumour cells infected with HPV over- express p16INK4a, as such p16INK4a has been used as a surro- gate of HPV infections. Objective: To evaluate the prognostic factor of p16INK4a over- expression in penile cancer. Methods: Retrospective analysis of patients diagnosed with penile cancer, submitted to surgery in a Portuguese Oncological Institution in the last 20 years (n = 35). Histological review of surgical pieces and immunohistochemi- cal identification of p16INK4a. Relation between p16INK4a and the following factors were studied: age, histological subtype, tumour dimensions, grade, TNM stage, perineural invasion, perivascular invasion, disease free survival (DFS) and cancer specific survival (CSS). Results: p16INK4a was positive in 8 patients (22.9%). Identification of p16INK4a did not correlate with none of the histopathological factors. In this work we identified a better DFS and CSS in patients positive for p16INK4a (DFS at 36 months was 100.0% vs. 66.7%; CSS at 36 months was 100.0% vs. 70.4%), although without statistical significance (p > 0.05). In multivariate analysis of histopathological factors studied, only N staging correlated with DFS and CSS (p = 0.017 and p = 0.014, respectively). Discussion: the percentage of cases positive for p16INK4a is smaller than the one found in literature, which can suggest a less relevant part of HPV infection in the oncogenesis of penile cancer in the studied population. Identification of p16INK4a did not relate with other clinicopathological factors. Tendency for a more favourable prognosis in patients with p16INK4a agrees with results found in literature. The most relevant factor for prognosis is nodal staging. Conclusions: penile cancer positive for p16INK4a shows a trend for better survival, although the most relevant factor is nodal staging. KEY WORDS: Penile cancer; HPV; p16; Prognosis. Submitted 29 August 2019; Accepted 1 September 2019 Summary No conflict of interest declared. DOI: 10.4081/aiua.2020.1.11 INTRODUCTION In industrialized countries, penile cancer is rare, having an incidence of approximately 1/100000 in Europe and the United States of America, accounting for less than 1% of all male cancers (1-3). However, in other regions of the world, particularly South America, Southeast Asia and parts of Africa, the incidence of penile cancer is much greater, accounting for about 2% of all male cancers, although in some countries it can reach 10% (4, 5). The incidence of penile cancer increases with age, reach- ing a peak in the six decade, although it can occur in much younger patients (1, 6). Penile cancer is most common in areas with high preva- lence of HPV, with a third of cases attributed to the car- cinogenic effects of HPV (1, 7). Other risk factors iden- tified where: phimosis (8, 9), chronic penile inflamma- tion/ lichen sclerosus (10), psoralene and phototherapy with ultraviolet radiation A (11), smoking (8), residing in rural areas/low socioeconomical level (12, 13) and multiple sexual partners (8). In relation to penile cancer, HPV DNA was identified in 30-40% of cases, varying in accordance to histological subtype. The histological subtypes most associated with HPV are Basaloid Penile Squamous Cells Carcinoma (PSCC) (76%), mixed Warty-basaloid PSCC (82%) and Warty PSCC (39%). The Usual PSCC and Papillary PSCC are not associated with HPV (14, 15). Although the classic PSCC is normally characterized as non-related to HPV, a recent metanalysis identified an association in over 30% of cases (16). The subtypes of HPV most commonly associated with penile cancer are 16 and 18 (17). The World Health Organization (WHO), utilizing the hypothesis of independent pathways of carcinogenesis, categorizes PSCC regarding HPV (18, 19). The prognos- tic value of the association with HPV is still controver- sial, with recent studies showing a better outcome in HPV associated penile cancer (20-22), while others do not show significant differences (18, 23). Various methods can be used to detect HPV in tumour cells, such as PCR amplification to detect HPV DNA. Due to the overexpression of p16INK4a in HPV infected cells, p16INK4a expression can be used as a surrogate of active HPV infections (16). In cervical cancer and in other squa- mous cell carcinomas, expression of p16INK4a is used as a marker for the presence of high-risk HPV (17-19). Progression and regression of low grade intraepithelial cervical cancer can be estimated utilizing p16INK4a and mark a better cancer specific survival (CSS). However, the Lourenco_Stesura Seveso 01/04/20 18:52 Pagina 11 Archivio Italiano di Urologia e Andrologia 2020; 92, 1 M. Pereira-Lourenço, D. Vieira e Brito, M, Eliseu, N. Castelo-Branco, J.P. Peralta, R. Godinho, P. Conceição, M. Reis, C. Rabaça, A. Sismeiro 12 correlation between expression of p16INK4a and HPV infection in penile cancer is still controversial (24). The main aim of this paper is to evaluate the prognostic value of p16INK4 expression in penile cancer. Other goals are to evaluate the epidemiologic association between HPV and penile cancer in Portugal (indirect assessment by assessing expression of p16INK4a) and to evaluate the association between HPV and histological subtypes of PSCC and the initial staging of the disease. METHODS Patient selection and data collection Retrospective analysis of all patients with the primary diagnosis of penile cancer treated in a Portuguese onco- logical institution, in the last 20 years. Retrospective eval- uation of patient data. Pathological and immunohistochemistry evaluation All surgical specimens of the identified patients were revaluated for this study. The material was fixed in 10% formol, embedded in paraffin and stained with haematoxilin-eosine, and was reviewed by a genitourinary pathologist that determined the histologic subtyping and the pathological grade using the morphologic criteria presented in the WHO classifi- cation of tumours of the penis of 2016 and tumour stag- ing was made according the AJCC cancer staging manu- al of 2017 (19, 25). Immunohistochemical analysis was performed on the BenchMark-Ultra platform (Ventana R). Antigenic retrieval was performed using the Ultraview Universe Dab Detection Kit (Ventana R). Slides were then incubated with monoclonal antibody to P16ink4a (mouse clone E6H4, CINtec R p16 Histology, Ventana R). The Bond Polymer Refine detection system (Ventana) was used for secondary antibody and visualization. Cervical squamous cell carcinoma was used as positive control, and benign skin as negative control. Cases were scored by a genitourinary pathologist. To define the expression patterns of p16INK4a, the classification of Cubila et al. (26) was adapted, and overexpression of p16INK4a was defined as diffuse, continuous, and strong nuclear and cytoplas- mic staining of the neoplastic cells. Discontinuous, focal and weak staining as well the absence of staining was interpreted as negative for p16INK4a overexpression. Statistical analysis The program SPSS 21 was used for statistical analysis. We used the Mann-Whitney test to assess the relation between clinical and pathological characteristics and p16INK4a. Survival related to each individual factor were calculated by the Kaplen-Meyer curves. Multivariate analysis utilizing Cox regression was utilized for the impact of clinical and pathological factors on survival. RESULTS Clinicopathological data The total number of patients was 35, the median age was 69 (range 33-90 years) and the median tumour size was of 2.5 cm (range, 0.4-12.0). Eight patients (22.9%) pre- sented with positive p16INK4a test. Relating to T staging, 1 (2.9%) presented with Tis, 13 (37.1%) T1, 11 (31.4%) T2 and 10 (28.6%) T3. The clinicopathological results are summarized in Table 1. P16INK4a immunoexpression The relation between p16INK4a expression and the remain- ing clinicopathological results are summarized in Table 2. P16INK4a immunoexpression did not correlate in a signifi- cant way (p > 0.05) with none of studied factors. P16INK4a immunoexpression and prognosis The median follow-up was 63 months (range 6-204). The Kaplan-Meyer curves of disease-free survival (DFS) and cancer specific survival (CSS) in relation to P16INK4a immunoexpression are presented in Figures 1, 2, respec- tively. Although a tendency to a longer survival with pos- itive P16INK4a immunoexpression, this was not statically significant (DFS: p = 0.219; CSS: p = 0.067). The DFS and CSS at 3 years for patients with positive P16INK4a immunoexpression were 100.0% and 100.0%, respec- tively. The DFS and CSS at 3 years for patients with neg- ative P16INK4a immunoexpression were 66.7% and 70.4%, respectively. Other clinicopathological factors and prognosis The disease-free survival and cancer specific survival in relation to T stage, N stage, tumour grade, perineural invasion and perivascular invasion were evaluated. In relation to DFS, the following factors were associated with higher survival: T stage T ≤ 1 (p = 0.002) and N = 0 (p < 0.001). Table 1. Clinicopathological results. N (%) Age (years) • < 65 13 (37.1%) • ≥ 65 22 (62.9%) PSCC histologic subtype • Usual 28 (80%) • Warty 3 (8.6%) • Verrucous 2 (5.7%) • Mixed Warty-Basaloid 2 (5.7%) Dimension (cm) • < 4 26 (74.8%) • ≥ 4 9 (25.7%) p16INK4a • Positive 8 (22.9%) • Negative 25 (78.1%) Differentiation grade • G1 14 (40%) • G2 15 (42.9%) • G3 6 (17.1%) T stage • ≤ 1 14 (40.0%) • > 1 21 (60.0%) Lymph node metastasis • No 26 (74.3%) • Yes 9 (25.7%) Died of the disease • No 26 (74.3%) • Yes 9 (25.7%) Lourenco_Stesura Seveso 01/04/20 18:52 Pagina 12 Analysing CSS the following factors presented statistical- ly significant improved survival: age < 65 years (p = 0.042), stage T ≤ 1 (p = 0.005), stage N = 0 (p < 0.001). In a multivariate Cox regression analysis with the previ- ously described factors and HPV, the model was statisti- cally significant in relation with DFS and CSS, although only N stage presented with statically relevance (p = 0.017 and p = 0.014, respectively). DISCUSSION In this study, we identified 22.9% of P16INK4a positive PSCC, a value smaller than the one calculated by a recent metanalysis, which identified P16INK4a in 42.6% (95% CI; 36.2-47.0) worldwide (2995 cases) and 44.9% (95% CI; 38.4-51.1) in Europe (16). A Spanish study (Bar - celona), interesting to compare due to the geographic proximity with Portugal, identified a P16INK4a positivity in 34.0% of 72 cases (22). In order to understand this result, it is essential to comprehend the meaning of pos- itive P16INK4a and its correlation with the physio-patho- logical role of HPV infection in penile cancer. The most sensitive method for the detection of HPV in tumoral tis- sue is PCR amplification (27). Due to the strong correla- tion between active HPV and P16INK4a overexpression in neoplastic cells, it has been used as a surrogate marker for HPV (28). The incorporation of high-risk HPV (HR- HPV) in the host genome, leads to the overexpression of oncoproteins (E7 and E6). The protein E7 binds to retinoblastoma protein, leading to the increased expres- sion of p16 (tumour suppressing protein). This overex- pression can be used as a reliable marker for high-risk HPV infection (26). Sensitivity and specificity of P16INK4a expression in HR-HPV was 100% and 57%, respectively (29). This lack of specificity leads some authors to defend that identification of HPV DNA is fundamental (30). According to Cubilla et al., positivity for P16INK4a in penile cancer has a strong correlation with the presence of HR-HPV (26). In the presence of a negative P16INK4a, infection with a low risk HPV genotype or absence of HPV infection can be suspected (24). In the previously addressed metanalysis by Olesen et al. (16), 79.6% of HPV positive cases presented with a pos- itive P16INK4a, while 18.5% of HPV negative cases also presented with positive P16INK4a. One of the explana- 13Archivio Italiano di Urologia e Andrologia 2020; 92, 1 p16INK4a overexpression in penile cancer Figure 1. Disease free survival for p16INK4a positive and negative patients. Figure 2. Cancer specific survival for p16INK4a positive and negative patients. Table 2. Relation between p16INK4a expression and the remaining clinicopathological results. N (%) Age (years) • < 65 2 (25.0%) 11 (40.7%) 0.425 • ≥ 65 6 (75.0%) 16 (59.3%) PSCC histologic subtype • Usual 6 (75%) 22 (81.5%) 0.800 • Warty 1 (12.5%) 2 (7.4%) • Verrucous 0 (0.0%) 2 (7.4%) • Mixed Warty-Basaloid 1 (12.5%) 1 (3.7%) Dimension (cm) • < 4 6 (75.0%) 20 (74.1%) 0.318 • ≥ 4 2 (25.0%) 6 (22.2%) Differentiation grade • G1 4 (50.0%) 10 (37.0%) 0.510 • G2 3 (37.5%) 12 (44.4%) • G3 1 (12.5%) 5 (18.5%) Perineural invasion • No 7 (87.5%) 4 (14.8%) 0.871 • Yes 1 (12.5%) 23 (85.2%) Lymphovascular invasion • No 7 (87.5%) 24 (88.9%) 0.915 • Yes 1 (12.5%) 3 (11.1%) T stage • ≤ 1 3 (37.5%) 11 (40.7%) 0.871 • > 1 5 (62.5%) 16 (59.3%) Lymph node metastasis • No 8 (100.0%) 18 (66.7%) 0.062 • Yes 0 (0.0%) 9 (33.3%) Died of the disease • No 8 (100.0%) 18 (66.7%) 0.062 • Yes 0 (0.0%) 9 (33.3%) Lourenco_Stesura Seveso 01/04/20 18:52 Pagina 13 Archivio Italiano di Urologia e Andrologia 2020; 92, 1 M. Pereira-Lourenço, D. Vieira e Brito, M, Eliseu, N. Castelo-Branco, J.P. Peralta, R. Godinho, P. Conceição, M. Reis, C. Rabaça, A. Sismeiro 14 tions for this variation and apparent incoherence may be the cut-off value used to consider a positive P16INK4a, although Olesen et al. did not find a significant difference between different cut-offs (16). Two recent reviews iden- tified a prevalence of HPV positive PSCC (identified by PCR amplification) of 33.1% (31) and 39.4% (32). Nevertheless, various authors consider that a tumour can only be considered HPV positive if it presents with dou- ble positivity for HPV and P16INK4a (16, 18, 33). In an interesting way, positivity for P16INK4a correlates with the presence of high-risk HPV subtypes (HPV 16, HPV18 e HPV59) (18). In this work there was no correlation between P16INK4a and histologic subtype. In the case of Usual PSCC, 6 (21.4%) were P16INK4a positive. Concerning Warty PSCC, Mixed PSCC (Basaloid + Warty) and Verrucous PSCC, the number of P16INK4a positive patients was 1 (33.3%), 1 (50%) and 0 (0%) respectively. The last World Health Organization (WHO), divides PSCC in tumours related to HPV and non-related to HPV, as there may be prognostic importance in this division (19). Curiously, usual PSCC is identified as non-related with HPV (as are Papillary, Verrucous, Sarcomatous and oth- ers), although data collected from literature indicates a prevalence of HPV DNA in Usual PSCC of 32.2% and positive P16INK4a of 36.9% (16). Our results and data from analysed literature, indicates that the classification Usual PSCC as independent of HPV is limited. In relation to Warty PSCC and mixed Basaloid-Warty PSCC (both classified as tumours related to HPV), literature indicates positivity for P16INK4a in > 90% of cases (16). The low number of Warty and Mixed Basaloid-Warty PSCC in our series does not allow for sustained comparations, although they corroborate the limitations present on the suggest classification presented by the WHO. In our work we did not directly study the presence of HPV, as such we cannot logically study the different HPV subtypes associated with PSCC. In developed and unde- veloped countries, the predominant HRHPV associated with PSCC is HPV-16 as shown by several studies. Although uncommon in European countries, HPV-18 is the second most prevalent in PSCC in the World (22, 34). In the metanalysis conducted by Olesen et al., HPV16 (68.3%), followed by HPV6 (8.1%%) and HPV18 (6.9%). were the predominant oncogenic subtypes (16). In our study, we did not find any relation between P16INK4a and other histologic characteristics. Pone et al. did not find a relation between P16INK4a and other histo- logic characteristics (size, clinical stage, histological grade, or lymphatic or perineural invasion), although identified a relation with histologic subtype (24). Our series did not present with any Basaloid tumour, although literature indicates a relation between positive P16INK4a and this histological subtype (26). Ferrándiz-Pulido et al. identified a connection between positive P16INK4a and histological differentiation (P16INK4a was associated with G3/4) and histological subtype (22). Some works distin- guish between penile epithelial neoplasia (PEN) and PSCC in evaluating the importance of HPV and P16INK4a, as most of PEN (> 70%) are HPV+. We decided not to exclude the single patient with PEN from our work, as positivity for P16INK4a between PEN and PSCC are very similar (49.5% vs. 41.6%, respectively) (16). Analysing prognosis, we did not find, in this work, a significant sta- tistical relation between P16INK4, DSF and CSS. Nevertheless, there is a clear trend for a better outcome in patients positive for P16INK4a with only one patient presenting with recurrence and no case of disease relat- ed mortality. The absence of statistical significance is probably related with the low number of P16INK4a posi- tive tumours in our sample. Various works have studied the effect of HPV and P16INK4a in the prognosis of PSCC. Regarding the effect HPV in DFS, Afonso et al. (112 patients, median follow-up of 20 months) and Lorenzo et al. (30 patients, median follow-up of 24 months) did not detect significant differences (35, 36). Scheiner et al. (72 patients) reported a better DFS at 5 years, although with- out statistical significance (37). Concerning the effects of HPV in CSS, in the review by Sand et al. (649 patients, 174 HPV+) a better CSS for patients HPV positive was noted (HR 0.61; 95% CI: 0.38-0.98) (32). Analysing the effects of HPV in Overall Survival (OS), studies did not show a significant relation (32, 38). Tang et al. described a better DFS in patients positive for P16INK4a (119 patients, 59 P16INK4a positive, median fol- low-up of 30 months) (44). However, other works did not show a relation between P16INK4a and DFS. The effect of P16INK4a in CSS was studied by Sand et al. (review of 414 patients, 191 positive for P16INK4a) with a HR of 0.45 (95% CI: 0.30- 0.69) for patients positive for P16INK4a (32). The percentage of patients alive 4 or 5 years after diagnosis range from 69% to 100% for P16INK4a positive and from 51% to 77% if P16INK4a neg- ative (22, 23, 29, 39-43). All the study-specific HRs are below 1 and ranging from 0.21 to 0.81, however only one (40) was statistically significant. Regarding OS, Pone et al. reported a better OS in patients positive for P16INK4a, with a HR of 0.88 (95%CI: 0.49-1.59) (24). Zargar-Shoshtari et al. found that men with penile cancer positive for P16INK4a had a significant better OS com- pared with negative P16INK4a (HR = 0.33; 95% CI: 0.13- 0.85) in a multivariable model adjusting for pathological nodal status, adjuvant chemotherapy and age (42). Tang et al. did not find a connection between P16INK4a and OS (44). In this work, due to the discharge from follow-up of some patients and limitation in the quality of data col- lection outside our institution, we did not calculate OS. In general, bibliography demonstrates a better survival for patients positive for HPV, as reported in other tumours related with HPV (vulvar, oropharyngeal) (32). Sand et al., in the previously referred metanalysis, that analysed the HR of CSS of P16INK4a and CSS of HPV pos- itive patients, discovered that the HR of CSS P16INK4a positive patients was lower than that of HPV positive patients. This could suggest that P16INK4a expression may be a stronger predictor of CSS than HPV, similar to studies of neck and head cancer (32). The prognostic value of HPV is still uncertain. Some have suggested that the presence of a viral infection (HPV), might increase immune surveillance, making HPV positive cancer less aggressive than non-viral cancers (21). In univariate analysis with other clinicopathological fac- tors, a significant relation was found between T staging and N staging with DFS and CSS, while age > 65 years Lourenco_Stesura Seveso 01/04/20 18:52 Pagina 14 presented with lower CSS. In multivariate analysis, only N staging correlated with survival. A work by Wen et al. (135 patients), reported a relation between N staging (clinical and pathologic) and CSS. In multivariate analy- sis, only pathologic N staging related with CSS (absence of relation between CSS and age, presence of phimoses, smoking, type of surgery, T stage or grade) (46). Our study presented with some limitations. Our series presents a limited number of patients, with only eight P16INK4a positive patients, which limits statistical results. In our work we did not evaluate the presence of HPV DNA, which can be relevant to corroborate the know connection between P16INK4a status. 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Correspondence Mário Pereira-Lourenço, MD (Corresponding Author) mariolourenco88@gmail.com Duarte Vieira e Brito, MD João Pedro Peralta, MD Ricardo Godinho, MD Paulo Conceição, MD Mário Reis, MD Carlos Rabaça, MD Amílcar Sismeiro, MD Urology Department Portuguese Institute of Oncology Coimbra, Coimbra (Portugal) Rua Maria Bourbon Bobone, n57, RE/Esq, Coimbra, 3030-481, Portugal Miguel Eliseu, MD Urology and Kidney transplant Department Coimbra Hospital University Centre, Coimbra (Portugal) Noémia Castelo-Branco, MD Department Portuguese Institute of Oncology Coimbra, Coimbra (Portugal) Lourenco_Stesura Seveso 01/04/20 18:52 Pagina 16