The role of human high-risk papillomavirus infection as a causative agent in the development of penile squamous cell carcinoma is well established [
20]. However, the impact of HPV-related tumourigenesis on tumour morphology as well as aggressiveness is still subject to research and has led to contradictory results [
15-
17]. Moreover, it is still not clear to which extent immunostaining for p16
INK4a might be regarded as a surrogate marker for HPV infection, since site-specific differences in sensitivity and specificity have been reported [
10,
21-
23]. The observed variety of staining patterns for p16
INK4a further complicates histologic evaluation [
24,
25]. Therefore, the aim of this study was to evaluate p16
INK4a staining patterns and results from HPV DNA subtyping with histopathological and clinical characteristics in a cohort of 58 patients with penile squamous cell carcinoma (pSCC). Among the study cohort, 91% of pSCCs were classified as conventional-type (keratinizing pSCC) because various degrees of keratinization could be observed in these cases. This is a higher proportion than has been described in earlier studies, while the rate of basaloid-type tumours was comparable to data from literature [
11,
16,
26]. HPV DNA (of any subtype) was detected in 31% of all cases, with HR-HPV DNA (HPV-16 and HPV-45) being present in 28% of conventional-type and 40% of basaloid-type carcinomas. Thus, despite a specificity of 95% and a positive predictive value of 40%, the resulting sensitivity of basaloid tumour differentiation for the presence of HR-HPV DNA was only 11%. Due to the significant proportion of conventional-type (keratinizing) tumours that turned out HPV-positive in PCR-based assays, our results do not support the correlation between presence of HR-HPV DNA and basaloid tumour subtype that has been previously described by other authors. This might be due to the fact that distinguishing one histological type from another can be challenging, and overlapping differentiation patterns as well as intratumoural heterogeneity exist [
27]. Therefore, we would not recommend to rely solely on basaloid tumour subtype when assessing hints for HPV-driven tumourigenesis as proposed by some authors but rather use a combination of criteria as proposed in a 2014 study by Chaux et al. [
28,
29]. Positive immunostaining for p16
INK4a, a tumour suppressor protein that is regarded as a surrogate marker for HPV-associated tumours in other organs [
7,
30], was observed in 59% of specimens. There was a wide variety from scattered-focal over confluent-intense to almost exclusively nuclear staining patterns. P16
INK4a immunostaining correlated significantly with the presence of HR-HPV DNA with good sensitivity (100%), but lacked specificity (60%) when all staining patterns were considered (PPV, 53%). Considering only those specimens with intense nuclear positivity for p16
INK4a in all tumour cells improved the specificity for the presence of HR-HPV DNA to 85% (PPV, 75%). P16
INK4a was present in all HPV-positive cases; however, lack of p16
INK4a immunostaining in HPV-associated tumours due to loss of heterozygosity near the
CDKN2A locus and/or hypermethylation of the
CDKN2A promoter has been described previously [
31]. It is therefore well conceivable that these genetic aberrations accumulate stepwise during tumour progression, and could not be observed here due to the relatively large proportion of early/noninvasive HPV-positive tumours in our study. Also, it has to be stated that we did not examine p16
INK4a expression in corresponding metastases, but a 2014 study showed identical immunohistochemical or HPV in situ hybridization profiles between primary pSCCs and their corresponding metastases [
32]. However, the question remains if there is a prognostic value of HR-HPV-driven tumourigenesis in pSCC at all. To address this, we investigated whether presence of HR-HPV DNA was associated with tumour aggressiveness or cancer-specific survival in pSCC. We found that HR-HPV as well as p16
INK4a positivity was significantly associated with non-invasive tumour growth (pTis/pTa stage). This finding is in contrast to a proposed proinvasive role for HPV oncoproteins that has been recently described in head and neck as well as cervical cancer, but is supported by data for penile cancer published by other authors [
13,
16,
33-
35]. Our findings therefore add to the growing evidence that there are site-specific differences in the role of HPV regarding the gain of an invasive phenotype. These differences may be linked to interaction of HPV-derived oncoproteins with β-integrin localization and signalling [
36]. For nodal or distant metastasis, there was no significant correlation with presence of HR-HPV DNA, p16
INK4a staining or histologic grade in our sample set; similar results have been previously described [
37]. Furthermore, we detected no significant differences in cancer-specific survival with regard to histologic differentiation grade, or p16
INK4a positivity. This is in line with some previous studies that failed to confirm a proposed association between histopathologic grade and lymph node metastasis or overall survival in pSCC, while it has to be stated as a clear limitation of our study that follow-up data was only obtainable for 35 patients (60.3%) [
38-
40]. Considering the substantial interobserver variability that has been described for histopathologic grading in pSCC (between 59-87% with ĸ = 0.38-0.69 [
41]), we think that the current histopathologic grading is of limited value due to an obvious lack of prognostic relevance; this conclusion is also supported by other authors [
42]. For the presence of HR-HPV we observed a trend towards better prognosis that failed to reach statistical significance; further investigations in larger cohorts might therefor be indicated.