miRNA Expression Characterizes Histological Subtypes and Metastasis in Penile Squamous Cell Carcinoma

Although microRNAs are described as promising biomarkers in many tumor types, little is known about their role in PSCC. Thus, we attempted to identify miRNAs involved in tumor development and metastasis in distinct histological subtypes considering the impact of HPV infection. In a first step, microarray analyses were performed on RNA from formalin-fixed, paraffin-embedded tumor (22), and normal (8) tissue samples. Microarray data were validated for selected miRNAs by qRT-PCR on an enlarged cohort, including 27 tumor and 18 normal tissues. We found 876 significantly differentially expressed miRNAs (p ≤ 0.01) between HPV-positive and HPV-negative tumor samples by microarray analysis. Although no significant differences were detected between normal and tumor tissue in the whole cohort, specific expression patterns occurred in distinct histological subtypes, such as HPV-negative usual PSCC (95 differentially expressed miRNAs, p ≤ 0.05) and HPV-positive basaloid/warty subtypes (247 differentially expressed miRNAs, p ≤ 0.05). Selected miRNAs were confirmed by qRT-PCR. Furthermore, microarray data revealed 118 miRNAs (p ≤ 0.01) that were significantly differentially expressed in metastatic versus non-metastatic usual PSCC. The lower expression levels for miR-137 and miR-328-3p in metastatic usual PSCC were validated by qRT-PCR. The results of this study confirmed that specific miRNAs could serve as potential diagnostic and prognostic markers in single PSCC subtypes and are associated with HPV-dependent pathways

Evaluation of Prognostic Parameters to Identify Aggressive Penile Carcinomas

Background: Advanced penile carcinoma is characterized by poor prognosis. Most data on prognostic factors are based on small study cohorts, and even meta-analyses are limited in patient numbers. Therefore, there is still a lack of evidence for clinical decisions. In addition, the most recent TNM classification is questionable; in line with previous studies, we found that it has not improved prognosis estimation. Methods: We evaluated 297 patients from Germany, Russia, and Portugal. Tissue samples from 233 patients were re-analyzed by two experienced pathologists. HPV status, p16, and histopathological parameters were evaluated for all patients. Results: Advanced lymph node metastases (N2, N3) were highly significantly associated with reductions in metastasis-free (MFS), cancer-specific (CS), and overall survival (OS) rates (p = <0.001), while lymphovascular invasion was a significant parameter for reduced CS and OS (p = 0.005; p = 0.007). Concerning the primary tumor stage, a significant difference in MFS was found only between pT1b and pT1a (p = 0.017), whereas CS and OS did not significantly differ between T categories. In patients without lymph node metastasis at the time of primary diagnosis, lymphovascular invasion was a significant prognostic parameter for lower MFS (p = 0.032). Histological subtypes differed in prognosis, with the worst outcome in basaloid carcinomas, but without statistical significance. HPV status was not associated with prognosis, either in the total cohort or in the usual type alone. Conclusion: Lymphatic involvement has the highest impact on prognosis in penile cancer, whereas HPV status alone is not suitable as a prognostic parameter. The pT1b stage, which includes grading, as well as lymphovascular and perineural invasion in the T stage, seems questionable; a revision of the TNM classification is therefore required

The prominent role of the S100A8/S100A9-CD147 axis in the progression of penile cancer

Currently, no established biomarkers are recommended for the routine diagnosis of penile carcinoma (PeCa). The rising incidence of this human papillomavirus (HPV)–related cancer entity highlights the need for promising candidates. The Calprotectin subunits S100A8 and S100A9 mark myeloid-derived suppressor cells in other HPV-related entities while their receptor CD147 was discussed to identify patients with PeCa at a higher risk for poor prognoses and treatment failure. We thus examined their expression using immunohistochemistry staining of PeCa specimens from 74 patients on tissue microarrays of the tumor center, the invasion front, and lymph node metastases. Notably, whereas the tumor center was significantly more intensively stained than the invasion front, lymph node metastases were thoroughly positive for both S100 subunits. An HPV-positive status combined with an S100A8+S100A9+ profile was related with an elevated risk for metastases. We observed several PeCa specimens with S100A8+S100A9+-infiltrating immune cells overlapping with CD15 marking neutrophils. The S100A8+S100A9+CD15+ profile was associated with dedifferentiated and metastasizing PeCa, predominantly of HPV-associated subtype. These data suggest a contribution of neutrophil-derived suppressor cells to the progression of HPV-driven penile carcinogenesis. CD147 was elevated, expressed in PeCa specimens, prominently at the tumor center and in HPV-positive PeCa cell lines. CD147+HPV+ PeCa specimens were with the higher-frequency metastasizing cancers. Moreover, an elevated expression of CD147 of HPV-positive PeCa cell lines correlated negatively with the susceptibility to IgA-based neutrophil-mediated tumor cell killing. Finally, stratifying patients regarding their HPV/S100A8/S100A9/CD15/CD147 profile may help identify patients with progressing cancer and tailor immunotherapeutic treatment strategies

DKK1 inhibits canonical Wnt signaling in human papillomavirus-positive penile cancer cells

Penile squamous cell cancer (PSCC) is the most frequent penile malignant disease. Infections with human papillomaviruses (HPV) are a major etiologic driver of PSCC. However, the molecular details of the underlying carcinogenesis are understudied because of rare clinical specimens and missing cell lines. Here, we investigated if the expression of high-risk HPV16 oncogenes causes an augmentation of the Wnt pathway using unique HPV-positive penile cancer (PeCa) cell lines in monolayer and organotypic 3D raft cultures as well as tissue micro arrays containing clinical tissue specimens. The HPV oncoproteins enhanced the expression of Leucine-rich repeat-containing G-protein coupled receptor 6 (LGR6) and the HPV-positive PeCa cells expressed a signature of Wnt target and stemness-associated genes. However, the notable lack of nuclear β-catenin in vitro and in situ raised the question if the enhanced expression of Wnt pathway factors is tantamount to an active Wnt signaling. Subsequent TOP-flash reporter assays revealed Wnt signaling as absent and not inducible by respective Wnt ligands in PeCa cell lines. The HPV-positive PeCa cells and especially HPV-positive PeCa specimens of the tumor core expressed the Wnt antagonist and negative feedback-regulator Dickkopf1 (DKK1). Subsequent neutralization experiments using PeCa cell line-conditioned media demonstrated that DKK1 is capable to impair ligand-induced Wnt signaling. While gene expression analyses suggested an augmented and active canonical Wnt pathway, the respective signaling was inhibited due to the endogenous expression of the antagonist DKK1. Subsequent TMA stainings indicated Dkk1 as linked with HPV-positivity and metastatic disease progression in PeCa suggesting potential as a prognostic marker

miRNA Expression Characterizes Histological Subtypes and Metastasis in Penile Squamous Cell Carcinoma

Although microRNAs are described as promising biomarkers in many tumor types, little is known about their role in PSCC. Thus, we attempted to identify miRNAs involved in tumor development and metastasis in distinct histological subtypes considering the impact of HPV infection. In a first step, microarray analyses were performed on RNA from formalin-fixed, paraffin-embedded tumor (22), and normal (8) tissue samples. Microarray data were validated for selected miRNAs by qRT-PCR on an enlarged cohort, including 27 tumor and 18 normal tissues. We found 876 significantly differentially expressed miRNAs (p ≤ 0.01) between HPV-positive and HPV-negative tumor samples by microarray analysis. Although no significant differences were detected between normal and tumor tissue in the whole cohort, specific expression patterns occurred in distinct histological subtypes, such as HPV-negative usual PSCC (95 differentially expressed miRNAs, p ≤ 0.05) and HPV-positive basaloid/warty subtypes (247 differentially expressed miRNAs, p ≤ 0.05). Selected miRNAs were confirmed by qRT-PCR. Furthermore, microarray data revealed 118 miRNAs (p ≤ 0.01) that were significantly differentially expressed in metastatic versus non-metastatic usual PSCC. The lower expression levels for miR-137 and miR-328-3p in metastatic usual PSCC were validated by qRT-PCR. The results of this study confirmed that specific miRNAs could serve as potential diagnostic and prognostic markers in single PSCC subtypes and are associated with HPV-dependent pathways