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Purpose: Our objective was to investigate the association between programmed death-ligand (PD-L1) immunoexpression measured as a combined positive score and clinical outcomes in penile SqCC., Materials and Methods: We retrospectively reviewed all penile SqCC cases diagnosed in our institution between 2018 and 2023. PD-L1 immunohistochemistry was performed as a qualitative assay. Immunoexpression in both tumor and immune cells equal or superior to 1 was considered positive., Results: A total of 34 patients with conventional penile SqCC were included. Eleven cases were HPV-associated (32.4%). Twelve cases were PD-L1 CPS < 1 and twenty-two were PD-L1 CPS ≥ 1. Nine cases (32.4%) were PD-L1 CPS ≥ 1 and p16 positive, but this did not translate in worse clinicopathological features. Larger tumors (3.0 cm in PD-L1 CPS ≥ 1 vs 2.5 cm in PD-L1 CPS < 1; p = 0.662), vascular invasion (36.4% in PD-L1 CPS ≥ 1 vs. 25.0% in PD-L1 CPS < 1; p = 0.705) and perineural invasion (40.9% in PD-L1 CPS≥1 vs. 16.7% in PD-L1 CPS < 1; p = 0.252) were associated with PD-L1 expression. Among the high-risk features, only lymph node involvement had statistical significance, with 14 out of 22 PD-L1 CPS ≥ 1 patients (63.6%) having lymph node metastases when lymphadenectomy was performed (p = 0.031). With a median follow-up of 16 months (IQR 27.5), PD-L1 CPS ≥ 1 patients had worse overall survival (53.4 months vs 75.9 months), but no statistical significance could be inferred (p = 0.188)., Conclusions: It is noteworthy the clinical significance of lymph node involvement in PD-L1 CPS ≥ 1 cases and a trend towards worse overall survival in this group of patients.

Background: To uncover the potential significance of JAK-STAT-SOCS1 axis in penile cancer, our study was the pioneer in exploring the altered expression processes of JAK-STAT-SOCS1 axis in tumorigenesis, malignant progression and lymphatic metastasis of penile cancer. Methods: In current study, the comprehensive analysis of JAK-STAT-SOCS1 axis in penile cancer was analyzed via multiple analysis approaches based on GSE196978 data, single-cell data (6 cancer samples) and bulk RNA data (7 cancer samples and 7 metastasis lymph nodes). Results: Our study observed an altered molecular expression of JAK-STAT-SOCS1 axis during three different stages of penile cancer, from tumorigenesis to malignant progression to lymphatic metastasis. STAT4 was an important dominant molecule in penile cancer, which mediated the immunosuppressive tumor microenvironment by driving the apoptosis of cytotoxic T cell and was also a valuable biomarker of immune checkpoint inhibitor treatment response. Conclusions: Our findings revealed that the complexity of JAK-STAT-SOCS1 axis and the predominant role of STAT4 in penile cancer, which can mediate tumorigenesis, malignant progression, and lymphatic metastasis. This insight provided valuable information for developing precise treatment strategies for patients with penile cancer., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Tumor-infiltrating lymphocytes (TILs) play a key role in regulating the host immune response and shaping tumor microenvironment. It has been previously shown that T cell infiltration in penile tumors was associated with clinical outcomes. However, few studies have reported the T cell receptor (TCR) repertoire in patients with penile cancer. In the present study, we evaluated the TCR repertoires in tumor and adjacent normal tissues from 22 patients with penile squamous cell carcinoma (PSCC). Analysis of the T cell receptor beta-variable (TRBV) and joining (TRBJ) genes usage and analysis of complementarity determining region 3 (CDR3) length distribution did not show significant differences between tumor and matched normal tissues. Moreover, analysis of the median Jaccard index indicated a limited overlap of TCR repertoire between these groups. Compared with normal tissues, a significantly lower diversity and higher clonality of TCR repertoire was observed in tumor samples, which was associated with clinical characteristics. Further analysis of transcriptional profiles demonstrated that tumor samples with high clonality showed increased expression of genes associated with CD8 + T cells. In addition, we analyzed the TCR repertoire of CD4 + T cells and CD8 + T cells isolated from tumor tissues. We identified that expanded clonotypes were predominantly in the CD8 + T cell compartment, which presented with an exhausted phenotype. Overall, we comprehensively compared TCR repertoire between penile tumor and normal tissues and demonstrated the presence of distinct T cell immune microenvironments in patients with PSCC., (© 2024. The Author(s).)

Background: Baculoviral IAP repeat containing 5 (BIRC5) is overexpressed and plays as a key regulator in the progression of various human carcinomas. The inflammatory tumor microenvironment (ITM) is closely associated with the development of cancers. However, the role of BIRC5 in penile cancer (PC) and the ITM-induced abnormal progression of PC is still obscure., Methods: In this study, serum and tissues of patients with PC were recruited to evaluate the expression profile of BIRC5. We used PC cell lines (Penl1 and Penl2) and constructed a PC xenograft mice model to explore the effects of the silencing of BIRC5 on proliferation, migration, invasion and tumor growth, as well as survival of mice. Besides, interferon (IFN)-γ was utilized to mimic the ITM of PC cells., Results: Our results showed that BIRC5 was dramatically upregulated in the serum and tissues of PC patients, as well as PC cell lines. Knockdown of BIRC5 inhibited the proliferation, migration and invasion of PC cells. Meanwhile, it suppressed PC xenograft tumor growth and improved mice survival. Moreover, IFN-γ significantly aggravated PC progression both in vivo and in vitro while the silencing of BIRC5 reversed these unfavorable effects., Conclusions: Taken together, our data revealed that BIRC5 silencing inhibited aggravation of PC cell processes and tumor development induced by ITM. This suggested that BIRC5 may function as a diagnosis and therapy target of PC in the future., (© 2022. The Author(s).)

In penile squamous cell carcinoma (pSCC), primary surgery aims to obtain oncologically safe margins while minimizing mutilation. Surgical guidance provided by receptor-specific tracers could potentially improve margin detection and reduce unnecessary excision of healthy tissue. Here, we present the first results of a prospective feasibility study for real-time intraoperative visualization of pSCC using a fluorescent mesenchymal-epithelial transition factor (c-MET) receptor targeting tracer (EMI-137). Methods: EMI-137 tracer performance was initially assessed ex vivo ( n  = 10) via incubation of freshly excised pSCC in a solution containing EMI-137 (500 nM). The in vivo potential of c-MET targeting and intraoperative tumor visualization was assessed after intravenous administration of EMI-137 to 5 pSCC patients scheduled for surgical resection using a cyanine-5 fluorescence camera. Fluorescence imaging results were related to standard pathologic tumor evaluation and c-MET immunohistochemistry. Three of the 5 in vivo patients also underwent a sentinel node resection after local administration of the hybrid tracer indocyanine green- 99m Tc-nanocolloid, which could be imaged using a near-infrared fluorescence camera. Results: No tracer-related adverse events were encountered. Both ex vivo and in vivo, EMI-137 enabled c-MET-based tumor visualization in all patients. Histopathologic analyses showed that all pSCCs expressed c-MET, with expression levels of at least 70% in 14 of 15 patients. Moreover, the highest c-MET expression levels were seen on the outside rim of the tumors, and a visual correlation was found between c-MET expression and fluorescence signal intensity. No complications were encountered when combining primary tumor targeting with lymphatic mapping. As such, simultaneous use of cyanine-5 and indocyanine green in the same patient proved to be feasible. Conclusion: Fluorescence imaging of c-MET receptor- expressing pSCC tumors after intravenous injection of EMI-137 was shown to be feasible and can be combined with fluorescence-based lymphatic mapping. This combination is unique and paves the way toward further development of this surgical guidance approach., (© 2022 by the Society of Nuclear Medicine and Molecular Imaging.)

Equine penile squamous cell carcinoma (epSCC) is the most frequent tumor of the external male genitalia, representing 67.5% of equine genital cancers. epSCC is associated with papilloma virus (PV) infection and has been recently proposed as a model for human PV-induced squamous cell carcinomas. It has already been suggested that epSCC might undergo epithelial-to-mesenchymal transition (EMT). This work aims to investigate in detail this process and the possible role of PV oncoproteins in epSCC. For this purpose, 18 penile SCCs were retrospectively selected and tested for both EcPV2 presence and oncoproteins (EcPV2 E6 and EcPV2 E7) expression. Moreover, immunohistochemical EMT characterization was carried out by analyzing the main epithelial markers (E-cadherin, β-catenin, and pan-cytokeratin AE3/AE1), the main mesenchymal markers (N-cadherin and vimentin), and the main EMT-related transcription factors (TWIST-1, ZEB-1). PCR analysis was positive for EcPV2 in 16 out of 18 samples. EMT was investigated in epSCC positive for EcPV2. The immunohistochemistry results suggested the presence of EMT processes in the neoplastic cells at the tumor invasive front. Moreover, the significant upregulation of RANKL , together with BCATN1 , LEF1 , and FOSL1 genes, might suggest a canonical Wnt pathway activation, similarly to what is reported in human penile squamous cell carcinomas.

Squamous penile cancer displays a rare human papillomavirus (HPV)-associated tumor entity. Investigations on the molecular pathogenesis of HPV-driven penile cancer are impaired by the rareness of clinical specimens and, in particular, are missing relevant cell culture models. Here, we identified in HPV-positive penile cancer cell lines that HPV16 oncoproteins control TP63 expression by modulating critical regulators, while integration into the TP63 open reading frame facilitates oncogene expression. The resulting feed-forward loop leads to elevated p63 levels that in turn enhance the release of the neutrophil-recruiting chemokine CXCL8. Remarkably, elevated CXCL8 amounts lead to the increased surface exposition of the Fc receptor of human IgA antibodies, FcαRI, on neutrophils and correlated with a higher susceptibility to antibody-dependent neutrophil-mediated cytotoxicity (ADCC) using an EGFR-specific IgA2 antibody. IHC staining of tissue microarrays proved that elevated expression of p63 together with neutrophil infiltration were significantly more frequent in HPV-positive penile cancer displaying a higher tumor grade. In summary, we identified a promising marker profile of patients with penile cancer at higher risk for worse prognosis. However, these patients may benefit from immunotherapeutic approaches efficiently engaging neutrophils for tumor cell killing., (©2020 American Association for Cancer Research.)

Background : Lymph node metastasis is the most unfavorable prognostic factor of penile squamous cell carcinoma (PSCC). However, patients with the same lymph node status have different outcomes, and molecular classifiers for precise prognostic assessments are lacking. Methods: Comprehensive genomic profiling and high-content proliferation screening were performed in eight PSCC and normal tissue pairs and in cell lines. BCL2A1 and AIM2 were selected and further evaluated by qPCR and Western blot. The clinical relevance and prognostic value of the target genes were validated via immunohistochemistry in a cohort of 220 PSCC patients with a defined pN stage. Finally, the biological functions and molecular mechanisms of BCL2A1 and AIM2 were investigated in vitro and in vivo . Results: BCL2A1 and AIM2 were both upregulated in PSCC tissues and associated mostly with cell proliferation. Staining for either BCL2A1 or AIM2 revealed that both are correlated with pN status, extranodal extension, clinical stage and cancer-specific survival (CSS). Compared to patients who are single-positive or double-negative for BCL2A1 and AIM2, those overexpressing both genes had a higher risk of tumor progression and the poorest survival in the pN0 (5-year CSS: 63.3% vs. 94.9% and 100.0%, respectively, p = 0.000) and pN+ subsets (5-year CSS: 24.1% vs. 45.7% and 55.1%, respectively, p = 0.035). Molecular biofunction and mechanistic studies demonstrated that BCL2A1 and AIM2 knockdown inhibited tumorigenesis via the AIM2/NF-κB/BCL2A1/MAPK/c-Myc signaling pathway. Conclusions: BCL2A1 and AIM2 promote PSCC progression. Integrating BCL2A1 and AIM2 as novel molecular classifiers with pN stage provides additional information for the prognosis and treatment of PSCC patients., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Penile cancer and its precursor lesions are morphologically and clinically heterogenous and they can be further characterized by immunohistochemical (IHC) and molecular genetic analyses. According to the current World Health Organization (WHO) classification, penile intraepithelial neoplasia (PeIN) and invasive penile carcinomas can be grouped into human papillomavirus (HPV)-related and non-HPV-related neoplasms. This distinction is clinically relevant for etiological and prognostic reasons. To gain insight into the current use of molecular testing and IHC in their diagnostics, a survey was held among the membership of the International Society of Urological Pathology (ISUP). About 250 pathologists from 51 countries answered the survey on the practice and use of IHC/molecular technique as aids in the diagnosis of penile squamous neoplasia. More than half (60%) of the respondents worked at an academic hospital. The questions focused on condylomas, precancerous squamous lesions, and squamous cell carcinoma (SCC). About 35% to 45% of the pathologists considered the use of IHC or molecular tests of value in the pathologic evaluation of precancerous and invasive neoplasms. The vast majority of the respondents do not use IHC for the diagnosis and subtyping of condylomas. There is emerging evidence that some condylomas may participate in the penile carcinogenesis process, especially the high-risk HPV-related atypical condylomas. We recommend the use of p16 in such cases. For most PeIN cases, about half of the responding pathologists make the diagnosis on hematoxylin and eosin slides only. For their subtyping, 50% to 55% of the pathologists use IHC in warty or basaloid PeINs and 40% in differentiated PeIN. To separate HPV-related PeIN from non-HPV-related PeIN, 80% reported using p16 and 20% Ki-67. On the basis of literature review and our practice, the ISUP working group recommends the use of Ki-67 to separate non-HPV-differentiated PeIN from squamous hyperplasia and the use of p16 to distinguish the pleomorphic variant of differentiated PeIN from HPV-related PeIN. With respect to SCCs, according to the survey, immunostaining is only applied in 15% of conventional invasive SCCs, the majority being diagnosed by hematoxylin and eosin. To separate HPV and non-HPV tumors, most (80%) would use p16 and 25% would use p53. For subtype classification, they consider IHC necessary to identify verrucous, papillary, warty, warty-basaloid, and basaloid carcinomas. p16 is used as a surrogate of polymerase chain reaction for the identification of high-risk HPV. We recommend the use of p16 immunostain in cases where the tumoral histologic features of the SCCs are not classical for HPV-related neoplasms, especially in poorly differentiated tumors. Because the majority of these neoplasms harbor high-risk HPV (HPV16), identifying the presence of the virus is rather more important than documenting its specific genotype.

Background: Penile squamous cell carcinoma (PSCC) is a rare malignancy with higher incidence in developing countries. Treatment options include surgery, radiation therapy, and systemic chemotherapy. However, effective treatments for advanced disease are lacking. To understand the biology underlying PSCC may help the development of new therapeutic strategies. The objective of this study was to evaluate immunohistochemical expression of programmed death-ligand 1 (PD-L1) and p16 in PSCC and its association with clinicopathologic features and outcomes., Patients and Methods: A cohort of 40 patients with PSCC from an academic institution in Brazil was analyzed. Clinicopathologic features and outcomes were retrospectively collected. PD-L1 and p16 immunohistochemical expression were performed in formalin-fixed paraffin-embedded specimens. PD-L1 was positive with any staining in more than 1% of tumor, and p16 was positive in more than 10%. Associations were performed using the Mann-Whitney and Fisher exact test. Kaplan-Meier curves were used to estimate survival rates with log-rank., Results: Of 35 patients, 5 were excluded, 4 owing to a lack of data and 1 owing to no tumor available; 18 (51.4%) patients were PD-L1-positive (PD-L1 + ). PD-L1 + was associated with larger tumors (P = .027). There was an association between PD-L1 + and p16 expression (P = .002). PD-L1 + was more frequent in grade II and III disease than grade I (77.8% vs. 22.2%) and was expressed in all patients with grade III disease. Lymph node involvement was associated with PD-L1 expression (69.2% PD-L1 + vs. 30.8% PD-L1-negative). The 5-year mortality was 37.1%., Conclusion: PD-L1 expression appears to be associated with p16 expression, larger tumors, and worse clinical outcomes in PSCC and may provide clinical data for new studies to evaluate anti-PD-L1 immune therapies., (Copyright © 2019 Elsevier Inc. All rights reserved.)

An 85-year-old asymptomatic man with suspected biochemical recurrence of prostate cancer underwent an F-fluciclovine PET/CT scan, which revealed a solitary suspicious tracer uptake in the dorsal right corporal body of the proximal pendulous penis. The patient underwent ultrasound-guided fine-needle aspiration of the penile lesion, which revealed metastatic prostate cancer. The patient had definitive external beam radiation therapy 3 years before the examination. At the time of scan, the prostatic-specific antigen (PSA) was only 1.0 ng/mL, although the PSA doubling time was 2.6 months. It is unusual to detect a solitary penile metastasis in a patient with a low level of PSA.

Penile squamous cell carcinoma (PSCC) accounts for over 95% of penile malignancies and causes significant mortality and morbidity in developing countries. Molecular mechanisms and therapies of PSCC are understudied, owing to scarcity of laboratory models. Herein, we describe a genetically engineered mouse model of PSCC, by co-deletion of Smad4 and Apc in the androgen-responsive epithelium of the penis. Mouse PSCC fosters an immunosuppressive microenvironment with myeloid-derived suppressor cells (MDSCs) as a dominant population. Preclinical trials in the model demonstrate synergistic efficacy of immune checkpoint blockade with the MDSC-diminishing drugs cabozantinib or celecoxib. A critical clinical problem of PSCC is chemoresistance to cisplatin, which is induced by Pten deficiency on the backdrop of Smad4/Apc co-deletion. Drug screen studies informed by targeted proteomics identify a few potential therapeutic strategies for PSCC. Our studies have established what we believe to be essential resources for studying PSCC biology and developing therapeutic strategies.

Purpose of Review: Whereas substantial advances have been made in systemic tumour therapy in the past decade, the prognosis of advanced squamous cell carcinoma (SCC) of the penis remains disproportionally poor. In this review, we aimed to present an update on systemic therapy of penile SCC highlighting the most recent data and future perspectives., Recent Findings: Lymph node metastases play a key role in treating and assessing the prognosis of patients with penile SCC. Data show longer overall survival with the use of adjuvant chemotherapy in patients with pelvic lymph node metastases and recent analyses lead to the development of a nomogramm predicting overall survival in connection with the use of perioperative chemotherapy. There are two pathways in the pathogenesis of penile cancer, including human papilloma virus related and unrelated, leading to many possible novel therapeutic targets. Other targeted therapies have been evaluated, which show promising results with the use of tyrosine kinase inhibitors., Summary: Chemotherapy has shown moderate activity in advanced stages of the disease, however, the ideal timing of chemotherapy in patients with lymph node metastases is not entirely clear. Potential targets for future therapies exist, and are already being tested in other malignancies. Owing to the rarity of this condition, a robust evidence is lacking and it is of great importance to pursue further research to unveil several aspects of this disease, particularly in patients with recurrence, lymph node metastases or metastatic disease.

The aim of this study is to investigate the expression of the luteinizing hormone/choriogonadotropin (LHCG) receptor in the human penis to see, if the luteinizing hormone (LH) effects are possible in the spongious and cavernous tissue of the penis. The number of men with erection disturbances increases significantly simultaneously with the elevated LH concentrations between 40 and 70 years. It is possible that the elevated LH concentrations may influence locally the erectile mechanisms. The precondition for this is the expression of LHCG receptors in the penis. Penile tissue was obtained from three patients undergoing total or partial penectomy due to a rectal cancer with secondary penile metastasis or squamous cell carcinoma of the penis. Immunohistochemistry was used for the detection of the LHCG receptor. Positive immunoreaction for LHCG receptors was discovered in the endothelial cells of cavernous spaces in the corpus cavernosum and corpus spongiosum penis, also in the endothelial cells of the capillary walls in all patients. Our results show that LHCG receptor is expressed in the spongious and cavernous tissue of the human penis. This finding suggests that LH can affect the spongious and cavernous tissue in human and play a significant role in the development of erectile dysfunction among the aging men.

Background: Inguinal lymph node involvement is the main prognostic factor in patients with penile cancer. However, there is a lack of marker/s for lymph node metastasis. microRNAs have been investigated as potential markers for prognosis of various types of cancer. Taking this into consideration, our main goal was to determine the association of miR-223-3p, miR-107, and miR-21-5p expression with clinicopathological characteristics, as well as presence of lymph node metastasis in patients with penile cancer., Methods: Formalin-fixed paraffin-embedded penile squamous cell carcinoma specimens from 50 patients, at diagnosis and prior to any cancer treatment, were obtained. Tissue samples comprising at least 70% malignant cells and adjacent non-tumor tissues were evaluated by using qRT-PCR for expression level of miR-223-3p, miR-107 and miR-21-5p. Additionally, molecular identification of HPV was performed by PCR, and the expression levels of PTEN were analyzed by immunohistochemistry., Results: Penile squamous cell carcinoma primary tumors presented higher expression of miR-223-3p, miR-107, and miR-21-5p when compared to non-tumor adjacent tissues. Upregulation of miR-223-3p was associated lymph node metastasis. Higher expression of miR-107 was associated with worsening of prognosis (as observed by histological grade II and III, tumors bigger than 2.0 cm, stage III and IV, and lower disease-free survival). In addition, higher expression of miR-107 and miR-21-5p was correlated to the absence of PTEN protein expression., Conclusions: Our data demonstrate that higher expression of miR-223-3p, miR-107, and miR-21-5p is correlated with poor prognosis in penile cancer. The upregulation of these microRNAs potentially affect critical cancer pathways and may be important for the prognosis and response to therapy in penile cancer.

Background: Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan (Trp) catabolism have been demonstrated to play an important role in tumor immunosuppression. This study examined the expression and catalytic activity of IDO1 in penile squamous cell carcinoma (PSCC) and explored their clinical significance., Methods: IDO1 expression level, serum concentrations of Trp and kynurenine (Kyn) were examined in 114 PSCC patients by immunohistonchemistry and solid-phase extraction-liquid chromatography-tandem mass spectrometry. The survival was analyzed using Kaplan-Meier method and the log-rank test. Hazard ratio of death was analyzed via univariate and multivariate Cox regression. Immune cell types were defined by principal component analysis. The correlativity was assessed by Pearson's correlation analysis., Results: The expression level of IDO1 in PSCC cells was positively correlated with serum Kyn concentration and Kyn/Trp radio (KTR; both P < 0.001) but negatively correlated with serum Trp concentration (P = 0.001). Additionally, IDO1 up-regulation in cancer cells and the increase of serum KTR were significantly associated with advanced N stage (both P < 0.001) and high pathologic grade (P = 0.008 and 0.032, respectively). High expression level of IDO1 in cancer cells and serum KTR were associated with short disease-specific survival (both P < 0.001). However, besides N stage (hazard radio [HR], 6.926; 95% confidence interval [CI], 2.458-19.068; P < 0.001) and pathologic grade (HR, 2.194; 95% CI, 1.021-4.529; P = 0.038), only serum KTR (HR, 2.780; 95% CI, 1.066-7.215; P = 0.036) was an independent predictor for PSCC prognosis. IDO1 expression was positively correlated with the expression of interferon-γ (IFNγ, P < 0.001) and immunosuppressive markers (programmed cell death protein 1, cytotoxic T-lymphocyte-associated protein 4 and programmed death-ligand 1 and 2; all P < 0.05), and the infiltration of immune cells (including cytotoxic T lymphocytes, regulatory T lymphocytes, tumor-associated macrophages, and myeloid-derived suppressor cells; all P < 0.001) in PSCC tissues. Furthermore, the expression of IDO1 was induced by IFNγ in a dose-dependent manner in PSCC cells., Conclusions: IFNγ-induced IDO1 plays a crucial role in immunoediting and immunosuppression in PSCC. Additionally, serum KTR, an indicator of IDO1 catabolic activity, can be utilized as an independent prognostic factor for PSCC., (© 2020 The Authors. Cancer Communications published by John Wiley & Sons Australia, Ltd. on behalf of Sun Yat-sen University Cancer Center.)

Background: It has been hypothesized that PD-L1 expression in tumor cells and tumor-infiltrating immune (TII) cells may contribute to tumor progression by inhibiting antitumor immunity., Objective: To investigate the association between PD-L1 expression in tumor cells and TII cells and clinical outcomes in penile cancer., Design, Setting, and Participants: A cohort of 222 men treated for penile squamous cell carcinoma (SqCC) at Örebro University Hospital between 1984 and 2008 with long-term follow-up (median 34 mo) was evaluated for PD-L1 expression in tumor cells and TII cells via immunohistochemistry., Outcome Measurements and Statistical Analysis: Association between clinicopathological features and PD-L1 expression was estimated using χ 2 and Fisher's exact tests. For survival analyses, Kaplan-Meier curves with log-rank tests and multivariate Cox proportional hazards regression models were used., Results and Limitations: We found that 32.1% of the tumors and 64.2% of the TII cells expressed PD-L1. Our data demonstrate that penile SqCC patients with PD-L1-positive tumor cells or TII cells are at significant risk of lower cancer-specific survival and that the prognostic value of PD-L1 expression was strongest for tumor cell positivity. The use of tissue microarrays rather than whole sections may be viewed as a limitation., Conclusions: Tumor PD-L1 expression independently identifies penile SqCC patients at risk of poor clinical outcomes., Patient Summary: We investigated how many patients with penile cancer had tumors that manufactured PD-L1, a protein that decreases the ability of the immune system to fight cancer. We found that up to one-third of penile tumors make this protein. Patients whose tumors make PD-L1 have more aggressive penile cancer and worse clinical outcomes., (Copyright © 2018 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

Purpose: To assess the prognostic value of PI3K-AKT-mTOR signaling pathway up-regulation in a contemporary cohort of penile squamous-cell carcinoma (PSCC) patients., Patients and Methods: Tissue microarrays were constructed for 57 patients with invasive PSCC treated at our institution between 2000 and 2013. Immunohistochemical staining was performed for PTEN, AKT, and S6. Human papillomavirus (HPV) in-situ hybridization for high-risk subtypes was also performed. Biomarker expression was evaluated by a semiquantitative H score. Overall survival, disease-specific survival and recurrence-free survival stratified by biomarker expression (low vs. high) were estimated by the Kaplan-Meier method. Multivariable Cox regression models were used to determine predictors of mortality and recurrence., Results: HPV in-situ hybridization was positive in 23 patients (40%). PTEN was down-regulated in 43 patients (75%), while phosphorylated-AKT (p-AKT) and phosphorylated-S6 (p-S6) were up-regulated in 27 (47%) and 12 patients (21%), respectively. In multivariable Cox regression models, patients with low expression of p-AKT had an increased risk of recurrence (hazard ratio [HR] = 3.95; 95% confidence interval [CI], 1.47-10.59; P = .02), while those with low expression of p-S6 had an increased risk of overall mortality (HR = 6.15; 95% CI, 1.55-24.36; P = .01). HPV status was an independent predictor of overall survival (HR = 6.99; 95% CI, 2.42-20.16; P < .001) and disease-specific survival (HR = 6.74; 95% CI, 2.02-22.48; P = .002)., Conclusion: PI3K-AKT-mTOR signaling pathway up-regulation and HPV coinfection in PSCC are associated with favorable disease. mTOR pathway biomarkers along with HPV status may represent novel prognosticators for risk stratification of PSCC patients and may help guide treatment decisions and follow-up strategies. These findings require further investigation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objectives: To evaluate the expression of COX-2, E-cadherin, vimentin, 14-3-3σ, and Phosphatase and tensin homolog (PTEN) tumor-related proteins in equine penile papillomas (ePP) and squamous cell carcinomas (ePSCC), the occurrence of epithelial-mesenchymal transition (EMT) at the invasion front (IF) and compare our findings with current knowledge on human penile squamous cell carcinoma (hPSCC)., Material and Methods: We analyzed, by immunohistochemistry in 45 equine penile proliferative epithelial lesions, the expression of COX-2, E-cadherin, vimentin, 14-3-3σ, and PTEN using monoclonal antibodies. Tumors were histopathologically classified as well-differentiated or poorly differentiated using the IF grading scheme. Semiquantitative analysis was performed to determine down or up-regulation of the proteins and association with histopathological characteristics were statistically investigated using Mann-Whitney U test and/or Spearman's tests., Results: COX-2 was neo-expressed in 86.6% of the cases and expression progressively increased from ePP to ePSCC (P = 0.0003) and from well to poorly differentiated (P = 0.033). High COX-2 expression was associated with a high mitotic index (MI) (P = 0.026). In contrast to normal epidermis, ePSCC had very low E-cadherin expression in 64% of the cases (P = 0.0005). Vimentin was neo-expressed in 65% of poorly differentiated ePSCC at the IF indicating EMT. Cytoplasmic 14-3-3σ protein expression was reduced in 42% of the ePSCC and additionally, nuclear expression of 14-3-3σ in neoplastic keratinocytes and in the cytoplasm of stromal fibroblasts at the IF was features only found in ePSCC. PTEN protein showed a tendency to be decreased or lost in ePSCC., Conclusions: Our study provides evidence of molecular abnormalities in ePSCC similar to those reported for human PSCC. The occurrence of EMT at the IF is a common event in ePSCC. Naturally occurring ePSCC could serve as a valuable preclinical animal model to explore upcoming therapeutic options for hPSCC., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Androgen receptor (AR) is a member of the steroid and nuclear family receptor that acts as transcription factor. AR signaling plays pivotal role in the development and progression of prostate cancer. However, the role of AR in penile cancer (PeCa) is poorly explored. Our previous molecular studies unveiled frequent AR mRNA loss in PeCa, which was further predicted as a major driver alteration in this neoplasm. Herein, we assessed the AR protein expression in 59 usual PeCa tissues and 42 surrounding normal tissues (SNT) by immunohistochemistry using a tissue microarray. In a paired analysis, we found a total absence of nuclear AR expression in PeCa while 95.2% of SNT samples presented strong nuclear AR expression (P < 0.001). Interestingly, 17 of 42 PeCa presented weak or moderate cytoplasmic AR staining, contrasting with 5 of 42 SNT (P = 0.008). Increased levels of AR cytoplasmic expression were related with poor prognosis features including advanced clinical staging (P = 0.044), compromised surgical margins (P = 0.005), and pathological inguinal node status (P = 0.047). Furthermore, AR cytoplasmic expression was also related with shorter overall survival (P = 0.032). In conclusion, the frequent loss of nuclear AR protein levels suggests a potential function in PeCa development. Based on this result, the androgen deprivation therapy is not indicated for PeCa patients. In addition, the AR cytoplasmic expression found in a significant number of cases (40.5%) showed prognostic value and pathways activated by the non-genomic AR signaling may represent a promising therapeutic strategy.

It has been shown that human papillomavirus (HPV) and p16 status has prognostic value in some HPV-associated cancers. However, studies examining survival in men with penile cancer according to HPV or p16 status are often inconclusive, mainly because of small study populations. The aim of this systematic review and meta-analysis was to examine the association between HPV DNA and p16 status and survival in men diagnosed with penile cancer. Multiple electronic databases were searched. Twenty studies were ultimately included and study-specific and pooled HRs of overall survival and disease-specific survival (DSS) were calculated using a fixed effects model. In the analysis of DSS, we included 649 men with penile cancer tested for HPV (27% were HPV-positive) and 404 men tested for p16 expression (47% were p16-positive). The pooled HR HPV of DSS was 0.61 [95% confidence interval (CI), 0.38-0.98], and the pooled HR p16 of DSS was 0.45 (95% CI, 0.30-0.69). In conclusion, men with HPV or p16-positive penile cancer have a significantly more favorable DSS compared with men with HPV or p16-negative penile cancer. These findings point to the possible clinical value of HPV and p16 testing when planning the most optimal management and follow-up strategy. Cancer Epidemiol Biomarkers Prev; 27(10); 1123-32. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Background: Recently, P16 INK4a expression has been shown to be correlated with cancer-specific survival (CSS) in penile squamous cell carcinoma (SCC). Therefore, we performed this meta-analysis to clarify the prognostic value of P16 INK4a for penile SCC., Methods: A systematic search was performed in PubMed, Embase, and Web of Science to identify all relevant articles up to May 25, 2017. Hazard ratios (HRs) and corresponding 95% confidence intervals (CIs) of included studies were pooled to estimate the prognostic value. Trial sequential analysis (TSA) was performed to assess the quantity and strength of survival evidence., Results: Five retrospective studies consisting of 323 cases were finally included. We found that P16 INK4a positive expression was significantly associated with a better CSS of penile SCC (HR=0.45, 95%CI: 0.30-0.67, P<0.001). No heterogeneity or publication bias was noted among the included studies. Furthermore, TSA demonstrated that the findings were based on sufficient evidence., Conclusions: P16 INK4a positive expression is independently associated with improved CSS for patients with penile SCC.

Background: For penile cancer (PC) there are no known molecular predictors of lymphatic spread and/or chemoresistance., Objective: To identify functional biomarkers that can predict malignant progression and treatment responsiveness., Design, Setting, and Participants: We used four patient-derived PC cell lines and measured invasion and capillary tube formation, chemoresponsiveness, and mRNA and protein expression. Data were further validated in E2F1 transcription factor knockdown and overexpression experiments. We quantified E2F1 transcript levels in a set of nonmetastatic tumours (NM), metastasised primary tumours (PT), and lymph node metastases (M) from 24 patients. E2F1 immunohistochemistry was performed in another set of 13 PC biopsies., Outcome Measurements and Statistical Analysis: Relationships between different parameters were analysed using Student t tests. Transcript levels in patient samples were compared using Mann-Whitney U tests. Significance was set at p<0.05., Results and Limitations: In cell lines established from lymph node metastases, E2F1 was more abundantly expressed, pRB was inactivated, and CDK2, CDK4, and cyclins D and E were elevated in comparison to cells from primary PC. Overexpression of E2F1 enhanced migratory capacity and lymphatic endothelial tubule formation, while depletion reduced invasiveness and increased chemosensitivity. VEGFR-3 and VEGF-C and mesenchymal markers were upregulated by high E2F1. E2F1 was clearly upregulated in infiltrative and metastatic primary tumours and metastases (NM vs PT, p<0.05; NM vs M, p<0.0005). E2F1 Quick scores increased from grade I to grade III tumours. A limitation of the study is the small number of patients., Conclusions: E2F1 is a driver of invasion and lymphatic dissemination and promotes chemoresistance. E2F1-related biomarkers might assist in stratifying PC patients for different treatment regimens., Patient Summary: The availability of penile cancer cell lines allows molecular research on the mechanisms underlying metastasis and chemotherapy. A critical pathway involved in both features has been identified and may lead to better patient stratification for treatment selection., (Copyright © 2017 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

Background: Therapeutic targeting of the PI3K-AKT-mTOR pathway may benefit patients with advanced penile squamous cell carcinoma (PSCC)., Objectives: To determine the prevalence of PIK3CA copy number gain and correlate this with the activity status of PI3K-AKT-mTOR pathway in pre-malignant penile intraepithelial neoplasia (PeIN) and invasive PSCC., Materials and Methods: Archival tissue blocks were obtained from 58 PeIN and 244 primary PSCC patients treated at St George's Hospital. PIK3CA copy number status (CNS) was assessed by fluorescence in-situ hybridisation. High-risk HPV DNA was detected with INNO-LiPA assay. p16INK4A, p-AKT and p-mTOR protein expression were assessed using immunohistochemistry (IHC)., Results: Increased prevalence of PIK3CA copy number gain was seen in PSCC in comparison to PeIN (84/199 (42%) vs. 10/58 (17%); p = 0.0009). Analysis of the p-AKT and p-mTOR revealed a tendency to a more common expression of cytoplasmic p-AKT (p = 0.1318), nuclear p-AKT (p<0.0001) and cytoplasmic mTOR (p = 0.0006) in PeIN than PSCC. A significant association between p-AKT cytoplasmic immunoexpression and PIK3CA CNS (p = 0.0404) was found in PeIN., Conclusion: Overall, PIK3CA copy number gain correlated with activation of the PI3K-AKT-mTOR pathway in PeIN and activation of this pathway is primarily involved in early penile carcinogenesis. Based on these results therapeutic targeting of this pathway in advanced PSCC is unlikely to produce significant clinical benefit. Future studies will need to focus on alternative therapeutic targets., Competing Interests: The authors have declared that no competing interests exist.

Background: APOBECs (apolipoprotein B mRNA-editing catalytic polypeptides) are cytidine deaminases that have been implicated in the host defense against viruses by blocking viral replication. They have also been shown to play a role in genome hypermutation in several human cancers. An APOBEC3 hypermutation signature has been discovered in cervical cancer, which is intimately associated with infection by high-risk human papillomaviruses (HPVs). At the same time, HPV genomes themselves are subject to DNA editing by APOBECs. Similar to cervical cancer, a proportion of penile squamous cell carcinomas (SCCs) is etiologically driven by high-risk HPVs, but very little is known about the role of APOBECs in penile SCC development and progression., Methods: A series of 34 penile SCCs was analyzed for the expression of APOBEC3A protein by immunohistochemistry. HPV genotyping was carried out using a bead-based multiplex hybridization assay preceded by BSGP5+6+ primer-based amplification., Results: We found a frequent reduction of APOBEC3A protein expression in the invasive parts of the majority of HPV-negative penile SCCs. In contrast, the majority of HPV-positive penile SCCs retained APOBEC3A expression during malignant progression., Conclusion: Our results suggest that APOBEC3A expression is downregulated during progression towards invasiveness in HPV-negative penile SCC, but maintained in HPV-positive penile SCC. How high-risk HPV-infected tumor cells tolerate high APOBEC3A, which appears to exert tumor suppressive functions in HPV-negative penile SCCs, remains to be elucidated., (© 2017 S. Karger AG, Basel.)

Multiparametric MRI (mpMRI) is currently the test of choice for imaging sites of prostate cancer located within the pelvis. However, molecular imaging with PET radiotracers that bind to prostate-specific membrane antigen is increasingly being used for this purpose and may compliment mpMRI findings. We present a case of a 59-year-old man with elevated postprostatectomy serum prostate-specific antigen level who was found on both mpMRI and prostate-specific membrane antigen-targeted PET to harbor residual prostate cancer in the corpus spongiosum along the proximal urethra.

Penile cancer (PeCa) is a rare malignancy with potentially devastating effects. Squamous cell carcinoma is the most common variant with distinct precancerous lesions before development into invasive disease. Involvement of the inguinal lymph nodes is the most important prognostic factor in PeCa, and once disease is present outside the groin, prognosis is poor. Metastatic PeCa is challenging to treat and often requires multidisciplinary approaches in management. Due to its rarity, molecular understanding of the disease continues to be limited with most studies based on small, single center series. Thus far, it appears PeCa has diverse mechanisms of carcinogenesis affecting similar molecular pathways. In this review, we evaluate the current landscape of the molecular carcinogenesis of PeCa and explore ongoing research on potential actionable targets of therapy. The emergence of anti-epidermal growth factor receptor (EGFR) and other immunotherapeutic strategies may improve outcomes for PeCa patients., Competing Interests: The authors declare no conflict of interest.

Molecular data generation and their combination in penile carcinomas (PeCa), a significant public health problem in poor and underdeveloped countries, remain virtually unexplored. An integrativemethodology combin ing genome-wide copy number alteration, DNA methylation, miRNA and mRNA expression analysis was performed in a set of 20 usual PeCa. The well-ranked 16 driver candidates harboring genomic alterations and regulated by a set of miRNAs, including hsa-miR-31, hsa-miR-34a and hsa-miR-130b, were significantly associated with over-represented pathways in cancer, such as immune-inflammatory system, apoptosis and cell cycle. Modules of co-expressed genes generated from expression matrix were associated with driver candidates and classified according to the over-representation of passengers, thus suggesting an alteration of the pathway dynamics during the carcinogenesis. This association resulted in 10 top driver candidates (AR, BIRC5, DNMT3B, ERBB4, FGFR1, PML, PPARG, RB1, TNFSF10 and STAT1) selected and confirmed as altered in an independent set of 33 PeCa samples. In addition to the potential driver genes herein described, shorter overall survival was associated with BIRC5 and DNMT3B overexpression (log-rank test, P = 0.026 and P = 0.002, respectively) highlighting its potential as novel prognostic marker for penile cancer.

Purpose: PD-L1 (programmed death ligand 1) inhibits T-cell function and prevents tumor eradication. This is facilitated by PD-L1 positive tumor cells and PD-L1 positive immune cells, and can be prevented by anti-PD-1 (programmed death 1)/PD-L1 immunotherapy. In advanced penile cancer there is a need for new therapeutic strategies. We investigated PD-L1 expression in penile cancers and compared PD-L1 expression with disease specific survival, lymph node metastases at diagnosis and high risk HPV status in a large patient cohort., Materials and Methods: A total of 213 primary tumors were immunohistochemically stained for PD-L1 and scored for tumor (percentage), stroma (binary) and PD-L1 positive tumor infiltrating macrophages. Additionally, PD-L1 positive tumors were scored for expression pattern, that is diffuse or predominantly present at the tumor-stroma margin., Results: Staining was successful in 200 tumors, of which 75% were high risk HPV negative. Median followup was 62 months. Of 200 tumors 96 (48%) were PD-L1 positive (scored 1% or greater), of which 59 (62%) had a marginal expression pattern and 79 (82%) were high risk HPV negative (p = 0.03). Compared to PD-L1 negative tumors, the PD-L1 expression patterns had different prognostic values in the whole cohort as well as in the high risk HPV negative subgroup. On multivariable analyses a marginal expression pattern was associated with absent lymph node metastases (OR 0.4) while diffuse expression was associated with poor survival (HR 2.58). These results were more prominent in the high risk HPV negative subgroup (OR 0.25, HR 3.92)., Conclusions: PD-L1 was expressed in 48% of penile carcinomas and mainly in high risk HPV negative tumors. The pattern of expression was a prognostic factor as marginal expression was associated with absent lymph node metastases and diffuse expression was associated with poor survival., (Copyright © 2017 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Dysregulation of host microRNA expression has been involved in the development and progression of human papillomavirus (HPV)-related tumors. Analysis of miR-146a expression in a series of 59 penile squamous cell carcinomas (PSCCs) showed that its levels were lower in high-risk HPV-positive than in HPV-negative PSCCs and inversely correlated with expression of epidermal growth factor receptor (EGFR), a known target for miR-146a. Analysis of genotype distribution for rs2910164, a common functional polymorphism of miR-146a, did not identify correlations with miR-146a levels and EGFR expression in PSCCs. In vitro experiments demonstrated that E6 of HPV type 16, but not low-risk HPV-6, down-regulated miR-146a in human foreskin keratinocytes and up-regulated EGFR. Ectopic expression of miR-146a decreased expression of EGFR and inhibited proliferation of keratinocytes and cervical carcinoma cells. EGFR is commonly overexpressed in penile cancer and in other squamous cell carcinomas. Molecular mechanisms leading to EGFR overexpression and activation are known for HPV-negative cancers and include amplification or mutations of the EGFR gene. The results of this study indicate that down-regulation of miR-146a may represent another mechanism of EGFR overexpression in PSCCs, which can be mediated by high-risk HPV E6 in HPV-related tumors., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Ultraviolet rays are one of the leading factors in the development of melanoma (MM); however, ultraviolet rays seem not to play a role in non-sun-exposed MM, where systemic immunosuppression, anatomical, and physiological features may contribute toward the development of the malignancy. Our aim was to evaluate vitamin D receptor (VDR) expression in shield-site melanoma (ST-MM) and non-shield-site melanoma (NST-MM) to find features that could explain the different biological behavior of MM according to the area of onset. We reviewed 118 specimens of MM. VDR expression was assayed using immunohistochemistry by dividing the specimens according to the anatomical area. We included MM of the soles, feet, hands, gluteus, scrotum, skin of the penile shaft, and large vaginal labia in the ST-MM group. The NST-MM group was divided into two main categories: NST-MM of chronic sun-exposed areas, including MM of the face, scalp, neck, back of the hands, and NST-MM of intermittent sun-exposed areas, including MM of the trunk, lower, and upper limbs. In shield sites, 66.67% of MMs showed VDR expression; in intermittent sun-exposed areas, 33.3% showed VDR expression; and in chronic sun-exposed areas, only 4.66% showed VDR expression. A similar behavior was observed for Breslow's thickness, where VDR staining intensity was higher in thicker lesions, ranging between 60 and 100%. We found that VDR expression decreased from ST-MM to NST-MM. These findings confirm the hypothesis that different pathways are involved in ST-MM and NST-MM.

Background: Novel molecular markers are important diagnostic tools for the assessment of cancer progression and evaluation of effectiveness of the treatment. SOX9, a key regulator of developmental processes, is overexpressed in various neoplasms, such as prostate, breast, and colorectal cancers. However, the utilization of SOX9 as a biomarker for other urological cancers has not yet been investigated., Methods: In the present study, paired patient tissue microarrays were analyzed by immunohistochemistry, and the SOX9 protein expression was quantitated as immunoreactive scores in patients with renal cell carcinoma (RCC), bladder cancer (BCa), and penile cancer (PC)., Results: In comparison with normal tissues, SOX9 protein expression was significantly upregulated in RCC (p < 0.001) and BCa (p < 0.001), and significantly correlated with the advanced pathological grade (RCC: p = 0.023) and clinical stage (RCC: p = 0.022 and BCa: p = 0.046) of patients. Based on the mRNA level in the TCGA dataset, SOX9 was upregulated in RCC with gender (p = 0.027), advanced pathological grade (p = 0.003) and advanced clinical stage (p = 0.001). Kaplan-Meier survival curves revealed that RCC patients with high SOX9 levels had shorter survival (p < 0.001). Further, high SOX9 expression was an independent prognostic factor for RCC patients (hazard ratio 0.056, 95% confidence interval 0.607-1.184; p < 0.001)., Conclusion: These findings suggest that SOX9 may play an important role in tumor progression of RCC and BCa and it may be used as a biomarker of this malignancy., (© 2017 S. Karger GmbH, Freiburg.)

Background: Little is known about the outcomes and prognostic factors of adjuvant chemotherapy for locally advanced penile squamous cell carcinoma after regional lymphadenectomy (LAD)., Patients and Methods: We retrospectively reviewed the data from 21 patients who had received taxane, cisplatin, and 5-fluorouracil (TPF, every 3 weeks) in the adjuvant setting at our center. Univariable and multivariable Cox regression analyses were undertaken for disease-free (DFS) and overall survival (OS) of TPF., Results: The patients had received TPF from July 2004 to July 2012 after inguinal (n = 6) or inguinal plus pelvic LAD (n = 15), and the median follow-up was 52 months. Thirteen (61.9%) had pelvic and 5 (23.8%) bilateral inguinal nodal metastases. The median time from LAD to the start of TPF was 5.4 weeks (interquartile range [IQR], 4.1-7.3 weeks). Metastatic tumor tissue from 11 of 19 evaluable patients (57.9%) showed positive immunohistochemistry staining for p53. Univariably, only the expression of p53 showed a trend toward poorer DFS (hazard ratio [HR], 4.14; 95% confidence interval [CI], 0.87-19.68; P = .074) and OS (HR, 4.54; 95% CI, 0.95-21.56; P = .056). The same results were obtained multivariably for DFS (HR, 3.76; 95% CI, 0.78-17.96; P = .096) and OS (HR, 4.29; 95% CI, 0.89-20.57; P = .067). The median DFS was 8.9 months (IQR, 5.9-22.7 months) for p53-expressing patients versus not estimable for non-p53-expressing patients (P = .051) and the median OS was 17.2 months (IQR, 12.8-22.7 months) and not estimable, respectively (P = .037)., Conclusion: In patients who had received adjuvant TPF for node-positive penile squamous cell carcinoma, p53 IHC expression seemed to be associated with a poorer outcome, and further study is warranted in larger data sets to confirm these findings. This information might be useful to improve the prognostic allocation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: To describe the expression of tissue epidermal growth factor receptor (EGFR), excision-repair cross-complementation group 1 protein (ERCC1), and thymidylate synthase (TS) in patients with penile cancer and explore their association with stage and outcome., Methods: A total of 52 patients with penile squamous cell cancer who were treated at the University of Southern California from 1995 to 2010 were identified. Paraffin-embedded tissue underwent mRNA quantitation and immunohistochemistry for expression of EGFR, ERCC1, and TS. KRAS mutations were evaluated using polymerase chain reaction-based sequencing., Results: EGFR overexpression was common by mRNA (median, 5.09; range, 1.92-104.5) and immunohistochemistry. EGFR expression > 7 was associated with advanced stage and poor differentiation (P = .01 and .034 respectively) but not with survival in multivariate analysis. ERCC1 mRNA expression was a median of 0.65 (range, 0.21-1.87). TS expression was a median of 1.88 (range, 0.54-6.47). ERCC1 and TS expression were not associated with grade, stage, or survival. There were no KRAS mutations identified. A total of 17 men received chemotherapy; 8 (47%) had an objective response, including 1 with a pathologic complete response. There was a trend for lower expression of EGFR corresponding to a higher likelihood of response (response rate [RR]) to chemotherapy: 67% RR in EGFR mRNA < 7 versus 33% RR in EGFR > 7 (P = .31)., Conclusions: High expression of EGFR mRNA in squamous cell carcinoma of the penis is associated with advanced stage and poor differentiation, but not survival. In our small heterogeneous subset, molecular marker expression did not show a correlation with the likelihood of chemotherapy response. A prospective evaluation of the role of the EGFR pathway and its regulatory environment in penile cancer is warranted. Given the rarity of this cancer, collaborative prospective cohort evaluations and trials need to be encouraged., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: Our aims were to evaluate epithelial-mesenchymal transition (EMT) as a useful prognostic marker in penile carcinoma (PC), and establish an objective criterion to define EMT in PC specimens., Materials and Methods: A total of 149 consecutive cases surgically treated for PC were retrospectively selected. E-cadherin (E-CAD) and vimentin immunohistochemical expressions were evaluated. A combined analysis was performed using both markers to determine EMT status. To establish a normal control to E-CAD expression, we included 14 cases from circumcisions from patients without any neoplastic disease and 77 cases of tumor-free margins. The analyses of tumor samples were evaluated in 2 different areas of the tumor. The first one was in the tumor core. The second analyses were performed on the deepest infiltrative edge of the tumor, nominated invasion front. Cases were classified into EMT absent group, partial EMT group and complete EMT group. Overall survival (OS) and cancer-specific survival (CSS) were analyzed. Kaplan-Meier curves and the log-rank test were used. Cox proportional hazards model was used to determine which variables influenced survival., Results: Tumor specimens presented a significant loss of expression of E-CAD when compared with normal epithelium. Vimentin expression in more than 10% of tumor cells was observed in 50 cases. EMT status was associated with histologic grade, pattern of invasion, lymph node metastasis, and perineural and vascular invasion. Further, 10-year OS and CSS rates in patients with presence and absence of complete EMT status were 38.0% and 55.6%; and 48.0% and 91.9%, respectively. EMT status significantly affected CSS and OS rates even after patients were grouped based on lymph node involvement status. The presence of complete EMT status was associated with both CSS and OS rates. Patients in the complete EMT group had a higher risk of death from cancer (hazard ratio = 7.6, P<0.001) and overall death (hazard ratio = 3.0, P<0.001)., Conclusion: Our study represents an evidence of the prognostic effect of EMT in PC. We encourage the study of EMT markers in other centers to validate our findings and confirm its importance in such tumors., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Because of the low incidence of penile carcinoma (PC), the value of p16(ink4a), p53, and human papilloma virus (HPV) infection status in clinical practice remains unclear. Herein, we report our experience with potential clinical utility of these markers in men with PC treated at our institution., Patients and Methods: Tissue microarrays of 57 cases of invasive penile squamous cell carcinomas were immunohistochemically stained for p16 and p53. HPV in situ hybridization (ISH) for high-risk subtypes was also performed. Association between marker status, nodal disease, overall (OS) and cancer-specific survival (CSS) were assessed., Results: p16 and HPV ISH were positive in 23 (40%) and 24 (42%) of the cohort, respectively. The proportion of warty, basaloid, or mixed warty basaloid tumor subtypes were significantly greater in the p16-positive patients (48% vs. 3%; P < .01). p53 expression was negative in 31 (54%) cases. Only in p16-negative patients, positive p53 status was associated with pN+ disease (odds ratio, 4.4 [95% confidence interval (CI), 1.04-18.6]). In Kaplan-Meier analysis, the unadjusted estimated OS was insignificantly longer in p16-positive patients (median OS, 75 vs. 27 months; P = .27) and median CSS was not reached (P = .16). In a multivariable Cox proportional hazard model, when controlling for pathological nodal status and adjuvant chemotherapy, p16 status was a significant predictor for improved CSS (hazard ratio, 0.36 [95% CI, 0.13-0.99]). The worst CSS was seen in pN+ patients with double negative p16 and p53 expression (8 vs. 34 months; P = .01)., Conclusion: In this current cohort, p53 and p16 status showed clinical utility in predicting nodal disease as well as survival., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Purpose: To determine the predictive value and feasibility of the new outcome prediction model for Chinese patients with penile squamous cell carcinoma., Results: The 3-year disease-specific survival (DSS) survival (DSS) was 92.3% in patients with < 8.70 mg/L CRP and 54.9% in those with elevated CRP (P < 0.001). The 3-year DSS was 86.5% in patients with a BMI < 22.6 Kg/m2 and 69.9% in those with a higher BMI (P = 0.025). In a multivariate analysis, pathological T stage (P < 0.001), pathological N stage (P = 0.002), BMI (P = 0.002), and CRP (P = 0.004) were independent predictors of DSS. A new scoring model was developed, consisting of BMI, CRP, and tumor T and N classification. In our study, we found that the addition of the above-mentioned parameters significantly increased the predictive accuracy of the system of the American Joint Committee on Cancer (AJCC) anatomic stage group. The accuracy of the new prediction category was verified., Methods: A total of 172 Chinese patients with penile squamous cell cancer were analyzed retrospectively between November 2005 and November 2014. Statistical data analysis was conducted using the nonparametric method. Survival analysis was performed with the log-rank test and the Cox proportional hazard model. Based on regression estimates of significant parameters in multivariate analysis, a new BMI-, CRP- and pathologic factors-based scoring model was developed to predict disease--specific outcomes. The predictive accuracy of the model was evaluated using the internal and external validation., Conclusions: The present study demonstrated that the TNCB score group system maybe a precise and easy to use tool for predicting outcomes in Chinese penile squamous cell carcinoma patients.

Mucin core protein (MUC) 5AC is a gel-forming glycoprotein that is expressed in different types of tumour cells. MUC5AC expression in cultured cells is regulated through the extracellular matrix and through remodelling by other membranous proteins such as type IV collagen (COL4) and E-cadherin. However, it has not been elucidated whether COL4 and E-cadherin affect MUC5AC expression in tumours in vivo. Here, by analysing a single individual with concomitant neoplasms in the skin [extramammary Paget disease (EMPD)] and the stomach (gastric cancer), we show that MUC5AC expression is reduced in COL4 and membranous E-cadherin-expressing EMPD specimens whereas MUC5AC is not abolished in gastric cancer with COL4 negativity and E-cadherin cytoplasmic localization. As the EMPD and gastric cancer specimens were derived from a single patient, each specimen had the same genetic background. These in vivo results support previous in vitro studies which showed that COL4 and E-cadherin downregulated MUC5AC expression. Our study suggests that concomitant neoplasms in different organs of the same individual can serve as a strong tool for uncovering functional diversity in tumour markers in distinct cancer cells., (© 2015 British Association of Dermatologists.)

There is a lack of studies on somatic gene mutations and cell signaling driving penile carcinogenesis. Our objective was to analyze somatic mutations in genes downstream of EGFR in penile squamous cell carcinomas, especially the mTOR and RAS/MAPK pathways. We retrospectively analyzed somatic mutations in 10 in situ and 65 invasive penile squamous cell carcinomas by using Sequenom's Mass Spectrometry iPlex Technology and Oncocarta v1.0 Panel. The DNA was extracted from FFPE blocks and we identified somatic missense mutations in three in situ tumors and in 19 invasive tumors, mostly in PIK3CA, KRAS, HRAS, NRAS, and PDGFA genes. Somatic mutations in the PIK3CA gene or RAS family genes were neither associated with tumor grade, stage or outcome, and were equally often identified in hrHPV positive and in hrHPV negative tumors that showed no p53 expression. Mutations in PIK3CA, KRAS, and HRAS are frequent in penile squamous cell carcinoma and likely play a role in the development of p53-negative tumors. Although the presence of these mutations does not seem to correlate with tumoral behavior or outcome, they could be biomarkers of treatment failure with anti-EGFR mAb in patients with penile squamous cell carcinoma., (© 2015 Wiley Periodicals, Inc.)

Metastases to the penis are extremely rare events. Most frequently, penile metastases come from the urogenital system (bladder, prostate) or the rectum-sigmoid colon. Usually painful, penile lesions may be asymptomatic, making diagnosis more challenging. Hence, we report the adding value of (18)F-fludeoxyglucose-positron emission tomography/computed tomography ((18)F-FDG PET/CT) in the detection of penile metastases originating from urothelial carcinoma of the bladder. Arguably, penile metastases must be considered as an advanced disease requiring essentially palliative care. Therefore, accurate staging of clinically localized muscle-invasive bladder cancer is crucial to avoid useless curative intent radical surgery.

Purpose: The SLC8A1 gene, which encodes the Na(+)/Ca(2+) exchanger, has a key role in calcium homeostasis. Our previous gene expression oligoarray data revealed SLC8A1 under expression in penile carcinoma. We investigated whether dysregulation of SLC8A1 expression is associated with apoptosis and cell proliferation in penile carcinoma via modulation of the calcium concentration. The underlying mechanisms of SLC8A1 under expression were also explored, focusing on copy number alteration and miRNA., Materials and Methods: Transcript levels of the SLC8A1 gene and miR-223 were evaluated by quantitative polymerase chain reaction to compare penile carcinoma samples with normal glans tissue. SLC8A1 copy number was evaluated by microarray based comparative genomic hybridization. In normal and tumor samples we investigated caspase-3 and Ki-67 immunostaining as well as calcium distribution by laser ablation imaging inductively coupled plasma mass spectrometry., Results: SLC8A1 under expression was detected in penile carcinoma samples (p = 0.001), confirming our previous data. It was not associated with gene copy number loss. In contrast, miR-223 over expression (p = 0.002) inversely correlated with its putative repressor SLC8A1 (r = -0.426, p = 0.015). SLC8A1 under expression was associated with decreased calcium distribution, high Ki-67 and low caspase-3 immunoexpression in penile carcinoma compared to normal tissue., Conclusions: Down-regulation of the SLC8A1 gene, most likely mediated by its regulator miR-223, can lead to decreased calcium in penile carcinoma and consequently to suppressed apoptosis and increased tumor cell proliferation. These data suggest that the miR-223-NCX1-calcium signaling axis may represent a potential therapeutic approach to penile carcinoma., (Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

ARID1A, a member of the chromatin remodeling genes family, has been suggested as a novel tumor suppressor gene in gynecologic malignancies. However, its role in penile cancer has yet to be determined. This study assesses the immunohistochemical expression of ARID1A in penile squamous cell carcinoma (SCC) and its association with pathologic features, human papillomavirus (HPV) status, and previously reported mammalian target of rapamycin pathway markers in the same cohort. Four tissue microarrays were constructed from 112 cases of formalin-fixed, paraffin-embedded penile SCC from Paraguay. Each tumor was sampled 3 to 12 times. ARID1A expression was evaluated by immunohistochemistry using a polyclonal rabbit anti-ARID1A (BAF250A) antibody. An H score was calculated in each spot as the sum of expression intensity (0-3+) by extent (0%-100%). Median H score per case was used for statistical analysis. ARID1A expression was observed in all cases, ranging from 3% to 100% of tumor cells (median, 95%). In 96 cases (86%), ARID1A expression was observed in 90% or more tumor cells. HPV DNA was detected in 20 (38%) of 52 analyzed samples. There was a significant trend of association between ARID1A and histologic grade. ARID1A expression was not associated with histologic subtype (P = .61) or HPV status (P = .18). ARID1A expression decreased with decreasing levels of PTEN expression (P = .01). ARID1A was expressed in penile SCC, in most cases at high levels. A significant trend of association was found between histologic grade and ARID1A expression, with lower ARID1A expression, lower histologic grades, and decreased PTEN expression., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Approximately 50% of penile squamous cell carcinomas (SCC) are associated with high-risk human papillomavirus (HR-HPV) infection. We evaluated the correlation of p16(INK4a) expression and HR-HPV with clinicopathological features and outcome in a cohort of patients with penile SCC. Two tissue microarrays were constructed from 53 invasive penile SCC at our hospital. p16(INK4a) expression was assessed by immunohistochemistry (CINtec Kit). High-risk human papillomavirus status was assessed by in situ hybridization (INFORM HPV III family 16 probe B cocktail). High-risk human papillomavirus was detected in 8 cases (15%), and p16(INK4a) overexpression was found in 23 cases (44%). Both markers showed a significant association with histologic subtype (P = .017 and P = .01, respectively) and lymphovascular invasion (P = .015 and P = .015, respectively). Regarding outcome analyses, neither HPV infection nor p16(INK4a) overexpression significantly predicted overall survival or cancer-specific survival using Cox proportional hazards regression model. High-risk human papillomavirus positivity and p16(INK4a) overexpression were significantly associated with histologic subtype and presence of lymphovascular invasion. Human papillomavirus status was not predictive of outcome in our cohort., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: to investigate the role of CD- 44 immunohistochemical expression within tumoural and non-tumoural tissue, aiming to understand if it can help us to predict the need of performing inguinal lymph nodes dissection to complete surgery of the penis., Materials and Methods: CD44 immunohistochemical expression was investigated in tissue specimens from 39 patients with squamous cell carcinoma of the penis who underwent partial or total penectomy between 1987 and 2008. Patient age, tumour size, and grade; CD44 intensity score, cytological expression, topographic and distribution pattern were evaluated by immunohistochemistry on archived material and correlated with disease-specific survival., Results: mean patients age was 67.7 years; mean followup was 130.44 months. Bilateral inguinal lymphadenectomy was performed in 14 patients; there were 8 N+ patients (23.5%). pTis-pT1 vs. &gt; pT1 and the EAU classification of risk group resulted to be predictive of lymph nodal metastases at univariate analysis (respectively p = 0.006 and p = 0.045), but not the grading. The intensity score, cytological expression, topographic and distribution pattern of CD44 staining did not correlate with stage, grade and lymph nodes metastases. All disease related deaths occurred only in patients showing an high CD44 intratumoral expression, but this correlation is not statistically significant. Multivariate analysis showed that only lymph node metastasis was an independent prognostic factor predictive of lymph nodes metastases., Conclusions: CD44 expression in patients with squamous cell carcinoma of the penis is not able to predict the need of performing inguinal lymphadenectomy; staging and the EAU classification of risk group resulted to be predictive of lymph nodal metastases.

Meta-analyses show that approximately half of all squamous cell carcinomas (SCCs) of the penis are associated with a human papillomavirus (HPV) infection. As data about the tumour microenvironment of HPV-positive and HPV-negative penile carcinomas is scarce and conflicting, we examined tumour-infiltrating lymphocyte populations in such cases. The HPV status of 28 penile SCCs was determined by polymerase chain reaction, while the number and distribution of different lymphocyte populations were analysed by immunohistochemistry on whole sections of paraffin-embedded tumour specimens. The average number of tumour-infiltrating T cells in HPV-associated SCC was higher than in HPV-negative SCC, and their phenotype showed strong polarization towards a T helper 1 and cytotoxic immune response. In addition, we identified more tumour-infiltrating regulatory T cells in HPV-positive carcinomas, which might represent a mechanism of immune evasion. The present study provides further evidence that the tumour microenvironment of HPV-positive carcinomas differs from that of HPV-negative carcinomas.

Penile squamous cell carcinoma (PSCC) is a rare tumor associated with high-risk human papillomavirus (HR-HPV) infection in 30% to 60% of cases. Altered expression of miRNAs has been reported in HPV-related cervical and head and neck cancers, but such data have not been available for PSCC. We analyzed a series of 59 PSCCs and 8 condylomata for presence of HPV infection, for p16(INK4a), Ki-67, and p53 immunohistochemical expression, and for expression of a panel of cellular miRNAs (let-7c, miR-23b, miR-34a, miR-145, miR-146a, miR-196a, and miR-218) involved in HPV-related cancer. HR-HPV DNA (HPV16 in most cases) was detected in 17/59 (29%) PSCCs; all penile condylomata (8/8) were positive for low-risk HPV6 or HPV11. HR-HPV(+) PSCCs overexpressed p16(INK4a) in 88% cases and p53 in 35% of cases, whereas HR-HPV(-) PSCCs were positive for p16(INK4a) and p53 immunostaining in 9% and 44% of cases, respectively. Among the miRNAs investigated, expression of miR-218 was lower in PSCCs with HR-HPV infection and in p53(-) cancers. Hypermethylation of the promoter of the SLIT2 gene, which contains miR-218-1 in its intronic region, was frequently observed in PSCCs, mainly in those with low miR-218 expression. Epigenetic silencing of miR-218 is a common feature in HR-HPV(+) PSCCs and in HR-HPV(-) PSCCs without immunohistochemical detection of p53., (Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)