Genome-Wide Profiling Reveals HPV Integration Pattern and Activated Carcinogenic Pathways in Penile Squamous Cell Carcinoma.

Human papillomavirus (HPV) is a significant etiologic driver of penile squamous cell carcinoma (PSCC). The integration pattern of HPV and its carcinogenic mechanism in PSCC remain largely unclear. We retrospectively reviewed 108 PSCC cases who received surgery between 2008 and 2017. Using high-throughput viral integration detection, we identified 35 HPV-integrated PSCCs. Unlike cervical cancer, the HPV E2 oncogene was not prone to involvement in integration. Eleven of the 35 (31.4%) HPV-integrated PSCCs harbored intact HPV E2; these tumors had lower HPV E6 and E7 expression and higher expression of p53 and pRb proteins than those with disrupted E2 did (p < 0.001 and p = 0.024). Integration breakpoints are preferentially distributed in or near host genes, including previously reported hotspots (KLF5, etc.) and newly identified hotspots (CADM2, etc.), which are mainly involved in oncogenic signaling pathways (MAPK, JAK/STAT, etc.). Regarding the phosphorylation levels of JNK, p38 was higher in HPV-positive tumors with MAPK-associated integration than those in HPV-positive tumors with other integration and those in HPV-negative tumors. In vitro, KLF5 knockdown inhibited proliferation and invasion of PSCC cells, while silencing CADM2 promoted migration and invasion. In conclusion, this study enhances our understanding of HPV-induced carcinogenesis in PSCC, which may not only rely on the E6/E7 oncogenes, but mat also affect the expression of critical genes and thus activate oncogenic pathways.

Pelvic Lymph Node Dissection in Penile Cancer With Inguinal Lymph Node Extranodal Extension: A Multicenter Experience.

Background: The aim of this study is to determine the necessary extent of penile lymph node dissection (PLND) in penile cancer patients with inguinal lymph node extracapsular extension (ILN-ENE). Methods: Penile cancer patients who underwent PLND in 15 centers from January 2006 to April 2020 were retrospectively analyzed. PLND was performed in patients with ILN-ENE. Results: Sixty-two patients with ILN-ENE were included in the analysis. A total of 51.6% (32/62) of the patients were confirmed to have pelvic lymph node metastasis (PLNM), and 31.3% (10/32) of patients were confirmed to have multiple PLNMs. Of the patients with metastases, 59.4% (19/32) had bilateral inguinal lymph node metastasis (ILNM). According to the anatomical structure, 71.9% (23/32) of the patients had PLNM in the external iliac region, and 56.2% (18/32) had PLNM in the obturator region. Among those with oligo-PLNM, 65.1% (28/43) of the patients had PLNM in the external iliac region and 38.9% (15/43) had PLNM in the obturator region. A significant overall survival difference was observed between patients with the bilateral ILNM and unilateral ILNM (36-month: 21.2 vs. 53.7%, respectively, P = 0.023). Patients with bilateral ILNM had relatively poor metastasis-free survival compared with unilateral ILNM (36-month: 33.0 vs. 13.9%, respectively, P = 0.051). Conclusions: The external iliac and obturator region were the most commonly affected regions in patients with ILN-ENE, and these regions were the only affected regions in patients with oligo-PLNM. Patients with bilateral ILNM had a high risk of PLNM and worse survival.

Up-regulation of indoleamine 2,3-dioxygenase 1 (IDO1) expression and catalytic activity is associated with immunosuppression and poor prognosis in penile squamous cell carcinoma patients.

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.

EGFR mono-antibody salvage therapy for locally advanced and distant metastatic penile cancer: Clinical outcomes and genetic analysis.

PURPOSE: There are limited therapeutic options for patients with advanced penile squamous cell carcinoma (PSCC) after chemotherapy failure. Thus, we evaluated the feasibility of salvage treatment using the epidermal growth factor receptor (EGFR) mono-antibody nimotuzumab in chemotherapy-failed PSCC patients and explored potential response or resistance biomarkers. MATERIALS AND METHODS: Six chemotherapy-failed PSCC patients with locally advanced disease or distant metastasis were enrolled consecutively to nimotuzumab treatment. Clinical responses and side effects were evaluated, and genetic characteristics of cancer specimens were analyzed through the next-generation sequencing of hotspot regions in cancer-related genes. RESULTS: Two of 6 patients showed partial responses, one was identified as having stable disease, while the other 3 had disease progression after nimotuzumab therapy. Side effects were all welltolerated. Genetic analysis revealed that TP53, CDKN2A, RB1, SMAD4, FLT3, and PIK3CA were the most frequently mutated genes in PSCC specimens, while altered KRAS, HRAS, EGFR, ERBB2, and FLT3 may be correlated with nimotuzumab resistance. Furthermore, 3 patients that were human papillomavirus-positive each showed clinical response or stable disease. CONCLUSIONS: EGFR mono-antibody may be a potential modality for locally advanced PSCC patients after chemotherapy failure. Further large-scale clinical studies are needed to elucidate the role of human papillomavirus status and critical gene mutations in the clinical response to EGFR-targeted therapy.

Elevated serum LAMC2 is associated with lymph node metastasis and predicts poor prognosis in penile squamous cell carcinoma.

PURPOSE: Molecular biomarkers, especially serologic factors, have been widely applied in cancer diagnosis and patient follow-up. However, there are few valuable prognostic factors in penile squamous cell carcinoma (PSCC). Here, the authors investigated whether laminin gamma 2 (LAMC2) expression, especially serum LAMC2 (sLAMC2) level, was a suitable prognostic factor that could aid in the prediction of survival in PSCC. PATIENTS AND METHODS: This study included 114 PSCC patients. Reverse transcription-quantitative polymerase chain reaction, Western blotting, and immunohistochemistry were performed to detect LAMC2 expression; enzyme-linked immunosorbent assays were used to test sLAMC2 concentration; and a Transwell assay and an in vivo experiment in nude mice were used to test PSCC cell migration, invasion, and metastasis. The chi-squared test was used to analyze the association between LAMC2 level and clinical parameters, the Cox proportional hazards regression model was used to evaluate the hazard ratio for death, and Kaplan-Meier analysis with a log-rank test was used for the survival analysis. RESULTS: LAMC2 was overexpressed in PSCC tissues, and the LAMC2 expression level was higher in metastatic lymph node (LN) tissues than in primary cancer tissues; moreover, the LAMC2 levels in primary cancer tissues and sLAMC2 were higher in patients with LN metastasis than in those without LN metastasis. Upregulated LAMC2 facilitated the migration, invasion, and epithelial-to-mesenchymal transition of PSCC cells in vitro and promoted LN metastasis of PSCC cells in nude mice. Elevated LAMC2 levels were strongly correlated with advanced clinicopathologic parameters, especially LN metastasis, in PSCC patients and predicted shorter disease-specific survival. The predictive value of sLAMC2 is superior to that of C-reactive protein and squamous cell carcinoma antigen previously reported in PSCC patients, and a stratification analysis revealed that the level of sLAMC2 had a higher predictive value for disease-specific survival in early penile cancer (especially at the N0/X stage) than in later-stage penile cancer. CONCLUSION: These findings suggest that sLAMC2 is a potential serologic prognostic marker in PSCC and could aid in risk stratification in early-stage PSCC patients.

Molecular characterization and integrative genomic analysis of a panel of newly established penile cancer cell lines.

Cell line models are essential tools to study the molecular mechanisms underlying tumor initiation and progression. There are limited treatment options for penile squamous cell carcinoma (PSCC), accounting for 1-2% of male tumors in developing countries, and limited progress in preclinical research in PSCC due to lacking available models with identified genomic characteristics. Here, biological and molecular characteristics and whole-genomic alterations were analyzed in a panel of PSCC cell lines newly established in our laboratory. These cell lines were all human papillomavirus (HPV)-negative, epithelial-like, immortalized, and tumorigenic in nude mice, whereas they displayed different proliferation, migration and invasion capacities in vitro, and tumorigenic ability in nude mice. They were all cisplatin sensitive, anti-EGFR therapy resistant, and androgen irresponsive. Whole-genomic sequecing analysis revealed that transition mutations (C:G>T:A and T:A>C:G) were the most common substitution types in these cell lines, whereas ERCC5, TP53, PTH1, CLTCL1, NOTCH2, MAP2K3, CDK11A/B, USP6, ADCH5, BCLAF1, CDKN2A, FANCD2, HRAS, and NOTCH1 were the most frequently altered genes. Amplifications of MYC, PLAG1, NCOA2, RUNX1T1, COX6C, and EGFR and losses of FBXW7, TET2, XPC, and FANCE were frequently observed in cell lines. The exomic variations between cell lines and their corresponding cancer tissues were highly consistent. Genetic variations were mainly involved in the MAPK, Jak-STAT, TGF-beta, Notch, and apoptosis signaling pathways. Conclusively, these panel of PSCC cell lines established in our laboratory harbor some common or specific biological characteristics and genomic variations, and they may serve as optimal models to investigate the molecular mechanisms underlying the progression, metastasis, relapses, and treatment resistance of PSCC and to develop effective treatment strategy.

More precise prediction in Chinese patients with penile squamous cell carcinoma: protein kinase CK2α catalytic subunit (CK2α) as a poor prognosticator.

PURPOSE: In this study, we assess the CK2α expression in human penile squamous cell carcinoma (SCC) and its clinical significance. METHODS: A total of 157 human penile SCC tissue samples were immunohistochemically analyzed. In addition, 12 human penile SCC and adjacent normal tissues were examined for CK2α protein and mRNA expression by Western blotting and real-time quantitative PCR, respectively. Survival was analyzed using the Kaplan-Meier test and the log-rank test. Multivariate Cox proportional hazard regression analysis was performed to determine the impacts of CK2α expression and the clinicopathological features on patient disease-specific survival (DSS). Likelihood ratios (LRs), Akaike information criterion (AIC) values, and concordance indexes (C-indexes) were investigated to evaluate the accuracies of the factors. Bootstrap-corrected C-indexes were used for internal validation (with sampling 1000 times). RESULTS: A significant difference in the distribution of CK2α was observed between the normal and penile carcinoma tissues (P<0.001). CK2α expression was associated with the pathological T and N stages in the penile cancer tissues (P<0.001). High CK2α expression was with significantly poorer DSS compared with low expression one (P<0.001). Western blotting and real-time quantitative PCR also confirmed that CK2α expression was increased in the penile cancer tissues. In multivariate Cox regression analysis, CK2α overexpression still was one of independent prognostic factors for penile SCC (P=0.005). The predictive accuracy of CK2α was verified by analysis of the C-indexes. CONCLUSION: High protein kinase CK2α expression is associated with several prognostic factors and is thus a significant indicator of poor prognosis for penile cancer.

Bilateral pelvic lymph node dissection for Chinese patients with penile cancer: a multicenter collaboration study.

BACKGROUND: Current guidelines recommend pelvic lymphadenectomy (PLND) for patients with pelvic lymph node metastasis and special state. However, these data and recommendations do not distinguish the role of PLND in different patient groups and confirm the final benefits. The aim of this study was to confirm the efficacy of pelvic lymphadenectomy (PLND) for the different groups of patients. METHODS: Data obtained from 7 centers were retrospectively analyzed. Of the patients, 190 pN2-3 penile carcinoma patients confirmed by bilateral inguinal lymph node excision were included in this study. Sixty-nine and 121 of these patients did and did not undergo bilateral PLND, respectively. The baseline differences from the patients were matched by propensity score analysis. RESULTS: In this study, the Kaplan-Meier estimated disease-specific survival (DSS) was not significantly different between the PLND and no-PLND groups (P = 0.796). According to the propensity score matching for T stage, N stage, grade, adjuvant therapies, and lymph node stage (number of inguinal lymph node metastasis and extranodal extension), 48 patients were selected for each group. Among the pN2 patients, the PLND group showed higher DSS rates than the no-surgery group (P = 0.030). However, even after matching, survival did not differ between the PLND and no-PLND patients among all patients (P = 0.609) and pN3 patients (P = 0.417) with comparable DSS. CONCLUSION: Bilateral PLND may improve survival in pN2 patients. Men with pN3 may not benefit from bilateral PLND.

Development of a new outcome prediction model for Chinese patients with penile squamous cell carcinoma based on preoperative serum C-reactive protein, body mass index, and standard pathological risk factors: the TNCB score group system.

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.

Development of a New Classification Method for Penile Squamous Cell Carcinoma Based on Lymph Node Density and Standard Pathological Risk Factors: The ND Staging System.

OBJECT: In this study, we evaluated the role of lymph node density (LND) and validated whether LND increases the accuracy of survival prediction when combined with the American Joint Committee on Cancer (AJCC) pathological node (N) staging system for penile cancer (7(th) edition). METHODS: A total of 270 Chinese penile cancer patients treated between March 1999 and October 2014 were retrospectively analyzed. LND was analyzed as a trichotomous variable for the prediction of DSS in this cohort. We developed a new prediction model, which we refer to as the ND staging system, that is based on LND and pathological N staging. The predictive accuracy of this model was further assessed using the concordance index. RESULTS: LND was correlated with the laterality of lymph node metastasis, extranodal extension, pelvic lymph node metastases, and pathologic tumor (T) and N stages (P<0.05). In separate multivariate Cox regression models, the LND (hazard ratio [HR], 1.966, 95% confidence interval [CI], (1.112-3.473, P=0.020) yielded independent effects on the outcome. According to the LND classification, the 3-year disease-specific survival (DSS) rates for patients with LNDs <7.0%, 7.0 to 16.9%, and ≥17.0% were 90.9%, 66.5%, and 22.2%, respectively (P<7.0%; 7.0%-16.9% =0.006; P7.0-16.9%; ≥17.0% =0.001). The corresponding rates were 95.7%, 76.7%, and 28.1% for the ND1, ND2, and ND3 patients, respectively (PND1-ND2 =0.047; PND2-ND3 <0.001). The indexes indicated that the accuracy of the pathological ND category that incorporated LND was significantly increased. CONCLUSION: LND was associated with some prognosticators and is thus a prognostic factor. The ND staging system that incorporates the LND better reflects the prognoses of penile cancer patients.

Clinical significance of preoperative C-reactive protein and squamous cell carcinoma antigen levels in patients with penile squamous cell carcinoma.

OBJECTIVE: To evaluate the relevance of C-reactive protein (CRP) and squamous cell carcinoma antigen (SCC-Ag) levels in relation to clinicopathological factors and prognosis in penile cancer. PATIENTS AND METHODS: A total of 124 Chinese patients with penile squamous cell carcinoma (SCC), treated between November 2007 and October 2014, were analysed retrospectively. Receiver-operating characteristic curves were used to identify the combination of markers with the best sensitivity and specificity for prognosis prediction. Statistical data analysis was performed using a non-parametric method, and survival analysis was performed using the log-rank test and Cox proportional hazard model. RESULTS: Levels of CRP ≥4.5 mg/L and SCC-Ag ≥1.4 ng/mL were both significantly associated with lymph node metastasis (LNM) laterality (chi-squared trend test, P = 0.041), extranodal extension (chi-squared trend test, P < 0.001), pelvic LNM (chi-squared trend test, P = 0.024), pathological tumour status (chi-squared trend test, P = 0.002), pathological nodal status (chi-squared trend test, P < 0.001), and disease-specific survival (DSS; log-rank test, P < 0.001). Moreover, the influence of CRP and SCC-Ag levels on DSS (P = 0.033, hazard ratio 3.390, 95% confidence interval 1.104-10.411) remained after adjusting for smoking history, phimosis, tumour status, tumour cell differentiation and nodal status. CONCLUSIONS: The present study shows that the combined measurement of preoperative CRP and SCC-Ag levels may serve as an independent biomarker for LNM, advanced tumour stage and DSS in patients with penile SCC.