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.

VISTA and PD-L1 synergistically predict poor prognosis in patients with extranodal natural killer/T-cell lymphoma.

Although PD-1/PD-L1 blockade therapy confers salutary effects across cancer types, their efficacy in Extranodal Natural killer/T-cell lymphoma (ENKTCL) patients is limited and unpredictable. Here, we comprehensively evaluated the expression profile of a panel of immune-regulatory makers to identify novel prognostic biomarkers and/or therapeutic targets for this malignancy. Using immunohistochemistry and multiplex immunofluorescence, we found that the expression of VISTA (88.1%) was predominantly in CD68+ macrophages and much higher than PD-L1 expression (68.7%) in ENKTCL. B7-H4 and HHLA2 proteins were not detected in ENKTCL. B7-H3 was expressed in minority of ENKTCL patients (13.7%) and mainly colocalized with CD31. A close correlation was detected between VISTA and PD-L1, but they were not co-expressed in the same cells. High expressions of VISTA or PD-L1 were significantly associated with detrimental clinicopathological characteristics, dismal prognosis, and high density of CD8+ TILs, and high VISTA expression was also significantly associated with high density of Foxp3+ TILs. VISTA combined with PD-L1 was an independent prognostic factor for PFS and OS. Moreover, the patients with high VISTA showed a poor response to PD-1 blockades in ENKTCL. In conclusion, these findings provide a rationale for VISTA as an ideal immunotherapeutic target next to PD-L1 for ENKTCL.

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.

HHLA2 and PD-L1 co-expression predicts poor prognosis in patients with clear cell renal cell carcinoma.

BACKGROUND: Although clear cell renal cell carcinoma (ccRCC) is well known as a highly immunogenic tumor, only a small subset of patients could benefit from current immunotherapy, which might be due to the heterogeneity of immune microenvironment in ccRCC. So, it is meaningful to explore novel immunotherapy or combination therapy for improving therapeutic efficacy. HHLA2, a newly discovered B7 family member, is prevalently expressed in numerous tumors, including ccRCC. This study aimed to investigate the prognostic impact of HHLA2/PD-L1 co-expression and its relationship with tumor-infiltrating lymphocytes (TILs). METHODS: The expression levels of HHLA2, PD-L1, CD8, and CD4 in cancer tissues from cases (206 in the training cohort and 197 in the validation cohort) with surgically resectable primary ccRCC were evaluated by immunohistochemistry. RESULTS: The positive rates of HHLA2 were much higher than those of PD-L1 in ccRCC tissues. HHLA2-positive expression was significantly associated with necrosis, microvascular invasion, advanced Fuhrman nuclear, and TNM stage and indicated a shorter progression-free survival (PFS) and overall survival (OS) in both cohorts. Moreover, patients with HHLA2/PD-L1 co-expression suffered the highest risk of disease progression and death by a significant margin. Besides, HHLA2/PD-L1 co-expression was significantly associated with a high density of CD8+ and CD4+ TILs. Notably, a new immune classification, based on HHLA2/PD-L1 co-expression and TILs, successfully stratified PFS and OS, especially in patients with TILs positivity. CONCLUSIONS: The expression of HHLA2 is more frequent than PD-L1 in ccRCC. HHLA2/PD-L1 co-expression had an adverse impact on the prognoses of patients with ccRCC; this finding provides a rationale for combination immunotherapy with anti-HHLA2 and PD-L1 blockage for patients with ccRCC in the future.

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.

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.