Testicular Germ-Cell Tumors with Spermatic Cord Involvement: A Retrospective International Multi-Institutional Experience.

The 8th Edition of the American Joint Committee on Cancer (AJCC) Staging Manual designates discontinuous involvement of spermatic cord soft tissue by testicular germ cell tumors as a metastatic deposit. We conducted a retrospective international multi-institutional study to validate the current recommendations. Thirty-three (72%) nonseminomatous and 13 (28%) seminomatous testicular germ cell tumors were collected from 15 institutions in America, Europe, and Asia. Testicular tumor size ranged from 1.3 to 18.0 cm (mean: 6.1). Cases were classified as discontinuous involvement of spermatic cord soft tissue (n = 26), continuous cord involvement (n = 17), or cord lymphovascular invasion (n = 3). The mean follow-up was 39 months. Clinical stage for discontinuous involvement of spermatic cord soft-tissue patients was I (local disease) in 2/24 (8%), II (regional disease) in 6/24 (25%), and III (distant disease) in 16/24 (67%) cases; 16 (67%) patients presented with distant metastasis. Clinical stage for continuous cord involvement patients was I in 9/17 (53%), II in 4/17 (23%), and III in 4/17 (23%); 4 (23%) patients presented with distant metastasis. Disease progression was seen in 4 patients with discontinuous involvement of spermatic cord soft tissue and 5 with continuous cord-involvement (p = 0.699). When comparing discontinuous and continuous cord involvement, a significant difference was found in cord margin status (p = 0.044), spermatic cord tumor size (p = 0.016), lymph-node involvement (p = 0.037), distant metastasis (p = 0.010), individual clinical stage (p = 0.003), and nonadvanced vs. advanced disease (p = 0.003) at presentation. In multivariate analysis, after adjusting for age, histology, testicular tumor size, percent of embryonal carcinoma, lymphovascular invasion, and cord margin status, discontinuous involvement of spermatic cord soft tissue was significantly associated (p = 0.011) with advanced clinical stage at presentation. Our findings support the designation of metastatic disease for discontinuous involvement of spermatic cord soft tissue, as introduced by the 8th edition of the AJCC staging.

Tumour immune microenvironment in primary and metastatic papillary renal cell carcinoma.

AIMS: Among renal cell carcinoma (RCC) the tumour immune microenvironment has been best characterised in clear cell RCC. In this study we investigated the expression of several immune markers, including PD-L1, FoxP3 and CD8 in primary and metastatic papillary RCC. METHODS AND RESULTS: Three tissue microarrays were constructed from 78 cases with primary papillary RCC and paired metastatic tumour (24 cases) from 78 patients treated between 1982 and 2014. Immunohistochemistry analysis was performed using commercially available antibodies for PD-L1 (clone E1L3N), FoxP3, CD8 and Ki-67. Markers expression level in tumour and/or associated immune cells was analysed by tissue type (non-tumour versus primary tumour versus metastatic tumour) and correlated to clinicopathological features and outcome. CONCLUSION: We found PD-L1 expression in up to one-quarter of primary and metastatic papillary RCC. On univariate analysis, CD8/FoxP3 ratio >1 was associated with favourable outcome, whereas papillary RCCs with high numbers of dual CD8/Ki-67-positive lymphocytes showed an increased likelihood for tumour progression and overall and cancer-related mortality. The association of CD8/FoxP3 ratio >1 and high count of CD8/Ki-67 with outcome remained significant on multivariate analysis when adjusting for stage, grade and patient's age.

Expression of Nectin-4 and PD-L1 in Upper Tract Urothelial Carcinoma.

Enfortumab vedotin is a novel antibody-drug conjugate targeting Nectin-4, which is highly expressed in urothelial carcinoma. However, the expression status of Nectin-4 in upper tract urothelial carcinoma (UTUC) remains unclear. The relationship between Nectin-4 and Programmed Death Ligand 1 (PD-L1) in UTUC is also ambiguous. We performed immunohistochemical analysis of 99 UTUC tissue microarray to assess the expression of Nectin-4 and PD-L1 in UTUC. Nectin-4-positivity was detected in 65 (65.7%) samples, and PD-L1 was detected in 24 (24.2%) samples. There was no correlation between the expression of Nectin-4 and PD-L1. Patients with strong Nectin-4-expressing tumors had a significantly higher risk of progression (p = 0.031) and cancer-specific mortality (p = 0.036). Strong Nectin-4 expression was also an independent predictor of disease progression in the high-risk group (pT3 ≤ or presence of lymphovascular invasion or lymph node metastasis) (Hazard ratio, 3.32 [95% confidence interval, 1.20-7.98; p = 0.027]). In conclusion, we demonstrated that Nectin-4 expression rate in UTUC was 65.7% and independent of PD-L1 expression. Strong Nectin-4 expression was associated with worse progression-free survival in high-risk UTUC. These findings suggested that enfortumab vedotin may be effective in a broad range of patients with UTUC, regardless of PD-L1 expression.

Incidence and distribution of UroSEEK gene panel in a multi-institutional cohort of bladder urothelial carcinoma.

Noninvasive approaches for early detection of bladder cancer are actively being investigated. We recently developed a urine- based molecular assay for the detection and surveillance of bladder neoplasms (UroSEEK). UroSEEK is designed to detect alterations in 11 genes that include most common genetic alterations in bladder cancer. In this study, we analyzed 527 cases, including 373 noninvasive and 154 invasive urothelial carcinomas of bladder from transurethral resections or cystectomies performed at four institutions (1991-2016). Two different mutational analysis assays of a representative tumor area were performed: first, a singleplex PCR assay for evaluation of the TERT promoter region (TERTSeqS) and second, a multiplex PCR assay using primers designed to amplify regions of interest of 10 (FGFR3, PIK3CA, TP53, HRAS, KRAS, ERBB2, CDKN2A, MET, MLL, and VHL) genes (UroSeqS). Overall, 92% of all bladder tumors were positive for at least one genetic alteration in the UroSEEK panel. We found TERT promoter mutations in 77% of low-grade noninvasive papillary carcinomas, with a relatively lower incidence of 65% in high-grade noninvasive papillary carcinomas and carcinomas in situ; p = 0.017. Seventy-two percent of pT1 and 63% of muscle-invasive bladder tumors harbored TERT promoter mutations with g.1295228C>T alteration being the most common in all groups. FGFR3 and PIK3CA mutations were more frequent in low-grade noninvasive papillary carcinomas compared with high-grade noninvasive papillary carcinomas and carcinomas in situ (p < 0.0001), while the opposite was true for TP53 (p < 0.0001). Significantly higher rates of TP53 and CDKN2A mutation rates (p = 0.005 and 0.035, respectively) were encountered in muscle-invasive bladder tumors compared with those of pT1 stage. The overwhelming majority of all investigated tumors showed at least one mutation among UroSEEK assay genes, confirming the comprehensive coverage of the panel and supporting its potential utility as a noninvasive urine-based assay.

Vascular architectural patterns in clear cell renal cell carcinoma and clear cell papillary renal cell carcinoma.

Renal cell carcinomas (RCC) are well-vascularized tumors. Although clear cell RCC (CCRCC) show a characteristic vascular network, some cases show overlapping features with other RCC. We aimed to evaluate vascular architectural patterns, microvessel density (MVD), and endothelial cell density (ECD) in CCRCC compared to clear cell papillary RCC (ccpRCC). Thirty-four RCC (17 CCRCC and 17 ccpRCC) were included in the study. CD34 was used to evaluate vascular architectural patterns by microscopic estimation in all cases. CD34, ERG, and Bioquant Osteo 2019 Imaging Analysis Software were used to evaluate MVD and ECD in 17 CCRCC and 15 ccpRCC. Mean MVD was 526.63 in CCRCC vs. 426.18 in ccpRCC (p = 0.16); mean ECD was 937.50 in CCRCC vs. 1060.21 in ccpRCC (p = 0.25). CD34 highlighted four distinct vascular architectural patterns: pseudoacinar, Golgi-like, lacunae, and scattered. Lacunae and pseudoacinar was the most frequent combination in CCRCC; lacunae and Golgi-like was the predominant combination among ccpRCC. Pseudoacinar was most extensive in CCRCC and least in ccpRCC; Golgi-like was predominant in ccpRCC and uncommon in CCRCC. The extent of pseudoacinar and Golgi-like vascular architectural patterns was significantly different between CCRCC and ccpRCC (p < 0.05). Pathologists acquainted with these different vascular architectural patterns may utilize them as an additional tool in the distinction of CCRCC from ccpRCC.

Well-differentiated neuroendocrine tumors of the lower urinary tract: biologic behavior of a rare entity.

The spectrum of neuroendocrine (NE) tumors in the genitourinary tract ranges from the aggressive large and small cell carcinomas to the often benign paraganglioma and well-differentiated neuroendocrine tumor (WD-NET). At least 15 pure lower urinary tract (LUT) WD-NETs have been described. Owing to the rarity of WD-NET in the LUT and the limited number of reported cases, a better definition of their biologic long-term behavior is warranted. Herein, we aim to describe 10 new cases of WD-NET arising in the LUT and expand on follow-up findings. Ten consultation cases were identified and included 6 men and 4 women who ranged from 45 to 73 years of age. Seven cases arose in the bladder with one located in the bladder neck, 1 arose in the prostatic urethra, 1 arose in the female urethra, and 1 arose in the left ureteral orifice. All lesions were confined to the lamina propria, and tumor architecture was pseudoglandular in all cases. Associated cystitis cystica et glandularis was identified in 5 cases; urothelial papilloma and florid von Brunn's nests were found in 2 additional cases. Immunohistochemical staining for synaptophysin and chromogranin was diffusely positive in 9 cases and focal in 1 case, and the Ki-67 proliferation index was 5% or less in all tumors. Follow-up ranged from 37 to 137 months (mean = 82; median = 77), and there was no evidence of residual disease or recurrence in any of the 10 patients during the follow-up period.

Rare MDM2 amplification in a fat-predominant angiomyolipoma.

Angiomyolipomas (AMLs) are triphasic tumors (smooth muscle, vascular and adipocytic components) with myomelanocytic differentiation, arising most commonly in the kidneys, which can show predominant epithelioid morphology and fat-predominant or fat-poor variants. Fat-predominant AMLs can show areas of hypercellularity and lipoblast-like cells, and these features can mimic well-differentiated liposarcoma (WDLS). To date, only one documented metastatic epithelioid AML showed unequivocal MDM2 amplification by fluorescence in situ hybridization. We describe our findings in a series of 35 AMLs including epithelioid, fat-poor, and fat-predominant variants, following interrogation of the MDM2 locus by FISH and CISH assays. MDM2 amplification was detected in 1 fat-predominant AML. Our findings demonstrate that rare MDM2 amplifications can occur in AMLs. We favor that this finding likely represents a "molecular bystander" event since these tumors are mainly driven by aberrations in the TSC1/TSC2 genes. Nevertheless, the presence of MDM2 amplification in a fat-predominant AML could present a potential diagnostic pitfall, particularly when confronted with the differential diagnosis of fat-predominant AML and WDLS in limited material from the retroperitoneum.

Immune checkpoint status and tumor microenvironment in vulvar squamous cell carcinoma.

Vulvar squamous cell carcinoma accounts for 5% of cancers of the female genital tract. Current guidelines recommend wide local excision with negative surgical margins as the standard treatment. However, the extent of the tumor-free resection margin after wide local excision is still controversial in many cases. Drugs targeting immune checkpoints such as PD-1 or its ligand PD-L1 have potential clinical utility in these patients. We examined the expression of PD-L1 in tumor cells and immune cells, as well as the proportion of PD-1, CD8, and FOXP3 positive lymphocytes. Twenty-one cases of invasive vulvar squamous cell carcinomas were reviewed. Whole slides of representative formalin-fixed, paraffin-embedded archival material were used for analysis. Odds ratios (OR) and hazard ratios (HR) were used to estimate risk for disease recurrence, overall mortality, and cancer mortality. PD-L1 expression was found in 43% of tumor cells, with higher proportions in intratumoral (67%) and peritumoral (81%) immune cells. OR and HR for disease recurrence and cancer mortality were higher in tumors with higher CD8 expression. OR and HR for overall mortality were also higher in tumors with higher PD-L1 and CD8 expression. In conclusion, nearly half of cases were PD-L1 positive in tumor cells with over two-third of cases demonstrating PD-L1 positivity in immune cells. Immunohistochemical expression of PD-L1 and CD8 could be used to suggest higher risk of disease recurrence, overall mortality, and cancer mortality. Furthermore, our data contributes to the growing evidence that targeting the PD-1/PD-L1 pathway may be beneficial in vulvar squamous cell carcinomas.

Performance of novel non-invasive urine assay UroSEEK in cohorts of equivocal urine cytology.

Urine cytology is an essential element of the diagnostic work up of hematuria. A significant proportion of cases continue to be placed in the "atypical" or "suspicious" categories of the Paris system for urine cytology, posing difficulty in patient management. We report on the performance of our recently described urine-based assay "UroSEEK" in cases with equivocal diagnosis in patients who are investigated for bladder cancer. Urine samples were collected from two cohorts. The first consisted of patients who presented with hematuria or lower urinary tract symptoms (early detection cohort) and the second of patients that are in follow-up for prior bladder cancer (surveillance cohort). Urine samples were analyzed for mutations in 11 genes and aneuploidy. In the early detection setting, we found high sensitivity and specificity (96% and 88%, respectively) and a strong negative predictive value of 99%. The assay performance was less robust in the surveillance cohort (sensitivity of 74%, specificity of 72%, and negative predictive value of 53%). UroSEEK demonstrated a notable lead time to cancer diagnosis. Seven cases in the early detection cohort and 71 surveillance cases were detected at least 6 months prior to clinical diagnosis. Our results suggest a potential role for UroSEEK assay in guiding management of patients with atypical urine cytology if confirmed in future prospective trials.

Expression and Role of Methylenetetrahydrofolate Dehydrogenase 1 Like (MTHFD1L) in Bladder Cancer.

Cancer cells utilize vitamin folate to fulfill their excessive demand for nucleotides and amino acids. Dihydrofolate reductase (DHFR), an enzyme involved in folate metabolism converts dihydrofolate into tetrahydrofolate, which is required for the de novo synthesis of purines, and certain amino acids. DHFR inhibitors are used as a chemotherapeutic agent. Cancer sequencing analysis has identified additional enzymes in folate metabolism that are dysregulated in cancer. Methylenetetrahydrofolate dehydrogenase 1 like (MTHFD1L), one such enzyme is overexpressed in bladder cancer. MTHFD1L is a mitochondrial enzyme involved in the folate cycle by catalyzing the reaction of formyl-tetrahydrofolate to formate and tetrahydrofolate (THF). THF is crucial for de novo purine and thymidylate synthesis and is also involved in the regeneration of methionine. Cancer cells rely on purines derived from the de novo pathway for the nucleotides whereas normal cells favor the salvage pathway. In this study we examined MTHFD1L expression in bladder cancer. By using publicly available cancer transcriptome data analysis web-portal UALCAN, we found overexpression of MTHFD1L in bladder cancer and expression was associated with overall survival. RT-PCR and immunoblot analysis confirmed the overexpression of MTHFD1L in muscle invasive bladder cancer tissues compared to normal urothelium. Furthermore, our investigations suggested a critical role for MTHFD1L in bladder cancer cell proliferation, colony formation and invasion. Thus, in this study, we show the significance of the folate metabolic enzyme MTHFD1L in aggressive bladder cancers and suggest that being an enzyme, MTHFD1L serves as a valuable therapeutic target.

Immunohistochemical assessment of basal and luminal markers in non-muscle invasive urothelial carcinoma of bladder.

The Cancer Genome Atlas project introduced genomic taxonomy of basal and luminal molecular subtypes in muscle invasive bladder cancer. Fewer studies have addressed the molecular classification in non-muscle invasive bladder cancer (NMIBC). Our aim is to assess the applicability of the proposed phenotypic classification for NMIBC. Three TMAs were constructed from 193 TURBT specimens of 60 bladder cancer patients treated at one of the authors' institutions (1998-2008). Follow-up data on recurrence, grade, or stage progression was obtained. Immunohistochemistry was performed using an automated Ventana System for markers indicative of luminal (GATA3, CK20, ER, Uroplakin II, and HER2/neu) and basal (CK5/6 and CD44) phenotype. Marker expression was evaluated by 3 urologic pathologists. Using unadjusted logistic regression, we found significant association between tumor recurrence at next biopsy and CD44 expression (OR = 2.51, P = 0.03), tumor recurrence at any subsequent biopsy and ER expression (OR = 0.24, P = 0.04), and tumor grade progression at any subsequent biopsy and HER2/neu expression (OR = 0.24, P = 0.04). After adjusting for pathologic stage, we found a significant association between CK5/6 expression and tumor stage progression at either next or any subsequent biopsy (OR = 0.94, P = 0.006; and OR = 0.97, P = 0.02, respectively). Our findings suggest that individual immunohistochemical markers may be of value as prognostic factors in NMIBC.

Tumor immune microenvironment in non-muscle-invasive urothelial carcinoma of the bladder.

Immunotherapy has gained significance in a variety of tumor types including advanced urothelial carcinoma. Noninvasive urothelial lesions have been treated with intravesical Bacillus-Calmette-Guerin (BCG) for decades. Given treatment failure in a subset of these tumors, ongoing clinical trials investigating the role of checkpoint inhibitors are actively pursued in this group of patients. The present study aims to delineate PD-L1, CD8, and FOXP3 expression in tumor microenvironment in non-muscle-invasive urothelial carcinoma samples obtained via sequential biopsies and to assess its potential role in predicting disease outcome. Cases with >1% and> 5% PD-L1 expression in tumor cells showed lower relative risk (RR) to recur at any subsequent biopsy compared with those with lower PD-L1 expression (RRs, 0.83 [P = .009] and 0.81 [P = .03], respectively). Cases with higher expression of FOXP3 in peritumoral lymphocytes were at lower risk for tumor grade progression at any biopsy (RR, 0.2; P = .02). Tumors with FOXP3/CD8 expression ratio of >1 in intratumoral lymphocytes had lower risk of grade progression (RR, 0.28; P = .04). Although higher number of FOXP3-, CD8-, and PD-L1-positive lymphocytes were encountered after BCG treatment, the findings did not reach statistical significance. In patients without BCG treatment, PD-L1 expression in tumor cells and peritumoral lymphocytes varied across serial biopsies, suggesting the need for additional approaches to assess eligibility for immunotherapy in non-muscle-invasive urothelial carcinoma patients.

Targeted sequencing of plasmacytoid urothelial carcinoma reveals frequent TERT promoter mutations.

Activating mutations in the promoter of the telomerase reverse transcriptase (TERT) gene are the most common genetic alterations in urothelial carcinoma (UC) of the bladder and upper urinary tract. Although the cadherin 1 (CDH1) gene is commonly mutated in the clinically aggressive plasmacytoid variant of urothelial carcinoma (PUC), little is known about their TERT promoter mutation status. A retrospective search of our archives for PUC and UC with plasmacytoid and/or signet ring cell features (2007-2014) was performed. Ten specimens from 10 patients had archived material available for DNA analysis and were included in the study. Intratumoral areas of nonplasmacytoid histology were also evaluated when present. Samples were analyzed for TERT promoter mutations with Safe-SeqS, a sequencing error-reduction technology, and sequenced using a targeted panel of the 10 most commonly mutated genes in bladder cancer on the Illumina MiSeq platform. TERT promoter mutations were detected in specimens with pure and focal plasmacytoid features (6/10). Similar to conventional UC, the predominant mutation identified was g.1295228C>T. In heterogeneous tumors with focal variant histology, concordant mutations were found in plasmacytoid and corresponding conventional, glandular, or sarcomatoid areas. Co-occurring mutations in tumor protein p53 (TP53, 2 cases) and kirsten rat sarcoma (KRAS) viral proto-oncogene (1 case) were also detected. TERT promoter mutations are frequently present in PUC, which provides further evidence that TERT promoter mutations are common events in bladder cancer, regardless of histologic subtype, and supports their inclusion in any liquid biopsy assay for bladder cancer.

A Role for De Novo Purine Metabolic Enzyme PAICS in Bladder Cancer Progression.

Genomic and transcriptome sequencing of bladder cancer (BLCA) has identified multiple molecular alterations during cancer progression. Many of these identified genetic and epigenetic changes play a role in the progression of this disease. Studies have identified molecular subtypes in muscle-invasive bladder cancer (MIBC) with different sensitivities to frontline therapy suggesting the heterogeneity in these tumors and the importance of molecular characterization of MIBC to provide effective treatment. Specifically, it has become increasingly evident, as demonstrated by The Cancer Genome Atlas project, that metabolic enzymes are commonly dysregulated in BLCA. Elevated expression of multiple metabolic enzymes is due to the increased demand from rapidly proliferating BLCA cells requiring extensive nucleotide synthesis. Cancer cells utilize the de novo purine and pyrimidine biosynthetic pathway as a source of their nucleotide needs. In this study, we show that phosphoribosyl aminoimidazole succinocarboxamide synthetase (PAICS), an enzyme involved in de novo purine biosynthetic pathway, is significantly overexpressed in BLCA. Immunohistochemical staining of paraffin-embedded tissue sections showed that PAICS is overexpressed in MIBC. Furthermore, we found that tumor suppressor miR-128 negatively regulated PAICS expression by binding to its 3'-untranslated region. We also found that PAICS induces EMT by positively regulating SNAI1 and by a reduction in E-cadherin expression. Additionally, our in vitro functional studies and in vivo chicken chorioallantoic membrane assay show that PAICS plays a critical role in BLCA cell proliferation, invasion, and tumor growth. Collectively, our data suggest that targeting PAICS may provide a therapeutic option in BLCA.

Non-invasive detection of urothelial cancer through the analysis of driver gene mutations and aneuploidy.

Current non-invasive approaches for detection of urothelial cancers are suboptimal. We developed a test to detect urothelial neoplasms using DNA recovered from cells shed into urine. UroSEEK incorporates massive parallel sequencing assays for mutations in 11 genes and copy number changes on 39 chromosome arms. In 570 patients at risk for bladder cancer (BC), UroSEEK was positive in 83% of those who developed BC. Combined with cytology, UroSEEK detected 95% of patients who developed BC. Of 56 patients with upper tract urothelial cancer, 75% tested positive by UroSEEK, including 79% of those with non-invasive tumors. UroSEEK detected genetic abnormalities in 68% of urines obtained from BC patients under surveillance who demonstrated clinical evidence of recurrence. The advantages of UroSEEK over cytology were evident in low-grade BCs; UroSEEK detected 67% of cases whereas cytology detected none. These results establish the foundation for a new non-invasive approach for detection of urothelial cancer.

Spectrum of genetic mutations in de novo PUNLMP of the urinary bladder.

Our group and others have previously demonstrated the presence of TERT promoter mutations (TERT-mut) in 60-80% of urothelial carcinomas and some of their histologic variants. Five other genes have been frequently implicated in bladder cancer: FGRF3, TP53, PIK3CA, HRAS, and CDKN2A. In the current study, we sought to determine the prevalence of mutations in TERT and these five other genes in de novo papillary urothelial neoplasms of low malignant potential (PUNLMP) of the urinary bladder. A retrospective search of our archives for PUNLMP was performed and 30 de novo cases were identified and included in the study. We found mutations in TERT (TERT-mut) and FGFR3 (FGFR3-mut) to be the most common alterations in the cohort (63 and 60%, respectively). The majority of the TERT-mut-positive tumors (84%) had a g.1295228C > T alteration with the remaining tumors demonstrating g.1295250C > T. Approximately one fourth of tumors had TP53 mutations. These findings support the potential utility of a uniform genetic mutation panel to detect bladder cancers of various subtypes.