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The 5th edition of the WHO Classification of Tumours of the Urinary and Male Genital Systems contains relevant revisions and introduces a group of molecularly defined renal tumour subtypes. Herein we present the World Health Organization (WHO) 2022 perspectives on papillary and chromophobe renal cell carcinoma with emphasis on their evolving classification, differential diagnosis, and emerging entities. The WHO 2022 classification eliminated the type 1/2 papillary renal cell carcinoma (pRCC) subcategorization, given the recognition of frequent mixed tumour phenotypes and the existence of entities with a different molecular background within the type 2 pRCC category. Additionally, emerging entities such as biphasic squamoid alveolar RCC, biphasic hyalinising psammomatous RCC, papillary renal neoplasm with reverse polarity, and Warthin-like pRCC are included as part of the pRCC spectrum, while additional morphological and molecular data are being gathered. In addition to oncocytomas and chromophobe renal cell carcinoma (chRCC), a category of 'other oncocytic tumours' with oncocytoma/chRCC-like features has been introduced, including emerging entities, most with TSC/mTOR pathway alterations (eosinophilic vacuolated tumour and so-called 'low-grade' oncocytic tumour), deserving additional research. Eosinophilic solid and cystic RCC was accepted as a new and independent tumour entity. Finally, a highly reproducible and clinically relevant universal grading system for chRCC is still missing and is another niche of ongoing investigation. This review discusses these developments and highlights emerging morphological and molecular data relevant for the classification of renal cell carcinoma.

Flow cytometry gating strategy is outlined. After digestion of tumor infiltrated bladders, samples yielded 1-2 million cells per bladder. A) Live T lymphocyte subpopulations were then gated from spleen tissue, and B) bladder T cells of interest were then selected.

Relationship between the expression levels of NF-κB/p65 versus p-NF-κB/p65 (A), AR versus NF-κB/p65 (B), and AR versus p-NF-κB/p65 (C) in bladder tumor specimens. Scatter plots show the distribution of IHC scores (0/1+/2+/3+) of two proteins. The Spearman's correlation coefficient (CC) and P value are indicated in each dot plot.

Intravesical bacillus Calmette–Guérin (BCG) immunotherapy is the standard of care in treating non–muscle-invasive bladder cancer, yet its mechanism of action remains elusive. Both innate and adaptive immune responses have been implicated in BCG activity. Although prior research has indirectly demonstrated the importance of T cells and shown a rise in CD4+ T cells in bladder tissue after BCG, T-cell subpopulations have not been fully characterized. We investigated the relationship between effector and regulatory T cells in an immune competent, clinically relevant rodent model of bladder cancer. Our data demonstrate that cancer progression in the N-methyl-N-nitrosourea (MNU) rat model of bladder cancer was characterized by a decline in the CD8/FoxP3 ratio, consistent with decreased adaptive immunity. In contrast, treatment with intravesical BCG led to a large, transient rise in the CD4+ T-cell population in the urothelium and was both more effective and immunogenic compared with intravesical chemotherapy. Whole-transcriptome expression profiling of posttreatment intravesical CD4+ and CD8+ T cells revealed minimal differences in gene expression after BCG treatment. Together, our results suggest that although BCG induces T-cell recruitment to the bladder, the T-cell phenotype does not markedly change, implying that combining T-cell–activating agents with BCG might improve clinical activity. Cancer Immunol Res; 5(7); 594–603. ©2017 AACR.

Supp Figure 3. Association of miRNA expression with water arsenic concentration and creatinine adjusted urine arsenic concentration (Mann-Whitney U test).

Univariate and multivariate Cox regression analyses in muscle-invasive tumors.

Supp Figure 1. Technical validation of Notch Signaling Pathway DNA Methylation PCR Array.

Supplementary Figure Legends

Supplementary Table S1: PD-L1+ RCC specimens used in this study. Supplementary Table S2: Genes differentially expressed in RCC based on whole genome microarray analysis, in patients responding or not to anti-PD-1 therapy (234 probe sets corresponding to 223 genes). Supplementary Table S3: Sixty genes included in custom multiplex qRT-PCR array to assess candidates from RCC whole genome microarray profiling.

(A) Rats 8 weeks post MNU were treated intravesically with MMC, MMC+BCG, and Cisplatin for 6 weeks (n=5 for each group). Bladders were harvested at week 16 and then digested into single cell suspensions and T cell populations analyzed with flow cytometry. (B) Flow Cytometry was then performed on digested bladders, and T cell subpopulations are reported as percentages, cell numbers, and (C) ratios. A "*" denotes statistical significance. All experiments were performed 2 times.

Exposure to toxicants leads to cumulative molecular changes that overtime increase a subject's risk of developing urothelial carcinoma. To assess the impact of arsenic exposure at a time progressive manner, we developed and characterized a cell culture model and tested a panel of miRNAs in urine samples from arsenic-exposed subjects, urothelial carcinoma patients, and controls. To prepare an in vitro model, we chronically exposed an immortalized normal human bladder cell line (HUC1) to arsenic. Growth of the HUC1 cells was increased in a time-dependent manner after arsenic treatment and cellular morphology was changed. In a soft agar assay, colonies were observed only in arsenic-treated cells, and the number of colonies gradually increased with longer periods of treatment. Similarly, invaded cells in an invasion assay were observed only in arsenic-treated cells. Withdrawal of arsenic treatment for 2.5 months did not reverse the tumorigenic properties of arsenic-treated cells. Western blot analysis demonstrated decreased PTEN and increased AKT and mTOR in arsenic-treated HUC1 cells. Levels of miR-200a, miR-200b, and miR-200c were downregulated in arsenic-exposed HUC1 cells by quantitative RT-PCR. Furthermore, in human urine, miR-200c and miR-205 were inversely associated with arsenic exposure (P = 0.005 and 0.009, respectively). Expression of miR-205 discriminated cancer cases from controls with high sensitivity and specificity (AUC = 0.845). Our study suggests that exposure to arsenic rapidly induces a multifaceted dedifferentiation program and miR-205 has potential to be used as a marker of arsenic exposure as well as a maker of early urothelial carcinoma detection. Cancer Prev Res; 8(3); 208–21. ©2015 AACR.

We investigated the role of NF-κB in the development and progression of urothelial cancer as well as cross-talk between NF-κB and androgen receptor (AR) signals in urothelial cells. Immunohistochemistry in surgical specimens showed that the expression levels of NF-κB/p65 (P = 0.015)/phospho-NF-κB/p65 (P < 0.001) were significantly elevated in bladder tumors, compared with those in nonneoplastic urothelial tissues. The rates of phospho-NF-κB/p65 positivity were also significantly higher in high-grade (P = 0.015)/muscle-invasive (P = 0.033) tumors than in lower grade/non–muscle-invasive tumors. Additionally, patients with phospho-NF-κB/p65-positive muscle-invasive bladder cancer had significantly higher risks of disease progression (P < 0.001) and cancer-specific mortality (P = 0.002). In immortalized human normal urothelial SVHUC cells stably expressing AR, NF-κB activators and inhibitors accelerated and prevented, respectively, their neoplastic transformation induced by a chemical carcinogen 3-methylcholanthrene. Bladder tumors were identified in 56% (mock), 89% (betulinic acid), and 22% (parthenolide) of N-butyl-N-(4-hydroxybutyl)nitrosamine-treated male C57BL/6 mice at 22 weeks of age. NF-κB activators and inhibitors also significantly induced and reduced, respectively, cell proliferation/migration/invasion of AR-positive bladder cancer lines, but not AR-knockdown or AR-negative lines, and their growth in xenograft-bearing mice. In both nonneoplastic and neoplastic urothelial cells, NF-κB activators/inhibitors upregulated/downregulated, respectively, AR expression, whereas AR overexpression was associated with increases in the expression levels of NF-κB/p65 and phospho-NF-κB/p65. Thus, NF-κB appeared to be activated in bladder cancer, which was associated with tumor progression. NF-κB activators/inhibitors were also found to modulate tumorigenesis and tumor outgrowth in AR-activated urothelial cells. Accordingly, NF-κB inhibition, together with AR inactivation, has the potential of being an effective chemopreventive and/or therapeutic approach for urothelial carcinoma. Mol Cancer Ther; 17(6); 1303–14. ©2018 AACR.

5 BCG treated rats, 5 untreated rats, and 20 controls rat were sacrificed and bladders were processed in single cell suspensions. CD4 and CD8 T cells were isolated for each sample and RNA was extracted. A gene expression analysis was subsequently performed on sorted CD4 and CD8 cells from each group using an Affymetrix Rat Gene 1.0 ST Array. MVA plot of sorted (A) CD8 and (B) CD4 T cells from MNU rats treated with BCG vs Untreated tumors. Genes selected from the Gene Ontology term "Immune Response" that have

Supp Figure 2A. Promoter methylation is inversely associated with gene expression

Supp Table 1, Supp Table 2. Supp Table 1: A.QMSP probe and primers, B.RT-PCR assay. Supplemental Table 2: The summary of notch methylation array findings

Figure S1: Extraction of paraffin-embedded PD-L1+ RCC tissues for RNA isolation. Figure S2: Molecules previously found to be up-regulated in PD-L1+ vs. PD-L1(-) melanomas are not differentially expressed in PD-L1+ RCCs from patients with divergent clinical outcomes after anti-PD-1 therapy. Figure S3: Gene expression in RCC cell lines. Figure S4: In silico analyses of TCGA RCC data do not demonstrate significant associations between UGT1A6 gene expression and overall survival or clinical stage.

Supp Figure 4. ROC curves for individual micro RNA expression in 177 normal and 32 bladder cancer urine samples were shown.

Supplementary Table 1: Relative Gene Expression of outlier "immune response" genes after BCG vs no treatment in sorted CD8+ cells from bladder cancer. Supplementary Table 2: Relative Gene Expression of outlier "immune response" genes after BCG vs no treatment in sorted CD4+ cells from bladder cancer.

Supplementary Materials and Methods

Supp Figure legends

PDF file - 82K, Supplementary Table 1: RB1 status (from high density SNP microarray, Reference 30) and Rb protein status across castrate resistant prostate cancer metastases. n = negative, p = positive, LOH = loss of heterozygosity.

Frequency of germline ASPN D-repeat-length in JHH prostate cancer cases and controls.

Positive controls of anti-mouse and anti-human antibodies for immunohistochemistry

Purpose: Given discrepancies between preclinical and clinical observations of mammalian target of rapamycin (mTOR) inhibition in prostate cancer, we sought to determine the pharmacodynamic effects of the mTOR/TORC1 inhibitor rapamycin in men with intermediate- to high-risk prostate cancer undergoing radical prostatectomy.Experimental Design: Rapamycin was given at 3 or 6 mg orally for 14 days before radical prostatectomy in men with multifocal Gleason sum ≥7 prostate cancer; 10 untreated control subjects were included. The primary outcome was inhibition of phosphorylation of ribosomal S6 in posttreatment radical prostatectomy versus pretreatment biopsy tumor tissue, evaluated using a Simon two-stage design for pharmacodynamic efficacy.Results: Thirty-two subjects were accrued: 20 at 3 mg, 2 at 6 mg, and 10 controls. No dose-limiting toxicities were observed at 3 mg; however, two of two men enrolled at 6 mg experienced dose-limiting toxicities including thrombocytopenia and fever with grade 3 stomatitis. Adverse events observed at 3 mg included stomatitis, rash, ileus, and neutropenia. Pharmacodynamic studies showed tumor S6 phosphorylation inhibition in 50% of 10 evaluable rapamycin-treated men with sufficient paired tissue [median 58% inhibition (P = 0.049) versus 2% inhibition in controls (P = 0.75)] with no significant effect on AKT activity. We observed no change in Ki-67 or caspase-3 cleavage but noted a reduction in cytoplasmic p27 staining with increased nuclear localization with rapamycin treatment. Prostate tissue rapamycin concentrations were 3- to 4-fold higher than blood.Conclusions: At 3 mg daily, rapamycin successfully and safely inhibited prostate cancer S6 phosphorylation and achieved relatively high prostate tissue concentrations. No effect on AKT phosphorylation, tumor proliferation, or apoptosis was observed. Clin Cancer Res; 16(11); 3057–66. ©2010 AACR.

Supplementary Figure S2: The ratio of CDKN2A/CCND1 is not as tightly correlated with Rb functional and genetic status in publicly available data from castate resistant prostate cancer (CRPC) metastases in rapid autopsy cohort.

Supplemental Table S1. Relative expression of stem cell-related genes using RT2 profile PCR array.

PDF file - 69KB, Correlation of PTEN protein status by immunohistochemistry (IHC) with PTEN genomic status by sequencing in NCI-60 cell line panel.

Purpose: To assess the clinical and pharmacodynamic activity of dovitinib in a treatment-resistant, molecularly enriched non–muscle-invasive urothelial carcinoma of the bladder (NMIUC) population.Experimental Design: A multi-site pilot phase II trial was conducted. Key eligibility criteria included the following: Bacillus Calmette-Guerin (BCG)-unresponsive NMIUC (>2 prior intravesical regimens) with increased phosphorylated FGFR3 (pFGFR3) expression by centrally analyzed immunohistochemistry (IHC+) or FGFR3 mutations (Mut+) assessed in a CLIA-licensed laboratory. Patients received oral dovitinib 500 mg daily (5 days on/2 days off). The primary endpoint was 6-month TURBT-confirmed complete response (CR) rate.Results: Between 11/2013 and 10/2014, 13 patients enrolled (10 IHC+ Mut−, 3 IHC+ Mut+). Accrual ended prematurely due to cessation of dovitinib clinical development. Demographics included the following: median age 70 years; 85% male; carcinoma in situ (CIS; 3 patients), Ta/T1 (8 patients), and Ta/T1 + CIS (2 patients); median prior regimens 3. Toxicity was frequent with all patients experiencing at least one grade 3–4 event. Six-month CR rate was 8% (0% in IHC+ Mut−; 33% in IHC+ Mut+). The primary endpoint was not met. Pharmacodynamically active (94–5,812 nmol/L) dovitinib concentrations in urothelial tissue were observed in all evaluable patients. Reductions in pFGFR3 IHC staining were observed post-dovitinib treatment.Conclusions: Dovitinib consistently achieved biologically active concentrations within the urothelium and demonstrated pharmacodynamic pFGFR3 inhibition. These results support systemic administration as a viable approach to clinical trials in patients with NMIUC. Long-term dovitinib administration was not feasible due to frequent toxicity. Absent clinical activity suggests that patient selection by pFGFR3 IHC alone does not enrich for response to FGFR3 kinase inhibitors in urothelial carcinoma. Clin Cancer Res; 23(12); 3003–11. ©2016 AACR.

Supplementary Table S2 shows bivariate and multivariable analyses of germline ASPN D13/14 compared to ASPN D13/13 for lymph node involvement and metastatic recurrence. Supplementary Table S3 show bivariate analyses of ASPN D genotypes/alleles for biochemical recurrence. Supplementary Table S4 shows multivariable analyses of ASPN D13/14 for biochemical recurrence.

Demographic and clinical-pathologic characteristics of eligible patients in intermediate/high risk radical prostatectomy cohort

Alterations in systemic and local prostate expression of IGF-1 due to Abx administration.

Detailed data were described.

Purpose: Small-cell neuroendocrine differentiation in prostatic carcinoma is an increasingly common resistance mechanism to potent androgen deprivation therapy (ADT), but can be difficult to identify morphologically. We investigated whether cyclin D1 and p16 expression can inform on Rb functional status and distinguish small-cell carcinoma from adenocarcinoma.Experimental Design: We used gene expression data and immunohistochemistry to examine cyclin D1 and p16 levels in patient-derived xenografts (PDX), and prostatic small-cell carcinoma and adenocarcinoma specimens.Results: Using PDX, we show proof-of-concept that a high ratio of p16 to cyclin D1 gene expression reflects underlying Rb functional loss and distinguishes morphologically identified small-cell carcinoma from prostatic adenocarcinoma in patient specimens (n = 13 and 9, respectively). At the protein level, cyclin D1, but not p16, was useful to distinguish small-cell carcinoma from adenocarcinoma. Overall, 88% (36/41) of small-cell carcinomas showed cyclin D1 loss by immunostaining compared with 2% (2/94) of Gleason score 7–10 primary adenocarcinomas at radical prostatectomy, 9% (4/44) of Gleason score 9–10 primary adenocarcinomas at needle biopsy, and 7% (8/115) of individual metastases from 39 patients at autopsy. Though rare adenocarcinomas showed cyclin D1 loss, many of these were associated with clinical features of small-cell carcinoma, and in a cohort of men treated with adjuvant ADT who developed metastasis, lower cyclin D1 gene expression was associated with more rapid onset of metastasis and death.Conclusions: Cyclin D1 loss identifies prostate tumors with small-cell differentiation and may identify a small subset of adenocarcinomas with poor prognosis. Clin Cancer Res; 21(24); 5619–29. ©2015 AACR.

Supplementary Data from A Pharmacodynamic Study of Rapamycin in Men with Intermediate- to High-Risk Localized Prostate Cancer

'Representative examples of primary tumors with discordant TP53 sequencing and p53 immunostaining results on tissue microarray. Two tumors with TP53 missense mutations (upper left and upper right) did not show appreciable nuclear accumulation of TP53. Note that the mutation in the tumor on the upper left (p.R282W) was detected in another sample from a different patient by IHC and the tumor on the upper right had 8 separate TP53 missense mutations detected, of unknown significance. Two additional tumors without missense mutations (one WT by sequencing on lower left, and one with an in-frame deletion on lower right) both showed nuclear accumulation of p53 protein by IHC in >10% of cells and were considered false positives. All photomicrographs are reduced from 200x.'

ASPN antibody validation.

Supplemental information includes Supplemental Figure S1. The characterization of SOX2 as an oncogene in bladder cancer, Supplemental Figure S2. Stem cell-related molecules associated with SOX2 expression, Supplemental Figure S3. CD24+/CD133+ as a potential marker for SOX2-expressing CSCs, Supplemental Figure S4. COX2/PGE2 signaling pathway in regulating SOX2, Supplemental Figure S5. The association of PGE2 expression with apoptosis in YAP1-LV or YAP1-sh cells, Supplemental Figure S6. Correlation among SOX2, YAP1, and COX2 expression in human UCB samples, Supplemental Figure S7. CSC expansion following CDDP treatment suppressed by combination treatment with verteporfin and celecoxib, Supplemental Figure S8. The EGFR-regulated YAP1 expression via PI3K signaling in basal-type UCB, and Supplementary Materials and Methods.