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Background TMPRSS2-ERG gene rearrangement, the most common E26 transformation specific (ETS) gene fusion within prostate cancer, is known to contribute to the pathogenesis of this disease and carries diagnostic annotations for prostate cancer patients clinically. The ERG rearrangement status in prostatic adenocarcinoma currently cannot be reliably identified from histologic features on H&E-stained slides alone and hence requires ancillary studies such as immunohistochemistry (IHC), fluorescent in situ hybridization (FISH) or next generation sequencing (NGS) for identification. Methods Objective We accordingly sought to develop a deep learning-based algorithm to identify ERG rearrangement status in prostatic adenocarcinoma based on digitized slides of H&E morphology alone. Design Setting, and Participants: Whole slide images from 392 in-house and TCGA cases were employed and annotated using QuPath. Image patches of 224 × 224 pixel were exported at 10 ×, 20 ×, and 40 × for input into a deep learning model based on MobileNetV2 convolutional neural network architecture pre-trained on ImageNet. A separate model was trained for each magnification. Training and test datasets consisted of 261 cases and 131 cases, respectively. The output of the model included a prediction of ERG-positive (ERG rearranged) or ERG-negative (ERG not rearranged) status for each input patch. Outcome measurements and statistical analysis: Various accuracy measurements including area under the curve (AUC) of the receiver operating characteristic (ROC) curves were used to evaluate the deep learning model. Results and Limitations All models showed similar ROC curves with AUC results ranging between 0.82 and 0.85. The sensitivity and specificity of these models were 75.0% and 83.1% (20 × model), respectively. Conclusions A deep learning-based model can successfully predict ERG rearrangement status in the majority of prostatic adenocarcinomas utilizing only H&E-stained digital slides. Such an artificial intelligence-based model can eliminate the need for using extra tumor tissue to perform ancillary studies in order to assess for ERG gene rearrangement in prostatic adenocarcinoma.

Summary: Sarcomatoid urothelial bladder cancer (SARC) displays a high propensity for distant metastasis and is associated with short survival. We report a comprehensive genomic analysis of 28 cases of SARC and 84 cases of conventional urothelial carcinoma (UC), with the TCGA cohort of 408 muscle-invasive bladder cancers serving as the reference. SARCs show a distinct mutational landscape, with enrichment of TP53, RB1, and PIK3CA mutations. They are related to the basal molecular subtype of conventional UCs and could be divided into epithelial-basal and more clinically aggressive mesenchymal subsets on the basis of TP63 and its target gene expression levels. Other analyses reveal that SARCs are driven by downregulation of homotypic adherence genes and dysregulation of the EMT network, and nearly half exhibit a heavily infiltrated immune phenotype. Our observations have important implications for prognostication and the development of more effective therapies for this highly lethal variant of bladder cancer. : Guo et al. report that sarcomatoid carcinoma of the bladder evolves by the progression of the basal subtype of conventional urothelial carcinoma with the enrichment of mutagenesis signature 1 and mutations of TP53, RB1, and PIK3CA. It is driven by the dysregulation of the EMT network and shows increased immune infiltrate with overexpression of PD-L1. Keywords: bladder cancer, sarcomatoid carcinoma, urothelial carcinoma, epithelial-mesenchymal transformation, genomic expression, chromatin remodeling, microRNA expression, molecular classification, basal subtype, immune phenotype

Neuroendocrine transdifferentiation of high-grade prostate cancer (PCA-NT) comprises a morphologic and immunophenotypic transition from conventional adenocarcinoma towards high-grade neuroendocrine/small cell carcinoma. This phenomenon is frequently observed post androgen deprivation and/or radiotherapy, but de novo instances are increasingly recognized. Herein, we report a series of de novo PCA-NT focusing on characteristic morphologic, immunophenotypic and clinical features. Treatment naive PCA-NT were identified. IHC for PSA, NKX3.1, Chromogranin, Synaptophysin, Cyclin D1, RB and Ki67 were performed. Radiology, treatment and follow-up data were reviewed. Sixteen patients were included. Apart from focal areas of high-grade prostate cancer with acinar features (reminiscent of Grade Group 5 disease), extensive areas with sheets of cells with deep amphophilic/basophilic cytoplasm, enlarged, hyperchromatic nuclei with granular chromatin and inconspicuous to prominent nucleoli with high mitotic activity were identified. Immunohistochemistry showed patchy NKX3.1, patchy PSA, variable Synaptophysin and Chromogranin; RB and CyclinD1 showed loss of expression. Ki67 showed high proliferative index, in most cases. Adverse radiologic findings and metastases were documented in most cases. Two patients died of disease. De novo PCA-NT exhibits high-grade nuclei, high mitotic activity, reduced PSA expression with high Ki67 and functional inactivation of RB1 pathway, suggesting transition from androgen-driven to proliferation-driven phenotype. Most cases presented at advanced stage with adverse radiological findings, metastasis at time of diagnosis, and high mortality. In light of their prognostic and therapeutic implications, pathologists may need to maintain a sensitive threshold for performing immunostains-in particular, Ki67 and CyclinD1-when presented with such cases in their day to day clinical practice.

Neuroendocrine transdifferentiation of high-grade prostate cancer (PCA-NT) comprises a morphologic and immunophenotypic transition from conventional adenocarcinoma towards high-grade neuroendocrine/small cell carcinoma. This phenomenon is frequently observed post androgen deprivation and/or radiotherapy, but de novo instances are increasingly recognized. Herein, we report a series of de novo PCA-NT focusing on characteristic morphologic, immunophenotypic and clinical features. Treatment naïve PCA-NT were identified. IHC for PSA, NKX3.1, Chromogranin, Synaptophysin, Cyclin D1, RB and Ki67 were performed. Radiology, treatment and follow-up data were reviewed. Sixteen patients were included. Apart from focal areas of high-grade prostate cancer with acinar features (reminiscent of Grade Group 5 disease), extensive areas with sheets of cells with deep amphophilic/basophilic cytoplasm, enlarged, hyperchromatic nuclei with granular chromatin and inconspicuous to prominent nucleoli with high mitotic activity were identified. Immunohistochemistry showed patchy NKX3.1, patchy PSA, variable Synaptophysin and Chromogranin; RB and CyclinD1 showed loss of expression. Ki67 showed high proliferative index, in most cases. Adverse radiologic findings and metastases were documented in most cases. Two patients died of disease. De novo PCA-NT exhibits high-grade nuclei, high mitotic activity, reduced PSA expression with high Ki67 and functional inactivation of RB1 pathway, suggesting transition from androgen-driven to proliferation-driven phenotype. Most cases presented at advanced stage with adverse radiological findings, metastasis at time of diagnosis, and high mortality. In light of their prognostic and therapeutic implications, pathologists may need to maintain a sensitive threshold for performing immunostains-in particular, Ki67 and CyclinD1-when presented with such cases in their day to day clinical practice.

272 Background: Prostate cancer (PCa) in the transition zone (TZ) accounts for approximately 30% of disease and tends to present with higher PSAs with a lower risk of seminal vesicle invasion, extra-capsular extension, and risk of biochemical recurrence compared with peripheral zone (PZ) tumors. The underlying biological mechanism for these differences is poorly understood. Here, we performed spatial transcriptomic profiling to elucidate the molecular differences between TZ and PZ PCa. Methods: We identified three patients who underwent radical prostatectomy for PCa (one each with PZ only, TZ only, and both PZ and TZ tumors) and used the NanoString’s Digital Spatial Profiling (DSP) platform to quantify whole transcriptomic gene expression data (17,128 genes) in multiple regions of interest (ROI) per patient (42 cancer and 8 normal samples). Four morphology markers to facilitate ROI selection in both cancer and normal areas (SYTO13 for nucleus, PanCK for epithelium, SMA for stroma and CD45 for immune cells) were selected. Raw counts for 17,128 genes were imported to R v.4.1.0 for downstream data analyses. Counts were Q3 normalized and scaled (Z-score) to enable plotting of all genes on the same axes. Differential gene expression analysis using a linear model fit by empirical Bayes moderation, gene set enrichment analysis (GSEA) by cancer hallmarks, XCell gene sets for pathway enrichment and immune cell deconvolution using CIBERSORT was performed. Results: There were grade group (GG) 4 (n=10) and 5 (n=10) tumors in PZ and GG 3 (n=10), 4 (n=11) and 5 (n=1) cancers in TZ regions. We observed distinct gene expression profiles between PZ (n = 20) and TZ (n=22) tumors. Interestingly, androgen receptor (AR) signaling was significantly higher in TZ PCa ROIs compared to PZ ROIs in both GSEA (false discovery rate < 5%) and the androgen subcomponent of the genomic prostate score (p

Genomic profiling studies have demonstrated that bladder cancer can be divided into two molecular subtypes referred to as luminal and basal with distinct clinical behaviors and sensitivities to frontline chemotherapy. We analyzed the mRNA expressions of signature luminal and basal genes in bladder cancer tumor samples from publicly available and MD Anderson Cancer Center cohorts. We developed a quantitative classifier referred to as basal to luminal transition (BLT) score which identified the molecular subtypes of bladder cancer with 80–94% sensitivity and 83–93% specificity. In order to facilitate molecular subtyping of bladder cancer in primary care centers, we analyzed the protein expressions of signature luminal (GATA3) and basal (KRT5/6) markers by immunohistochemistry, which identified molecular subtypes in over 80% of the cases. In conclusion, we provide a tool for assessment of molecular subtypes of bladder cancer in routine clinical practice.

Summary We report a comprehensive molecular analysis of 34 cases of small cell carcinoma (SCC) and 84 cases of conventional urothelial carcinoma (UC), with The Cancer Genome Atlas cohort of 408 conventional UC bladder cancers used as the reference. SCCs showed mutational landscapes characterized by nearly uniform inactivation of TP53 and were dominated by Sanger mutation signature 3 associated with loss of BRCA1/2 function. SCCs were characterized by downregulation of luminal and basal markers and were referred to as double-negative. Transcriptome analyses indicated that SCCs displayed lineage plasticity driven by a urothelial-to-neural phenotypic switch with a dysregulated epithelial-to-mesenchymal transition network. SCCs were depleted of immune cells, and expressed high levels of the immune checkpoint receptor, adenosine receptor A2A (ADORA2A), which is a potent inhibitor of immune infiltration. Our observations have important implications for the prognostication and development of more effective therapies for this lethal bladder cancer variant., Graphical Abstract, Highlights • SCCs show TP53/RB1 loss with mutational signature of BRCA1/2 loss of function • SCCs are driven by neural phenotypic switch with dysregulated EMT network • SCCs show depleted immune phenotype with upregulation of ADORA2A, Biological Sciences; Genomics; Cancer Systems Biology; Cancer; Transcriptomics

Background Progression of conventional urothelial carcinoma of the bladder to a tumor with unique microscopic features referred to as micropapillary carcinoma is coupled with aggressive clinical behavior signified by a high propensity for metastasis to regional lymph nodes and distant organs resulting in shorter survival. Objective To analyze the expression profile of micropapillary cancer and define its molecular features relevant to clinical behavior. Design, setting, and participants We retrospectively identified 43 patients with micropapillary bladder cancers and a reference set of 89 patients with conventional urothelial carcinomas and performed whole-genome expression messenger RNA profiling. Outcome measurements and statistical analysis The tumors were segregated into distinct groups according to hierarchical clustering analyses. They were also classified according to luminal, p53-like, and basal categories using a previously described algorithm. We applied Ingenuity Pathway Analysis software (Qiagen, Redwood City, CA, USA) and gene set enrichment analysis for pathway analyses. Cox proportional hazards models and Kaplan-Meier methods were used to assess the relationship between survival and molecular subtypes. The expression profile of micropapillary cancer was validated for selected markers by immunohistochemistry on parallel tissue microarrays. Results and limitations We show that the striking features of micropapillary cancer are downregulation of miR-296 and activation of chromatin-remodeling complex RUVBL1. In contrast to conventional urothelial carcinomas that based on their expression can be equally divided into luminal and basal subtypes, micropapillary cancer is almost exclusively luminal, displaying enrichment of active peroxisome proliferator-activated receptor γ and suppression of p63 target genes. As with conventional luminal urothelial carcinomas, a subset of micropapillary cancers exhibit activation of wild-type p53 downstream genes and represent the most aggressive molecular subtype of the disease with the shortest survival. The involvement of miR-296 and RUVBL1 in the development of micropapillary bladder cancer was identified by the analyses of correlative associations of genome expression profiles and requires mechanistic validation. Conclusions Micropapillary cancer evolves through the luminal pathway and is characterized by the activation of miR-296 and RUVBL1 target genes. Patient summary Our observations have important implications for prognosis and for possible future development of more effective therapies for micropapillary bladder cancer.

Background It has been suggested that bladder cancer can be divided into two molecular subtypes referred to as luminal and basal with distinct clinical behaviors and sensitivities to chemotherapy. We aimed to validate these subtypes in several clinical cohorts and identify signature immunohistochemical markers that would permit simple and cost-effective classification of the disease in primary care centers. Methods We analyzed genomic expression profiles of bladder cancer in three cohorts of fresh frozen tumor samples: MD Anderson (n = 132), Lund (n = 308), and The Cancer Genome Atlas (TCGA) (n = 408) to validate the expression signatures of luminal and basal subtypes and relate them to clinical follow-up data. We also used an MD Anderson cohort of archival bladder tumor samples (n = 89) and a parallel tissue microarray to identify immunohistochemical markers that permitted the molecular classification of bladder cancer. Findings Bladder cancers could be assigned to two candidate intrinsic molecular subtypes referred to here as luminal and basal in all of the datasets analyzed. Luminal tumors were characterized by the expression signature similar to the intermediate/superficial layers of normal urothelium. They showed the upregulation of PPARγ target genes and the enrichment for FGFR3, ELF3, CDKN1A, and TSC1 mutations. In addition, luminal tumors were characterized by the overexpression of E-Cadherin, HER2/3, Rab-25, and Src. Basal tumors showed the expression signature similar to the basal layer of normal urothelium. They showed the upregulation of p63 target genes, the enrichment for TP53 and RB1 mutations, and overexpression of CD49, Cyclin B1, and EGFR. Survival analyses showed that the muscle-invasive basal bladder cancers were more aggressive when compared to luminal cancers. The immunohistochemical expressions of only two markers, luminal (GATA3) and basal (KRT5/6), were sufficient to identify the molecular subtypes of bladder cancer with over 90% accuracy. Interpretation The molecular subtypes of bladder cancer have distinct clinical behaviors and sensitivities to chemotherapy, and a simple two-marker immunohistochemical classifier can be used for prognostic and therapeutic stratification. Funding U.S. National Cancer Institute and National Institute of Health., Highlights • Bladder cancer can be assigned to two intrinsic molecular subtypes referred to as luminal and basal. • Luminal tumors are characterized by the expression signature of intermediate/superficial layers of normal urothelium and they show up-regulation of PARγ target genes with enrichment for FGFR3, ELF3, CDKN1A, and TSC1 mutations. • Basal tumors show the expression signature similar to the basal layer of normal urothelium and they show the up-regulation of p63 target genes with the enrichment of TP53 and RB1 mutations. • Muscle invasive tumors of basal type are more aggressive when compared to luminal cancers. • The immunohistochemical expression of only two markers, luminal (GATA3) and basal (KRT5/6) is sufficient to identify the molecular subtypes of bladder cancer with over 90% accuracy. Our analyses of several large cohorts comprising of a total of 937 bladder cancer tumors samples with annotated clinical data showed that bladder cancers can be classified into molecular subtypes referred to as luminal and basal. Luminal tumors are similar in their gene expression pattern to more differentiated intermediate and superficial layers of normal urothelium. Basal cancers recapitulate the gene expression signature of the less differentiated basal layer of the urothelium. These molecular subtypes have distinct up-regulation of transcription factors that regulate the expression pattern of their target genes and are enriched for specific mutations. Basal tumors which deeply invade the bladder wall are more aggressive when compared to luminal types and they show distinct sensitivities to frontline chemotherapy. The classification of bladder cancer into its intrinsic molecular subtypes can be accomplished with two immunohistochemical markers such as GATA3 for luminal tumors and KRT5/6 for basal tumors which can be performed in primary care centers.

Renal cell carcinoma (RCC), the most common type of kidney cancer in adult, rarely metastasizes to the ovary or fallopian tube, and most cases published in the literature were case reports. Herein, we describe the clinicopathological features of 9 cases of RCC metastatic to the ovary (n = 8) or the fallopian tube (n = 1). The patients' age at the onset of primary renal tumor was available in 8 patients, ranging from 37 to 73 years (mean, 51 years; median, 50 years). Ovarian metastasis was detected prior to or concurrently with the primary renal tumors in 3 patients, and after the diagnosis of renal tumors in 6 patients. The histotypes of the RCCs were clear cell (n = 7), chromophobe (n = 1), and unclassified (n = 1). Immunohistochemical stainings were performed on the sections containing metastatic tumors in 4 cases. Interestingly, pagetoid intraepithelial spread in the tubal mucosa was observed in the case of RCC metastatic to the fallopian tube. Among the 8 patients with follow-up data, 5 died of disease and 3 were alive with disease, with a follow-up period ranging from 3.7 months to 17 years (mean, 77 months; median, 53 months) after the diagnosis of primary kidney tumors. Diagnostically, metastatic RCC may mimic primary ovarian tumors clinically, morphologically, or immunophenotypically. Pathologists should also keep in mind that both ovarian and kidney tumors express PAX8 and PAX2, the markers commonly used to diagnose metastatic RCC. In addition, chromophobe RCC only rarely metastasizes, but it can be a diagnostic challenge when it metastasizes to the ovary.

SUMMARYThe sarcomatoid variant of urothelial bladder cancer (SARC) displays a high propensity for distant metastasis and is associated with short survival. We report a comprehensive genomic analysis of 28 cases of SARCs and 84 cases of conventional urothelial carcinomas (UCs), with the TCGA cohort of 408 muscle-invasive bladder cancers serving as the reference. SARCs showed a distinct mutational landscape with enrichment ofTP53, RB1, and PIK3CAmutations. They were related to the basal molecular subtype of conventional UCs and could be divided into epithelial/basal and more clinically aggressive mesenchymal subsets based on TP63 and its target genes expression levels. Other analyses revealed that SARCs are driven by downregulation of homotypic adherence genes and dysregulation of cell cycle and EMT networks, and nearly half exhibited a heavily infiltrated immune phenotype. Our observations have important implications for prognostication and the development of more effective therapies for this highly lethal variant of bladder cancer.

Adenocarcinoma is an uncommon malignancy in the urinary bladder which may arise primarily in the bladder as well as secondarily from a number of other organs. Our aim is to provide updated information on primary and secondary bladder adenocarcinomas, with focus on pathologic features, differential diagnosis, and clinical relevance. Primary bladder adenocarcinoma exhibits several different growth patterns, including enteric, mucinous, signet-ring cell, not otherwise specified, and mixed patterns. Urachal adenocarcinoma demonstrates similar histologic features but it can be distinguished from bladder adenocarcinoma on careful pathologic examination. Secondary bladder adenocarcinomas may arise from the colorectum, prostate, endometrium, cervix and other sites. Immunohistochemical study is valuable in identifying the origin of secondary adenocarcinomas. Noninvasive neoplastic glandular lesions, adenocarcinoma in situ and villous adenoma, are frequently associated with bladder adenocarcinoma. It is also important to differentiate bladder adenocarcinoma from a number of nonneoplastic lesions in the bladder. Primary bladder adenocarcinoma has a poor prognosis largely because it is usually diagnosed at an advanced stage. Urachal adenocarcinoma shares similar histologic features with bladder adenocarcinoma, but it has a more favorable prognosis than bladder adenocarcinoma, partly due to the relative young age of patients with urachal adenocarcinoma.

INTRODUCTION AND OBJECTIVES: Advances in imaging technology and understanding differences in renal tumor histology have led to improved management of small renal masses. Several studies have suggested that papillary renal cell carcinoma Type 1 (T1-pRCC) and Type 2 (T2-pRCC) have distinct behaviors and that prediction of morphology may be possible by imaging. We investigated imaging characteristics of T1and T2-pRCC and their pathological correlates to determine utility of multiphase computerized tomography (CT), and the CT enhancement washout formula, in differentiating between T1 and T2-pRCC subtypes. METHODS: Retrospective analysis was performed on 39 patients (18 T1-pRCC/21 T2-pRCC) who underwent surgical extirpation and had multiphase CT at our institution from 12/2007-7/2012. Pathology was confirmed and data was analyzed between subgroups based on histology. Multiphase CT was analyzed and tumor size, morphology, and attenuation in Hounsfield Units (HU) were recorded. Change in HU (dHU) was calculated between noncontrast (NC), corticomedullary (CM), nephrographic (N) and delayed (D) phases. Enhancement washout was calculated by formula (N HU-D HU)/(N HU-NC HU). RESULTS: There was no difference in median tumor size (T1-pRCC 2.8 vs. T2-pRCC 2.0 cm, p1⁄40.520). Significantly greater proportion of high grade tumors (III/IV) were noted in pRCC-T2 (42.9%) vs. pRCC-T1 (5.6%) (p1⁄40.011). There were no imaging differences between pRCC subtypes with respect to: frequency of irregular borders (5.6% vs. 14.3%, p1⁄40.609), presence of calcifications (11.1% vs. 14.3%, p1⁄41.000), presence of necrosis (11.1% vs. 28.6%, p1⁄40.247), or heterogeneous enhancement (16.7% vs. 28.8%, p1⁄40.464). There was no difference in dHU between CM-NC (p1⁄40.126), and D-NC (p1⁄40.065). However, T2-pRCC had higher dHU between N-NC (42.7) vs. T1-pRCC (27.8, p1⁄40.036). Similar proportions of T1(61.1%) and T2-pRCC (52.4%) tumors had an enhancement washout

We report a case of CPPD crowned dens syndrome in an 87 year white old male with a known history of pseudogout, with clinical and radiological features characteristic of this syndrome. Interestingly, there was significant mass effect on the clivus, with clivus erosion and destruction, a finding that has not previously been described with this syndrome. The clinical and radiological characteristics of Crowned Dens syndrome, as well as CPPD are reviewed. We suggest that CPPD crowned dens syndrome may be included in the differential diagnosis when clivus destruction or erosion, in association with a soft tissue mass with calcification, is seen.

BACKGROUND: Recently we have proposed a molecular sub-classification of conventional urothelial carcinoma which could be divided into two major categories designated as basal and luminal similar to the ones defined for breast cancer. In addition, luminal cancers contain a group of cases characterized by upregulation of p53 regulatory genes and is referred to as p53-like. The purpose of this project is to verify whether such subtypes can be identified in several large independent cohorts of bladder cancers. DESIGN: Gene expression profiles and molecular subtyping were performed by using previously described analytical algorithms on the following bladder cancer patient cohorts: (1) The Cancer Genome Atlas (TCGA) cohort containing tumor tissues from muscle invasive high grade urothelial carcinomas of 129 patients; (2) the MD Anderson Cancer Center (MDA) cohort of fresh frozen tumor tissues which included 65 superficial papillary carcinomas and 77 invasive carcinomas; (3) the Swedish cohort containing fresh frozen tumor samples of 116 superficial papillary tumors and 192 invasive tumors. RESULTS: The three distinct molecular subtypes defined as luminal, p53-like and basal were identified in all three cohorts. The distribution of molecular subtypes in individual cohorts was as follows: the TCGA cohort contained 50 (39%) luminal, 30 (23%) p53-like and 49 (38%) basal tumors; the MDA cohort contained 54 (38%) luminal, 61 (43%) p53-like and 27 (19%) basal; the Swedish cohort contained 102 (33%) luminal, 129 (42%) p-53 like and 77 (25%) basal tumors. Interestingly, in the MDA and Swedish cohorts virtually all superficial tumors, of either low or high grade, were of luminal type. CONCLUSION: The conventional bladder cancer can be separated into three distinct molecular subtypes with luminal phenotype defining the vast majority of superficial bladder cancers while muscle invasive tumors can be separated into luminal and basal forms. A subset of luminal cancers is characterized by upregulation of p53 regulatory genes. The identification of subtypes may help to stratify the patients for targeted therapies. Citation Format: Vipulkumar Dadhania, Charles G. Guo, Tadeusz Majewski, Li Zhang, Jolanta Bondaruk, Shizhen Zhang, Woonyoung Choi, David McConkey, Colin Dinney, Keith Baggerly, Bogdan A. Czerniak. Validation of molecular subtypes of urothelial carcinoma of bladder in multi-institutional and public cohorts. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4809. doi:10.1158/1538-7445.AM2015-4809