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Background: Development of successful antibody-based immunotherapeutic and radioimmunotherapeutic strategies rely on the identification of cell surface tumor-associated antigens (TAA) with restricted expression on normal tissues. Desmoplastic small round cell tumor (DSRCT) is a rare and generally neglected malignancy that primarily affects adolescent and young adult males. New therapies capable of treating disseminated disease are needed for DSRCT, which is often widespread at diagnosis., Methods: We used immunohistochemistry (IHC) on fresh frozen surgical specimens and patient-derived xenograft (PDX) tumors and flow cytometry on DSRCT cell lines to evaluate expression of TAAs in these tumors. In vitro cytotoxicity assays were used to evaluate the efficacy of T cell-engaging bispecific antibodies (T-BsAbs) directed at these targets. In vivo , we used an intraperitoneal xenograft mouse model of DSRCT to test T-BsAbs against several TAAs., Results: In DSRCT specimens we found widespread expression of B7-H3, EGFR, GD2, HER2, mesothelin, and polysialic acid, clinical targets for which specific antibody therapeutics are available. The expression of B7-H3, EGFR, HER2, and mesothelin was confirmed on the cell surface of DSRCT cell lines. In vitro cytotoxicity assays confirmed the efficacy of T cell-engaging bispecific antibodies (T-BsAbs) directed at these targets against DSRCT cells. Remarkably, a HER2xCD3 T-BsAb was capable of completely shrinking established tumors in an intraperitoneal mouse model of DSRCT., Conclusions: We propose that these TAAs should be further investigated in preclinical models as targets for immunotherapy and radioimmunotherapy with the hope of providing a rationale to extend these therapies to patients with advanced DSRCT., Competing Interests: N-KC reports receiving commercial research grants from Y-mAbs Therapeutics. N-KC is the inventor and owner of issued patents licensed by MSKCC to Y-mAbs Therapeutics, Biotec Pharmacon/Lallemand, and Abpro-labs. MSKCC and N-KC have financial interest in Y-mAbs. N-KC reports receiving stock options from Eureka Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest, (Copyright © 2023 Espinosa-Cotton, Guo, Tickoo and Cheung.)

Cancers arising from the bladder urothelium often exhibit lineage plasticity with regions of urothelial carcinoma adjacent to or admixed with regions of divergent histomorphology, most commonly squamous differentiation. To define the biologic basis for and clinical significance of this morphologic heterogeneity, here we perform integrated genomic analyses of mixed histology bladder cancers with separable regions of urothelial and squamous differentiation. We find that squamous differentiation is a marker of intratumoral genomic and immunologic heterogeneity in patients with bladder cancer and a biomarker of intrinsic immunotherapy resistance. Phylogenetic analysis confirms that in all cases the urothelial and squamous regions are derived from a common shared precursor. Despite the presence of marked genomic heterogeneity between co-existent urothelial and squamous differentiated regions, no recurrent genomic alteration exclusive to the urothelial or squamous morphologies is identified. Rather, lineage plasticity in bladder cancers with squamous differentiation is associated with loss of expression of FOXA1, GATA3, and PPARG, transcription factors critical for maintenance of urothelial cell identity. Of clinical significance, lineage plasticity and PD-L1 expression is coordinately dysregulated via FOXA1, with patients exhibiting morphologic heterogeneity pre-treatment significantly less likely to respond to immune checkpoint inhibitors., (© 2022. The Author(s).)

Renal oncocytoma and chromophobe renal cell carcinoma were accepted as unique renal tumors in the late 1990s. Since their formal description, criteria for diagnosis have evolved and additional distinct tumor subtypes originally considered as one these two entities are now recognized. The last two decades have witnessed unprecedented interest in the spectrum of low grade oncocytic renal neoplasms in three specific areas: (1) histologic characterization of tumors with overlapping morphologic features between oncocytoma and chromophobe renal cell carcinoma; (2) description of potentially unique entities within this spectrum, such as eosinophilic vacuolated tumor and low-grade oncocytic tumor; and (3) better appreciation of the association between a subset of low grade oncocytic tumors and hereditary renal neoplasia. While this important work has been academically rewarding, the proposal of several histologic entities with overlapping morphologic and immunophenotypic features (which may require esoteric adjunctive immunohistochemical and/or molecular techniques for confirmation) has created frustration in the diagnostic pathology and urology community as information evolves regarding classification within this spectrum of renal neoplasia. Pathologists, including genitourinary subspecialists, are often uncertain as to the "best practice" diagnostic approach to such tumors. In this review, we present a practical clinically relevant algorithmic approach to classifying tumors within the low grade oncocytic family of renal neoplasia, including a proposal for compressing terminology for evolving categories where appropriate without sacrificing prognostic relevance., (© 2022. The Author(s), under exclusive licence to United States & Canadian Academy of Pathology.)

The morphologic spectrum of type 1 papillary renal cell carcinoma (PRCC) is not well-defined, since a significant proportion of cases have mixed type 1 and 2 histology. We analyzed 199 cases of PRCC with any (even if focal) type 1 features, with a median follow-up of 12 years, to identify clinicopathological features associated with outcome. Ninety-five tumors (48%) of the cohort contained some type 2 component (median amount: 25%; IQR: 10%, 70%). As a group they showed high rates of progression-free (PFS) and cancer-specific survival (CSS). Tumor size, mitotic rate, lymphovascular invasion, sarcomatoid differentiation, sheet-like architecture, and lack of tumor circumscription were significantly associated with CSS (p ≤ 0.015) on univariate analysis. While predominant WHO/ISUP nucleolar grade was associated with PFS (p = 0.013) and CSS (p = 0.030), the presence of non-predominant (<50%) nucleolar grade did not show association with outcome (p = 0.7). PFS and CSS showed no significant association with the presence or the amount of type 2 morphology. We compared the molecular alterations in paired type 1 and type 2 areas in a subset of 22 cases with mixed type 1 and 2 features and identified 12 recurrently mutated genes including TERT, ARID1A, KDM6A, KMT2D, NFE2L2, MET, APC, and TP53. Among 78 detected somatic mutations, 61 (78%) were shared between the paired type 1 and type 2 areas. Copy number alterations, including chromosome 7 and 17 gains, were similar between type 1 and 2 areas. These findings support that type 2 features in a PRCC with mixed histology represent either morphologic variance or clonal evolution. Our study underscores the notion that PRCC with any classic type 1 regions is best considered as type 1 PRCC and assigned the appropriate WHO/ISUP nucleolar grade. It provides additional evidence that type 2 PRCC as a separate category should be re-assessed and likely needs to be abandoned., (© 2021. The Author(s), under exclusive licence to United States & Canadian Academy of Pathology.)

Microphthalmia-associated transcription factor (MiT) family aberration-associated renal cell carcinoma (MiTF-RCC) is a subtype of renal cell carcinoma harboring recurrent chromosomal rearrangements involving TFE3 or TFEB genes. MiTF-RCC is morphologically diverse, can histologically resemble common RCC subtypes like clear cell RCC and papillary RCC, and often poses a diagnostic challenge in genitourinary clinical and pathology practice. To characterize the MiTF-RCC at the molecular level and identify biomarker signatures associated with MiTF-RCC, we analyzed RNAseq data from MiTF-RCC, other RCC subtypes and benign kidney. Upon identifying TRIM63 as a cancer-specific biomarker in MiTF-RCC, we evaluated its expression independently by RNA in situ hybridization (RNA-ISH) in whole tissue sections from 177 RCC cases. We specifically included 31 cytogenetically confirmed MiTF-RCC cases and 70 RCC cases suspicious for MiTF-RCC in terms of clinical and morphological features, to evaluate and compare TRIM63 RNA-ISH results with the results from TFE3/TFEB fluorescence in situ hybridization (FISH), which is the current clinical standard. We confirmed that TRIM63 mRNA was highly expressed in all classes of MiTF-RCC compared to other renal tumor categories, where it was mostly absent to low. While the TRIM63 RNA-ISH and TFE3/TFEB FISH results were largely concordant, importantly, TRIM63 RNA-ISH was strongly positive in TFE3 FISH false-negative cases with RBM10-TFE3 inversion. In conclusion, TRIM63 can serve as a diagnostic marker to distinguish MiTF-RCC from other renal tumor subtypes with overlapping morphology. We suggest a combination of TFE3/TFEB FISH and TRIM63 RNA-ISH assays to improve the accuracy and efficiency of MiTF-RCC diagnosis. Accurate diagnosis of MiTF-RCC and other RCC subtypes would enable effective targeted therapy and avoid poor therapeutic response due to tumor misclassification., (© 2021. The Author(s), under exclusive licence to United States & Canadian Academy of Pathology.)

Mucinous tubular and spindle cell carcinoma (MTSCC) is a rare subtype of renal cell carcinoma with characteristic histologic features and chromosomal alterations. Although typically indolent, a small subset of cases has been reported to exhibit aggressive clinical behavior. We retrospectively identified 33 patients with MTSCC, consisting of 10 cases of locally advanced/metastatic MTSCC (pT3 or N1 or M1) and 23 kidney-confined MTSCC (pT1/T2) without disease recurrence or progression. Utilizing a single-nucleotide polymorphism array and a targeted next-generation sequencing platform, we examined genome-wide molecular alterations in 24 cases, including 11 available samples from 8 patients with locally advanced/metastatic MTSCC. Ten patients with locally advanced/metastatic MTSCC were 8 females (80%) and 2 males (20%). At nephrectomy, 7 of these 10 cases (70%) were pT3 or pN1 while the remaining 3 (30%) were pT1/T2. Eight patients (80%) developed metastases and common sites included lymph node (4, 40%), bone (4, 40%), and retroperitoneum (3, 30%). Four patients died of disease (40%) during follow-up. Locally advanced/metastatic MTSCCs shared typical MTSCC genomic profiles with loss of chromosomes 1, 4, 6, 8, 9, 13, 14, 15, and 22, while some exhibited additional complex genomic alterations, most frequently a relative gain of 1q (7/8). Homozygous loss of CDKN2A/B was observed in 3 (38%) locally advanced/metastatic MTSCCs. Tumor necrosis, solid nested/sheet pattern, irregular trabecular/single-file infiltration in a desmoplastic stroma, lymphovascular space invasion, and increased mitotic activity were associated with locally advanced/metastatic MTSCCs (all p < 0.05). Our findings reveal that MTSCCs with aggressive clinical behavior have progressed through clonal evolution; CDKN2A/B deletion and additional complex genomic abnormalities may contribute to this process. Recognizing the morphologic presentation of high-grade MTSCC and evaluating adverse histologic features seen in these tumors can help establish a definitive diagnosis and stratify patients for treatment and prognostication.

Background: Immune checkpoint inhibitors are an important therapeutic option for urothelial carcinoma, but durable responses are achieved in a minority of patients. Identifying pre-treatment biomarkers that may predict response to these therapies or who exhibit intrinsic resistance, is of paramount importance., Objective: To explore the prevalence of PD-L1 copy number alteration in urothelial carcinoma and correlate with response to immune checkpoint inhibitors., Methods: We analyzed a cohort of 1050 carcinomas of the bladder and upper urinary tract that underwent targeted next generation sequencing, prospectively. We assessed PD-L1 protein expression, copy number status (next generation sequencing/FISH), and detailed treatment response., Results: We identified 9 tumors with PD-L1 amplification and 9 tumors with PD-L1 deletion. PD-L1 protein expression was the highest in PD-L1 amplified tumors. Of the 9 patients whose tumors harbored PD-L1 amplification, 6 received immunotherapy with 4 deriving clinical benefit, and two achieving durable response. Of the 9 patients whose tumors had PD-L1 copy number losses, 4 received immunotherapy with 3 experiencing disease progression., Conclusions: PD-L1 copy number alterations may serve as potential biomarkers of response to immunotherapy in urothelial carcinoma patients, if validated in larger cohorts.

Germ cell tumors (GCTs) are the most common cancer in men between the ages of 15 and 40. Although most patients are cured, those with disease arising in the mediastinum have distinctly poor outcomes. One in every 17 patients with primary mediastinal nonseminomatous GCTs develop an incurable hematologic malignancy and prior data intriguingly suggest a clonal relationship exists between hematologic malignancies and GCTs in these cases. To date, however, the precise clonal relationship between GCTs and the diverse additional somatic malignancies arising in such individuals have not been determined. Here, we traced the clonal evolution and characterized the genetic features of each neoplasm from a cohort of 15 patients with GCTs and associated hematologic malignancies. We discovered that GCTs and hematologic malignancies developing in such individuals evolved from a common shared precursor, nearly all of which harbored allelically imbalanced p53 and/or RAS pathway mutations. Hematologic malignancies arising in this setting genetically resembled mediastinal GCTs rather than de novo myeloid neoplasms. Our findings argue that this scenario represents a unique clinical syndrome, distinct from de novo GCTs or hematologic malignancies, initiated by an ancestral precursor that gives rise to the parallel evolution of GCTs and blood cancers in these patients.

Context.—: In prostate cancer, "tertiary" higher-grade patterns (TPs) have been associated with biochemical recurrence after radical prostatectomy., Objective.—: To determine variation regarding definition and application of TPs., Design.—: Online survey regarding TPs in a range of grading scenarios circulated to 105 experienced urologic pathologists., Results.—: Among 95 respondents, 40 of 95 (42%) defined TPs as "third most common pattern" and 55 (58%) as "minor pattern/less than 5% of tumor." In a tumor with pattern 3 and less than 5% pattern 4, of the 95 respondents, 35 (37%) assigned 3 + 3 = 6 with TP4, while 56 (59%) assigned 3 + 4 = 7. In a tumor with pattern 4 and less than 5% pattern 5, of the 95 respondents, 51 (54%) assigned 4 + 4 = 8 with TP5, while 43 (45%) assigned 4 + 5 = 9. Six scenarios were presented in which the order of most common patterns was 3, 4, and 5 (Group 1) or 4, 3, and 5 (Group 2) with varying percentages. In both groups, when pattern 5 was less than 5%, we found that 98% and 93% of respondents would assign 3 + 4 = 7 or 4 + 3 = 7 with TP5. In scenarios with 15% or 25% pattern 5, most respondents (70% and 80%, respectively) would include pattern 5 as the secondary grade, that is, 3 + 5 = 8 (Group 1) or 4 + 5 = 9 (Group 2). For 85 of 95 (89%), a TP would not impact Grade Group assignment., Conclusions.—: This survey highlights substantial variation in practice patterns regarding definition and application of "tertiary" grading in radical prostatectomy specimens. High consistency was observed in 3 + 4 = 7/4 + 3 = 7 scenarios with truly minor pattern 5. These findings should inform future studies assessing the standardization and predictive value of "tertiary" patterns.

Sarcomatoid clear-cell renal cell carcinomas (sRCC) are associated with dismal prognosis. Genomic alterations associated with sarcomatoid dedifferentiation are poorly characterized. We sought to define the genomic landscape of sRCC and uncover potentially actionable therapeutic targets. We assessed the genomic landscape of sRCC using targeted panel sequencing including patients with microdissected sarcomatoid and epithelial components. Along with common genomic alterations associated with clear-cell histology, we found that Hippo was one of the most frequently altered pathways in these tumours. Hippo alterations were differentially enriched in sRCC compared to non-sRCC. Functional analysis showed that Hippo members mutations were associated with higher nuclear accumulation of YAP/TAZ, core effectors of the Hippo pathway. In a NF2-mutant sRCC model, YAP1 knockdown and NF2 reconstitution suppressed cell proliferation, tumour growth and invasion, both in vitro and in vivo. Overall, we show that Hippo pathway alterations are a feature of sRCC, and enable the exploration of the Hippo pathway as a novel potential therapeutic target.

Amplifications of JAK2, PD-L1, and PD-L2 at 9p24.1 lead to constitutive expression of PD-L1. This, coupled with JAK2-activation dependent upregulation of PD-L1 and adaptive/induced expression leads to higher tumor PD-L1 expression and immune evasion. Renal tumors were therefore evaluated for 9p24.1 amplifications. A combination of next generation sequencing-based copy number analysis, fluorescence in situ hybridization for JAK2/INSL6 and PD-L1/PD-L2 and immunohistochemistry for phospho-STAT3 (downstream target of JAK2), PD-L1, PD-L2, and PD-1 was performed. In this study we interrogated a "Discovery" cohort of 593 renal tumors, a "Validation" cohort of 398 high-grade renal tumors, The Cancer Genome Atlas (879 cases) and other public datasets (846 cases). 9p24.1 amplifications were significantly enriched in renal tumors with sarcomatoid transformation (5.95%, 15/252) when compared to all histologic subtypes in the combined "Discovery", "Validation" and public datasets (16/2636, 0.6%, p < 0.00001). Specifically, 9p24.1 amplifications amongst sarcomatoid tumors in public datasets, the "Discovery" and "Validation" cohorts were 7.7% (6/92), 15.1% (5/33), and 3.1% (4/127), respectively. Herein, we describe 13 cases and amplification status for these was characterized using next generation sequencing (n = 9) and/or fluorescence in situ hybridization (n = 10). Correlation with PD-L1 immunohistochemistry (n = 10) revealed constitutive expression (mean H-score: 222/300, n = 10). Analysis of outcomes based on PD-L1 expression in tumor cells performed on 282 cases ("Validation" cohort) did not reveal a significant prognostic effect and was likely reflective of advanced disease. A high incidence of constitutive PD-L1 expression in tumor cells in the "Validation" cohort (H-Score ≥250/300) was noted amongst 83 rhabdoid (6%) and 127 sarcomatoid renal tumors (7.1%). This suggests additional mechanisms of constitutive expression other than amplification events. Importantly, two patients with 9p24.1-amplified sarcomatoid renal tumors showed significant response to immunotherapy. In summary, a subset of renal tumors with sarcomatoid transformation exhibits constitutive PD-L1 overexpression and these patients should be evaluated for enhanced response to immunotherapy.

Renal medullary carcinoma is a rare but highly aggressive type of renal cancer occurring in patients with sickle cell trait or rarely with other hemoglobinopathies. Loss of SMARCB1 protein expression, a core subunit of the switch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex, has emerged as a key diagnostic feature of these tumors. However, the molecular mechanism underlying this loss remains unclear. We retrospectively identified 20 patients diagnosed with renal medullary carcinoma at two institutions from 1996 to 2017. All patients were confirmed to have sickle cell trait, and all tumors exhibited a loss of SMARCB1 protein expression by immunohistochemistry. The status of SMARCB1 locus was examined by fluorescence in situ hybridization (FISH) using 3-color probes, and somatic alterations were detected by targeted next-generation sequencing platforms. FISH analysis of all 20 cases revealed 11 (55%) with concurrent hemizygous loss and translocation of SMARCB1, 6 (30%) with homozygous loss of SMARCB1, and 3 (15%) without structural or copy number alterations of SMARCB1 despite protein loss. Targeted sequencing revealed a pathogenic somatic mutation of SMARCB1 in one of these 3 cases that were negative by FISH. Tumors in the 3 subsets with different FISH findings largely exhibited similar clinicopathologic features, however, homozygous SMARCB1 deletion was found to show a significant association with the solid growth pattern, whereas tumors dominated by reticular/cribriform growth were enriched for SMARCB1 translocation. Taken together, we demonstrate that different molecular mechanisms underlie the loss of SMARCB1 expression in renal medullary carcinoma. Biallelic inactivation of SMARCB1 occurs in a large majority of cases either via concurrent hemizygous loss and translocation disrupting SMARCB1 or by homozygous loss.

Tubulocystic renal cell carcinoma, a unique tumor, was recently included as a new entity in the World Health Organization classification of renal tumors. It has variably been reported to be related to other renal cell carcinomas, including papillary renal cell carcinoma, fumarate hydratase-deficient carcinoma, and others, likely because many such carcinomas may show variable amounts of tubulocystic architecture. The published data characterizing the molecular features of these tumors are inconsistent. We studied nine "pure" tubulocystic renal cell carcinomas, as defined by International Society of Urologic Pathologists (ISUP) and World Health Organization (WHO), by targeted next-generation sequencing, and fluorescence in situ hybridization for X and Y chromosomes, to investigate if these show any unique characteristics or any overlap with known mutational/molecular profiles or copy number alterations in other subtypes of renal cell carcinoma. All nine tubulocystic carcinomas demonstrated combined losses at chromosome 9 and gains at chromosome 17, as well as, loss of chromosome Y (in 5/5). None of the tumors showed mutational profiles characteristic of other renal neoplasms, including those seen in fumarate hydratase-deficient renal cell carcinoma. Recurrent mutations in chromatin-modifying genes, KMT2C and KDM5C, were detected in two of nine tumors. Thus, tubulocystic renal cell carcinoma, if defined strictly, at the clinical and pathologic level, demonstrates genomic features distinct from other subtypes of renal cell carcinoma. These findings support the contention that tubulocystic renal cell carcinoma should be diagnosed only using strict morphological criteria and only when presenting in a "pure" form; presence of variable papillary, poorly differentiated, or other architectural patterns most likely do not belong to the category of tubulocystic renal cell carcinoma.

While genomic sequencing routinely identifies oncogenic alterations for the majority of cancers, many tumors harbor no discernable driver lesion. Here, we describe the exceptional molecular phenotype of a genomically quiet kidney tumor, clear cell papillary renal cell carcinoma (CCPAP). In spite of a largely wild-type nuclear genome, CCPAP tumors exhibit severe depletion of mitochondrial DNA (mtDNA) and RNA and high levels of oxidative stress, reflecting a shift away from respiratory metabolism. Moreover, CCPAP tumors exhibit a distinct metabolic phenotype uniquely characterized by accumulation of the sugar alcohol sorbitol. Immunohistochemical staining of primary CCPAP tumor specimens recapitulates both the depletion of mtDNA-encoded proteins and a lipid-depleted metabolic phenotype, suggesting that the cytoplasmic clarity in CCPAP is primarily related to the presence of glycogen. These results argue for non-genetic profiling as a tool for the study of cancers of unknown driver., Competing Interests: JX, ER, HL, GG, PJ, JS, AB, FS, CC, JH, LZ, PS, DK, ZT, JC, JT, NS, PJ, YD, WS, EC, PR, JC, EP, YC, VR, CS, SK, JH, CL, ST, AH No competing interests declared, SS This author is a former employee of Metabolon, Inc, (© 2019, Xu et al.)

We studied 137 primary testicular germ cell tumors (TGCTs) using high-dimensional assays of genomic, epigenomic, transcriptomic, and proteomic features. These tumors exhibited high aneuploidy and a paucity of somatic mutations. Somatic mutation of only three genes achieved significance-KIT, KRAS, and NRAS-exclusively in samples with seminoma components. Integrated analyses identified distinct molecular patterns that characterized the major recognized histologic subtypes of TGCT: seminoma, embryonal carcinoma, yolk sac tumor, and teratoma. Striking differences in global DNA methylation and microRNA expression between histology subtypes highlight a likely role of epigenomic processes in determining histologic fates in TGCTs. We also identified a subset of pure seminomas defined by KIT mutations, increased immune infiltration, globally demethylated DNA, and decreased KRAS copy number. We report potential biomarkers for risk stratification, such as miRNA specifically expressed in teratoma, and others with molecular diagnostic potential, such as CpH (CpA/CpC/CpT) methylation identifying embryonal carcinomas., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Chromophobe renal cell carcinoma (chRCC) typically shows ~7 chromosome losses (1, 2, 6, 10, 13, 17, and 21) and ~31 exonic somatic mutations, yet carries ~5%-10% metastatic incidence. Since extensive chromosomal losses can generate proteotoxic stress and compromise cellular proliferation, it is intriguing how chRCC, a tumor with extensive chromosome losses and a low number of somatic mutations, can develop lethal metastases. Genomic features distinguishing metastatic from nonmetastatic chRCC are unknown. An integrated approach, including whole-genome sequencing (WGS), targeted ultradeep cancer gene sequencing, and chromosome analyses (FACETS, OncoScan, and FISH), was performed on 79 chRCC patients including 38 metastatic (M-chRCC) cases. We demonstrate that TP53 mutations (58%), PTEN mutations (24%), and imbalanced chromosome duplication (ICD, duplication of ≥ 3 chromosomes) (25%) were enriched in M-chRCC. Reconstruction of the subclonal composition of paired primary-metastatic chRCC tumors supports the role of TP53, PTEN, and ICD in metastatic evolution. Finally, the presence of these 3 genomic features in primary tumors of both The Cancer Genome Atlas kidney chromophobe (KICH) (n = 64) and M-chRCC (n = 35) cohorts was associated with worse survival. In summary, our study provides genomic insights into the metastatic progression of chRCC and identifies TP53 mutations, PTEN mutations, and ICD as high-risk features.

The utility of cancer cell lines is affected by the similarity to endogenous tumour cells. Here we compare genomic data from 65 kidney-derived cell lines from the Cancer Cell Line Encyclopedia and the COSMIC Cell Lines Project to three renal cancer subtypes from The Cancer Genome Atlas: clear cell renal cell carcinoma (ccRCC, also known as kidney renal clear cell carcinoma), papillary (pRCC, also known as kidney papillary) and chromophobe (chRCC, also known as kidney chromophobe) renal cell carcinoma. Clustering copy number alterations shows that most cell lines resemble ccRCC, a few (including some often used as models of ccRCC) resemble pRCC, and none resemble chRCC. Human ccRCC tumours clustering with cell lines display clinical and genomic features of more aggressive disease, suggesting that cell lines best represent aggressive tumours. We stratify mutations and copy number alterations for important kidney cancer genes by the consistency between databases, and classify cell lines into established gene expression-based indolent and aggressive subtypes. Our results could aid investigators in analysing appropriate renal cancer cell lines.

PBRM1 is the second most commonly mutated gene after VHL in clear cell renal cell carcinoma (ccRCC). However, the biological consequences of PBRM1 mutations for kidney tumorigenesis are unknown. Here, we find that kidney-specific deletion of Vhl and Pbrm1, but not either gene alone, results in bilateral, multifocal, transplantable clear cell kidney cancers. PBRM1 loss amplified the transcriptional outputs of HIF1 and STAT3 incurred by Vhl deficiency. Analysis of mouse and human ccRCC revealed convergence on mTOR activation, representing the third driver event after genetic inactivation of VHL and PBRM1. Our study reports a physiological preclinical ccRCC mouse model that recapitulates somatic mutations in human ccRCC and provides mechanistic and therapeutic insights into PBRM1 mutated subtypes of human ccRCC., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Renal cell carcinomas with unclassified histology (uRCC) constitute a significant portion of aggressive non-clear cell renal cell carcinomas that have no standard therapy. The oncogenic drivers in these tumours are unknown. Here we perform a molecular analysis of 62 high-grade primary uRCC, incorporating targeted cancer gene sequencing, RNA sequencing, single-nucleotide polymorphism array, fluorescence in situ hybridization, immunohistochemistry and cell-based assays. We identify recurrent somatic mutations in 29 genes, including NF2 (18%), SETD2 (18%), BAP1 (13%), KMT2C (10%) and MTOR (8%). Integrated analysis reveals a subset of 26% uRCC characterized by NF2 loss, dysregulated Hippo-YAP pathway and worse survival, whereas 21% uRCC with mutations of MTOR, TSC1, TSC2 or PTEN and hyperactive mTORC1 signalling are associated with better clinical outcome. FH deficiency (6%), chromatin/DNA damage regulator mutations (21%) and ALK translocation (2%) distinguish additional cases. Altogether, this study reveals distinct molecular subsets for 76% of our uRCC cohort, which could have diagnostic and therapeutic implications.

Mutations, deletions, and changes in copy number of mitochondrial DNA (mtDNA), are observed throughout cancers. Here, we survey mtDNA copy number variation across 22 tumor types profiled by The Cancer Genome Atlas project. We observe a tendency for some cancers, especially of the bladder, breast, and kidney, to be depleted of mtDNA, relative to matched normal tissue. Analysis of genetic context reveals an association between incidence of several somatic alterations, including IDH1 mutations in gliomas, and mtDNA content. In some but not all cancer types, mtDNA content is correlated with the expression of respiratory genes, and anti-correlated to the expression of immune response and cell-cycle genes. In tandem with immunohistochemical evidence, we find that some tumors may compensate for mtDNA depletion to sustain levels of respiratory proteins. Our results highlight the extent of mtDNA copy number variation in tumors and point to related therapeutic opportunities.

Dysregulated metabolism is a hallmark of cancer, manifested through alterations in metabolites. We performed metabolomic profiling on 138 matched clear cell renal cell carcinoma (ccRCC)/normal tissue pairs and found that ccRCC is characterized by broad shifts in central carbon metabolism, one-carbon metabolism, and antioxidant response. Tumor progression and metastasis were associated with metabolite increases in glutathione and cysteine/methionine metabolism pathways. We develop an analytic pipeline and visualization tool (metabolograms) to bridge the gap between TCGA transcriptomic profiling and our metabolomic data, which enables us to assemble an integrated pathway-level metabolic atlas and to demonstrate discordance between transcriptome and metabolome. Lastly, expression profiling was performed on a high-glutathione cluster, which corresponds to a poor-survival subgroup in the ccRCC TCGA cohort., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Integrated sequencing analysis identified a group of tumors among clear cell renal cell carcinomas characterized by hotspot mutations in TCEB1 (a gene that contributes to the VHL complex to ubiquitinate hypoxia-inducible factor). We analyzed 11 tumors from two distinct cohorts with TCEB1 mutations along with an expanded cohort to assess whether these should be considered an entity distinct from clear cell renal cell carcinoma and clear cell papillary renal cell carcinoma. All tumors were characterized by hotspot mutations in TCEB1 Y79C/S/F/N or A100P. Morphological and immunohistochemical characteristics of the tumors were assessed by two experienced genitourinary pathologists. Clinical and pathological variables, copy number alterations, mutations, and expression signatures were compared with a cohort of TCEB1 wild-type tumors. All TCEB1-mutated tumors were VHL and PBRM1 wild type and contained distinct copy number profiles including loss of heterozygosity of chromosome 8, the location of TCEB1 (8q21.11). All tumors lacked the clear cell renal cell carcinoma signature 3p loss and contained distinct gene expression signatures. None of the clear cell papillary tumors harbored TCEB1 mutations. Pathologically, all TCEB1-mutated tumors shared characteristic features including thick fibromuscular bands transecting the tumor, pure clear cell cytology frequently with cells showing voluminous cytoplasm, and clear cell renal cell carcinoma-like acinar areas associated with infolding tubular and focally papillary architecture. The presence of voluminous cytoplasm, absence of luminal polarization of tumor nuclei, and lack of extensive cup-like distribution of carbonic anhydrase-IX expression distinguish it from clear cell papillary carcinoma. None of the patients developed metastases at last follow-up (median 48 months). In sum, TCEB1-mutated renal cell carcinoma is a distinct entity with recurrent hotspot mutations, specific copy number alterations, pathway activation, and characteristic morphological features. Further clinical follow-up is needed to determine whether these tumors are more indolent compared with the conventional clear cell renal cell carcinoma.

The International Consultations on Urological Diseases are international consensus meetings, supported by the World Health Organization and the Union Internationale Contre le Cancer, which have occurred since 1981. Each consultation has the goal of convening experts to review data and provide evidence-based recommendations to improve practice. In 2012, the selected subject was bladder cancer, a disease which remains a major public health problem with little improvement in many years. The proceedings of the 2nd International Consultation on Bladder Cancer, which included a 'Pathology of Bladder Cancer Work Group,' have recently been published; herein, we provide a summary of developments and consensus relevant to the practicing pathologist. Although the published proceedings have tackled a comprehensive set of issues regarding the pathology of bladder cancer, this update summarizes the recommendations regarding selected issues for the practicing pathologist. These include guidelines for classification and grading of urothelial neoplasia, with particular emphasis on the approach to inverted lesions, the handling of incipient papillary lesions frequently seen during surveillance of bladder cancer patients, descriptions of newer variants, and terminology for urine cytology reporting., Competing Interests: Disclosure/conflict of interest The authors declare no conflict of interest.

Context: While biopsies are now increasingly being performed for the diagnosis of renal cortical neoplasms, the influence of the rendered pathological diagnoses on the clinical management is only rarely documented., Objectives: To report our experience with consecutively performed renal biopsies and the potential impact of the diagnosis on subsequent clinical management., Design: Material from needle biopsies performed consecutively at our institution between 2006 and 2011 was reviewed. The influence of the reported pathology results on the clinical management was determined from patient follow-up medical record review., Results: In total, 218 percutaneous biopsies for renal masses were performed during this period. Among them, 181 (83%) yielded neoplastic tissue, including 81 clear cell renal cell carcinomas, 29 low-grade oncocytic neoplasms, 7 papillary renal cell carcinomas, 5 clear cell papillary renal cell carcinomas, 5 angiomyolipomas, and 14 urothelial carcinomas. Fourteen additional cases (6%) contained lesional material from clinically known nonneoplastic processes, for a total diagnostic yield of 89%. Twenty-three (11%) were nonrepresentative of lesional tissue. In 10 of these, repeat biopsies or resections established the diagnosis of renal tumors. Biopsy diagnosis was confirmed in 29 of 30 cases (97%) on subsequent nephrectomy. Following the biopsy diagnosis, there were significant differences in the clinical management; overall, 79% of clear cell renal cell carcinomas received therapeutic interventions, and 17% were put on active surveillance. In contrast, 77% of the benign or low-grade lesions were put on active surveillance., Conclusions: Accurate and specific diagnosis can be rendered on renal core biopsy in most renal tumors, and the biopsy diagnosis can have a definitive role in their clinical management.

We describe the landscape of somatic genomic alterations of 66 chromophobe renal cell carcinomas (ChRCCs) on the basis of multidimensional and comprehensive characterization, including mtDNA and whole-genome sequencing. The result is consistent that ChRCC originates from the distal nephron compared with other kidney cancers with more proximal origins. Combined mtDNA and gene expression analysis implicates changes in mitochondrial function as a component of the disease biology, while suggesting alternative roles for mtDNA mutations in cancers relying on oxidative phosphorylation. Genomic rearrangements lead to recurrent structural breakpoints within TERT promoter region, which correlates with highly elevated TERT expression and manifestation of kataegis, representing a mechanism of TERT upregulation in cancer distinct from previously observed amplifications and point mutations., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The 2004 World Health Organization classification of tumors defines epithelioid angiomyolipoma of kidney as a potentially malignant mesenchymal neoplasm with reported metastasis in approximately one-third of the cases. However, this conclusion was based primarily on individual case reports and small retrospective series. More recently reported larger series have shown varying results. We reviewed 437 consecutive renal angiomyolipomas with primary resection at three tertiary-care institutions with high nephrectomy volumes. Only tumors showing >80% epithelioid histology were included in this study. Tumors resected elsewhere and reviewed in consultation were not included. Twenty of these 437 (4.6%) were classified as epithelioid angiomyolipoma. The female to male ratio was 11:9, mean age 49.7 (range, 30-80) years, and mean tumor size 8.7 (range, 1-25) cm. Microscopic tumor necrosis was present in 10 (50%) tumors and mitotic activity (range, <1-5/10 high power fields) in 8 (40%); atypical mitoses were seen in only 1 (5%) tumor. Pleomorphic ganglion-like or multinucleated giant cells were seen in 18 (90%) tumors. With a mean follow-up of 82.5 (range, 1-356) months, seventeen patients were alive with no-evidence-of-disease at the time of last follow-up; two patients died of unrelated causes with no-evidence-of-disease, and one patient (5%) developed distant metastases. Our data, based on consecutively resected angiomyolipomas with long clinical follow-up, suggests that epithelioid angiomyolipomas constitute a small proportion of all angiomyolipomas, and the rate of aggressive behavior among epithelioid angiomyolipomas, even when showing morphologic features previously reported to portend aggressive clinical behavior, is very low.

Purpose: To investigate the impact of newly identified chromosome 3p21 epigenetic tumor suppressors PBRM1, SETD2, and BAP1 on cancer-specific survival (CSS) of 609 patients with clear cell renal cell carcinoma (ccRCC) from 2 distinct cohorts., Experimental Design: Select sequencing on 3p tumor suppressors of 188 patients who underwent resection of primary ccRCC at the Memorial Sloan-Kettering Cancer Center (MSKCC) was conducted to interrogate the genotype-phenotype associations. These findings were compared with analyses of the genomic and clinical dataset from our nonoverlapping The Cancer Genome Atlas (TCGA) cohort of 421 patients with primary ccRCC., Results: 3p21 tumor suppressors are frequently mutated in both the MSKCC (PBRM1, 30.3%; SETD2, 7.4%; BAP1, 6.4%) and the TCGA (PBRM1, 33.5%; SETD2, 11.6%; BAP1, 9.7%) cohorts. BAP1 mutations are associated with worse CSS in both cohorts [MSKCC, P = 0.002; HR 7.71; 95% confidence interval (CI)2.08-28.6; TCGA, P = 0.002; HR 2.21; 95% CI 1.35-3.63]. SETD2 are associated with worse CSS in the TCGA cohort (P = 0.036; HR 1.68; 95% CI 1.04-2.73). On the contrary, PBRM1 mutations, the second most common gene mutations of ccRCC, have no impact on CSS., Conclusion: The chromosome 3p21 locus harbors 3 frequently mutated ccRCC tumor suppressor genes. BAP1 and SETD2 mutations (6%-12%) are associated with worse CSS, suggesting their roles in disease progression. PBRM1 mutations (30%-34%) do not impact CSS, implicating its principal role in the tumor initiation. Future efforts should focus on therapeutic interventions and further clinical, pathologic, and molecular interrogation of this novel class of tumor suppressors.

Squamous cell carcinomas of the urinary bladder are rare in the Western world; the majority of cases are reported in countries endemic to Schistosoma parasitic infections. Unlike squamous tumors of the uterine cervix or oropharynx, the human papillomavirus (HPV) is not commonly associated with bladder squamous cell carcinomas. We report on two cases of HPV-positive urothelial carcinomas of the urinary bladder with extensive squamous differentiation showing the typical basaloid, poorly differentiated morphology of HPV-associated tumors. These occurred in patients with neurogenic bladders who had long-standing histories of self-catheterization with tumors that tested positive for HPV by in situ hybridization. A retrospective review of our institutional database revealed four additional patients with bladder tumors showing squamous differentiation arising in the setting of neurogenic bladder. Review of these cases showed the more common well-differentiated keratinizing appearance of squamous cell carcinomas of the bladder. These tumors showed only patchy positivity for p16 immunohistochemical stain (not the diffuse strong staining seen in HPV-positive tumors), and the one tested case was negative for HPV by in situ hybridization. HPV infection and neurogenic bladder have been independently associated with increased risk of developing carcinoma in the urinary bladder; however, this is the first report of squamous tumors arising in the setting of concurrent neurogenic bladder and HPV infection. The morphology of these tumors is similar to that of other high-risk HPV-associated squamous carcinomas with a basaloid, poorly differentiated appearance and little to no keratin formation.

Context: Cystic lesions of the kidney may be accompanied by a range of neoplasms with distinct prognoses and future risks of developing additional tumors. In addition, some renal tumors, with or without accompanying renal cysts, may show a prominent cystic component. In the adult population, neoplasms occurring in a background of renal cystic diseases and cystic renal neoplasms often pose diagnostic challenges because of their many overlapping features., Objective: To review the clinicopathologic characteristics of common entities in the spectrum of neoplastic and potential preneoplastic cystic lesions encountered in adults, with an emphasis on renal cystic diseases associated with tumor development and on renal neoplasms with predominantly cystic morphology., Data Sources: The relevant English-language literature was reviewed, accompanied by the authors' experience at their practicing institution., Conclusions: The presence of multiple renal cysts, both acquired and syndromic, can be associated with a variety of renal tumors. The morphology of the cysts and associated tumor types can help predict the genetic or acquired basis of the lesions, and particularly in specimens with no accompanying pertinent clinical history, such potential associations should be suggested in surgical pathology reports.

Over the past few years several investigators have independently described unique low-grade renal epithelial neoplasms with clear cytoplasm, focal to diffuse papillary architecture, and occasional leiomyomatous stromal metaplasia that are not currently recognized in the World Health Organization classification of renal tumors. These tumors have been referred to by a variety of names including clear-cell papillary renal cell carcinoma and recently "clear-cell tubulopapillary renal cell carcinoma". On the basis of the available data, such tumors are positive for cytokeratin 7 (CK7) and carbonic anhydrase IX (CA9), while being negative for CD10, α-methylacyl-CoA racemase (AMACR), and TFE3. These tumors reportedly lack trisomies of chromosomes 7 and 17, deletions of 3p25, von Hippel-Lindau (VHL) gene mutations, and VHL promoter hypermethylation. Herein, we report on nine cases of this tumor emphasizing detailed studies of the VHL gene and hypoxia-inducible factor (HIF) pathway. Molecular studies performed included VHL mutational analysis, copy number changes assessed using single-nucleotide polymorphism arrays, and qRT-PCR for VHL mRNA expression. Immunohistochemical stains for markers of HIF pathway activation (HIF-1α, CA9, and glucose transporter-1 (GLUT-1)) as well as other relevant markers (CK7, CD10, AMACR, and TFE3) were performed. None of our tumors harbored VHL gene mutations, losses of chromosomal region 3p25, or trisomies of chromosomes 7 or 17. VHL mRNA was overexpressed in our tumors relative to normal renal tissue and clear-cell renal cell carcinoma. All cases showed strong co-expression of CK7, HIF-1α, GLUT-1, and CA9. No expression of TFE3, CD10, or AMACR was seen. The morphological, immunophenotypic, and molecular features of these unique low-grade tumors are sufficiently distinct to allow separation from other renal cell carcinoma subtypes. The co-expression of CA9, HIF-1α, and GLUT-1 in the absence of VHL gene alterations in clear-cell papillary renal cell carcinoma suggests activation of the HIF pathway by non-VHL-dependent mechanisms.

TMPRSS2-ERG gene rearrangement is seen in about half of clinically localized prostate cancers, yet controversy exists with regard to its prognostic implications. Similarly, the relationship of TMPRSS2-ERG fusion to Gleason score and morphology remains uncertain. We assigned Gleason scores and recorded morphological features for 521 clinically localized prostate cancers sampled in triplicate and arrayed in eight tissue microarray blocks. Fluorescence in situ hybridization was performed to delineate TMPRSS2-ERG aberrations. Using maximum Gleason score, based on three core evaluation, and overall Gleason score, based on prostatectomy sections, Fisher's exact test was performed for tumors with TMPRSS2-ERG translocation/deletion, copy number increase (≥ 3) of the TMPRSS2-ERG region without translocation/deletion, and copy number increase and concomitant translocation/deletion. In all, 217 (42%) translocation/deletion and 30 (5.9%) copy number increase-alone cases were detected. Among 217 translocation/deletion cases, 32 had translocation/deletion with copy number increase. In all, 237, 200, and 75 cancers had maximum core-specific Gleason score of 6, 7, and 8-10, respectively. Tumors with translocation/deletion tended toward lower Gleason scores than those without (P=0.002) with similar results for overall Gleason score (P=0.02); copy number increase cases tended toward higher Gleason scores than those without (P<0.001). Gleason score of 8-10 tumors demonstrated lower odds of translocation/deletion (odds ratio (OR) 0.38; 95% CI 0.21-0.68) and higher odds of copy number increase alone (OR 7.33; 95% CI 2.65-20.31) or copy number increase+translocation/deletion (OR 3.03; 95% CI 1.12-8.15) relative to Gleason score of <7 tumors. No significant difference in TMPRSS2-ERG incidence was observed between patients with and without cribriform glands, glomerulations, signet-ring cells, or intraductal cancer (P=0.821, 0.095, 0.132, 0.375). TMPRSS2-ERG gene fusion is associated with lower core-specific and overall Gleason scores and not with high-grade morphologies. Conversely, TMPRSS2-ERG copy number increase, with or without rearrangement, is associated with higher Gleason score. These findings indicate that translocation/deletion of TMPRSS2-ERG is not associated with histological features of aggressive prostate cancer.

Fibrolamellar carcinoma is a rare malignant primary liver neoplasm with characteristic histological features that typically arises in young patients without viral hepatitis or cirrhosis. Previous studies on this entity have been limited by small numbers of patients. In contrast to classical hepatocellular carcinoma, individual cases of fibrolamellar carcinoma have been reported to express cytokeratin 7. In addition, ultrastructural and serological studies have suggested that fibrolamellar carcinoma may show neuroendocrine differentiation. The cellular differentiation of fibrolamellar carcinoma has not been studied and little is reported about its immunohistochemical profile. We studied 26 cases of fibrolamellar carcinoma and 62 cases of classical hepatocellular carcinoma by immunohistochemistry for HepPar1, glypican-3, pCEA, CD10, alpha-fetoprotein, cytokeratin 20, neuroendocrine markers, and surrogate markers for biliary differentiation (cytokeratin 7, cytokeratin 19, epithelial membrane antigen, EpCAM, mCEA, B72.3, and CA19.9). In situ hybridization for albumin mRNA was also performed. Tumor cells of fibrolamellar carcinoma and hepatocellular carcinoma showed positive signals for albumin mRNA by in situ hybridization in all cases. Both tumor types stained uniformly positively with HepPar1 and most showed a canalicular staining pattern for pCEA, confirming their hepatocellular differentiation. In addition, 39% of hepatocellular carcinoma cases and 59% of fibrolamellar carcinoma cases were positive for glypican-3. All 22 fibrolamellar carcinoma cases tested showed positive staining for cytokeratin 7 and epithelial membrane antigen, whereas less than one-third of hepatocellular carcinoma cases were positive for these markers (P<0.0001). Further, 36% of fibrolamellar carcinoma cases showed staining for B72.3, cytokeratin 19, EpCAM, or mCEA. Minimal evidence of neuroendocrine differentiation in either tumor was found with any of the usual immunohistochemical markers used for this purpose. Therefore, cytokeratin 7 and epithelial membrane antigen may be useful to differentiate between fibrolamellar carcinoma and hepatocellular carcinoma. On the basis of immunohistochemistry, fibrolamellar carcinoma seems to show both hepatocellular and bile duct differentiation.

Stem cell markers, OCT3/4, and more recently SOX2 and growth differentiation factor 3 (GDF3), have been reported to be expressed variably in germ cell tumors. We investigated the immunohistochemical expression of these markers in different testicular germ cell tumors, and their utility in the differential diagnosis of morphologically difficult-to-classify components of these tumors. A total of 50 mixed testicular germ cell tumors, 43 also containing difficult-to-classify areas, were studied. In these areas, multiple morphological parameters were noted, and high-grade nuclear details similar to typical embryonal carcinoma were considered 'embryonal carcinoma-like high-grade'. Immunohistochemical staining for OCT3/4, c-kit, CD30, SOX2, and GDF3 was performed and graded in each component as 0, negative; 1+, 1-25%; 2+, 26-50%; and 3+, >50% positive staining cells. The different components identified in these tumors were seminoma (8), embryonal carcinoma (50), yolk sac tumor (40), teratoma (40), choriocarcinoma (3) and intra-tubular germ cell neoplasia, unclassified (35). By immunohistochemistry, the staining patterns were OCT3/4 -3+, all seminomas, embryonal carcinomas and intra-tubular germ cell neoplasia; SOX2 -3+, all embryonal carcinomas and -2 to 3+, 11/14 (79%) primitive neuroectodermal components in immature teratomas; GDF3 -2 to 3+, all yolk sac tumors, seminomas and intra-tubular germ cell neoplasia and 1 to 2+, 40/50 embryonal carcinomas. A total of 34/43 (79%) of difficult-to-classify areas stained 3+ for OCT3/4, CD30, and SOX2, similar to embryonal carcinoma. Among these areas, only 'embryonal carcinoma-like high-grade' nuclear details were significantly associated with such an immunophenotype. Thus, SOX2 is expressed in embryonal carcinoma and primitive neuroectoderm of teratoma, and unlike OCT3/4, not in intra-tubular germ cell neoplasia and seminoma. Therefore, it may be useful in the distinction of seminoma from embryonal carcinoma, and potentially in diagnosing early carcinomatous differentiation in seminoma. GDF3 positivity, in the absence of OCT3/4 and CD30, combined with morphological features, is helpful in the diagnosis of yolk sac tumor. 'Embryonal carcinoma-like high-grade' nuclear details are the most important morphological criterion for the diagnosis of embryonal carcinoma in difficult-to-classify areas.

A significant number of prostate cancers have been shown to have recurrent chromosomal rearrangements resulting in the fusion of the androgen-regulated TMPRSS2 promoter to a member of the ETS transcription factor family, most commonly ERG. This results in ERG overexpression, which may have a direct causal role in prostate tumorigenesis or progression. However, the clinical significance of the rearrangement is unclear, and in particular, relationship to outcome has been inconsistent in recent reports. We analyzed TMPRSS2-ERG gene rearrangement status by fluorescence in situ hybridization in 521 cases of clinically localized surgically treated prostate cancer with 95 months of median follow-up and also in 40 unmatched metastases. Forty-two percent of primary tumors and 40% of metastases had rearrangements. Eleven percent had copy number increase (CNI) of the TMPRRS2-ERG region. Rearrangement alone was associated with lower grade, but not with stage, biochemical recurrence, metastases, or death. CNI with and without rearrangement was associated with high grade and advanced stage. Further, a subgroup of cancers with CNI and rearrangement by deletion, with two or more copies of the deleted locus, tended to be more clinically aggressive. DNA index assessment revealed that the majority of tumors with CNI of TMPRSS2-ERG had generalized aneuploidy/tetraploidy in contrast to tumors without TMPRSS2-ERG CNI, which were predominantly diploid. We therefore conclude that translocation of TMPRSS2-ERG is not associated with outcome, and the aggressive clinical features associated with CNI of chromosome 21 reflect generalized aneuploidy and are not due to CNI specifically of rearranged TMPRSS2-ERG.

Prostate-specific membrane antigen is a type II membrane glycoprotein that is expressed in benign and neoplastic prostatic tissue and has been recently shown to be also expressed in the neovasculature of various solid malignant tumors including renal cell carcinoma. Renal cell carcinoma is a heterogeneous group of tumors with distinct morphologic and genetic characteristics and clinical behaviors. We performed immunohistochemical studies on formalin-fixed, paraffin-embedded archival material from 75 nephrectomies, using antibodies 13D6 against prostate-specific membrane antigen and CD31 against endothelial cells. The study included 30 clear cell renal cell carcinomas, and 15 of each of papillary and chromophobe renal cell carcinoma and oncocytoma. The extent and intensity of staining were assessed semiquantitatively. In all cases, immunoreactivity was detected only in the tumor-associated neovasculature and not in tumor cells. Clear cell renal cell carcinoma showed the most diffuse staining pattern, where 24/30 cases or 80% had >50% reactive vessels, followed by chromophobe renal cell carcinoma (9/15; 60%) and oncocytoma (5/15, 33%). No diffuse staining was detected in any of the papillary renal cell carcinomas and only focal staining was detected in 11 cases (11/15; 73%). Staining intensity was the strongest in clear cell renal cell carcinoma (25/30; 83%) followed by chromophobe renal cell carcinoma (9/15; 60%), oncocytoma (8/15, 53%) and papillary renal cell carcinoma (5/15; 33%). In summary, prostate-specific membrane antigen is expressed in tumor-associated neovasculature of the majority of renal cortical tumors and is most diffusely and intensely expressed in clear cell renal cell carcinoma and least in papillary renal cell carcinoma. The differences in the expression of prostate-specific membrane antigen in renal cell carcinoma subtypes provide further evidence of the biological diversity of these tumors, and diagnostic and therapeutic applications of such expression can be expanded to include subtypes of renal cell carcinoma.

The presence of estrogen and progesterone-receptor-positive stroma is well known in renal mixed epithelial and stromal tumor, cystic nephroma, and angiomyolipoma with epithelial cysts. It has been suggested that the hormone receptor positivity in mixed epithelial and stromal tumor may be etiologically related to exogenous hormone intake-a phenomenon that has become more frequent in recent years. In the past few years, we have observed such stroma in some non-neoplastic kidneys, as well as in tumor-bearing kidneys away from the tumor. Herein we present our experience with 10 such cases. In a prospective manner, whenever we noted stroma resembling that in ovaries or müllerian organs (endometrial or cervical-like) in kidneys removed for any cause, immunohistochemical stains for estrogen and progesterone receptors were performed. There were eight males and two females among the group, with ages ranging from 11 months to 71 years. In six cases, the nephrectomies were performed for a non-functional kidney, and in three for tumors (one each of chromophobe, clear cell, and acquired cystic disease-associated renal cell carcinoma). One case was a partial nephrectomy for vesico-ureteric reflux, with upper pole hydronephrosis. Such stroma was present in nine cases as a non-mass forming proliferation around dilated, frequently inflamed pelvicalyceal system and collecting ducts. In one it was present at the periphery of an acquired cystic disease-associated renal cell carcinoma, as well as around non-tumorous cysts. The only common finding in all cases was a generalized or segmental hydronephrosis, or tumor compression-related focal obstruction. The stroma was positive for estrogen receptors in all 10 cases, and for progesterone receptors in seven. Thus, estrogen- and progesterone receptor-positive stroma can be present in the kidney, not only as a component of certain tumors, but also in association with non-neoplastic conditions. Its association with obstructive changes suggests that it may represent a metaplastic change in the renal interstitial cells surrounding these obstructed epithelial structures.

The Rex-1 (Zfp-42) gene encodes a zinc finger family transcription factor which is highly expressed in mouse and human embryonic stem cells. It is one of several gene markers used to identify human stem cells. While several organs are known to harbor adult human stem cells, the presence and distribution of stem cells in both the normal and neoplastic adult kidney remains largely unknown. In this study we evaluated Rex-1 mRNA and protein expression in normal and malignant kidney tissue specimens from human patients. Rex-1 mRNA expression was determined using both reverse transcription and real-time PCR. REX1 protein expression was assessed by western analysis and immunohistochemistry, using an affinity-purified, polyclonal antibody to the REX1 protein. We found that 14 of 15 (93%) non-tumor renal parenchymal specimens demonstrated Rex-1 mRNA, compared with 5 of 14 (36%) renal tumors (P < 0.005). REX1 protein expression was detected in 21 of 23 (91%) non-tumor and in 7 of 19 (37%) tumor specimens (P < 0.001). Furthermore, in six of these seven renal tumor specimens where REX1 protein expression was detected, the levels were at least 3-fold lower than those in adjacent, normal kidney tissue. There were no differences in Rex-1 mRNA or protein expression among the various histologic subtypes of renal tumors (clear cell carcinoma, papillary carcinoma, chromophobe carcinoma and oncocytoma). Immunohistochemical staining confirmed the absence of REX1 in three renal tumor specimens (two clear cell and one papillary carcinoma), while the REX1 protein was detected in a small percentage of proximal tubular cells in normal renal tissue. Immunohistochemical staining of another stem cell marker, OCT4, demonstrated a similar pattern of protein expression in a small percentage of normal renal proximal tubular cells. In summary, we were able to detect Rex-1 mRNA and protein expression in over 90% of normal renal parenchymal specimens and we observed a significant reduction in REX1 expression in renal tumor specimens of all histologic subtypes.

Metanephric adenoma (MA), a well-described renal neoplasm, usually behaves in a benign fashion. It may have areas that are morphologically similar to papillary renal cell carcinoma (RCC) type, or epithelial (tubular predominant) type Wilms' tumor. Prior immunohistochemical studies of MA have reported variable staining patterns. Alpha-methylacyl-CoA racemase (AMACR), a molecular marker for prostate carcinoma, has subsequently been found to be overexpressed in breast, colorectal and ovarian cancers, among others. Recent microarray analysis of renal tumors has shown an increase of AMACR mRNA levels in papillary RCC but not in other subtypes. We investigated the utility of immunohistochemical staining for AMACR, cytokeratin 7(CK7), CD57 and WT1 to differentiate between the above-mentioned three neoplasms. Immunohistochemical stains were performed on paraffin-embedded tissue sections from 25 papillary RCC, 10 MAs and eight Wilms' tumors. AMACR was positive in one (10%) of 10 MAs and 24 (96%) of 25 papillary RCC, while it was negative in all Wilms' tumors. CK7 was positive in 20 of 25 papillary RCCs, focally positive in one Wilms' tumor and was negative in all MAs. CD57 was positive in all six MAs that were stained, focally positive in one of 25 papillary RCC and one of eight Wilms' tumors. WT1 was positive in seven of 10 MAs, three of 25 papillary RCCs and all eight Wilms' tumors. In conclusion, diffuse and strong immunoreactivity for AMACR may be useful in differentiating papillary RCC from MA but a panel which includes AMACR, CK7 and CD57 is better in this differential diagnosis. AMACR is not helpful in differentiating MA from Wilms' tumor, but CD57 is helpful in this differential diagnosis. WT1 may be useful in separating Wilms' tumor from MA and papillary RCC but is not helpful in differentiating MA from papillary RCC.

Purpose: The Polycomb group gene, EZH2, functions as a transcriptional repressor involved in gene silencing. Amplification of EZH2 has been reported in several malignancies, including prostate, breast, and lymphoma. We evaluated EZH2 mRNA and protein expression in bladder specimens from patients and the EZH2 mRNA expression in five bladder cancer cell lines., Experimental Design: EZH2 mRNA expression was assessed by reverse transcription-PCR (RT-PCR) in 38 bladder tissue specimens. We also evaluated 39 bladder cancer specimens for EZH2 protein expression using immunohistochemistry with affinity-purified antibodies to human EZH2. In addition, five human bladder cancer cell lines were analyzed by RT-PCR for EZH2 mRNA expression., Results: Five of 14 (36%) nontumor bladder specimens versus 21 of 24 (88%) bladder tumors showed EZH2 mRNA expression (P=0.003). All of the invasive tumors (10 of 10) had detectable EZH2 mRNA expression, compared with 11 of 14 (79%) superficial tumors. In addition, EZH2 mRNA expression was noted in 100% (16 of 16) of high-grade bladder tumors versus 50% (4 of 8) of low-grade tumors (P=0.01). EZH2 protein expression, meanwhile, was increased in neoplastic tissue compared with nontumor urothelium (78% versus 69% of nuclei, P<0.005). There were no differences in EZH2 protein levels between superficial and invasive tumors. High-grade tumors had increased EZH2 staining compared with normal urothelium (78% versus 68%, P<0.005), whereas low-grade lesions did not. Four of five human bladder cancer cell lines expressed high levels of EZH2, whereas only low levels were detected in one cell line., Conclusions: We report a significant increase in EZH2 expression in transitional cell carcinoma of the bladder compared with normal urothelium. These data suggest that similar to other human malignancies, increased EZH2 expression correlates with oncogenesis of the bladder.

Background: E-cadherin, a cell surface protein involved in cell adhesion, is present in normal breast epithelium, benign breast lesions, and in breast carcinoma. Alterations in the gene CDH1 on chromosome 16q22 are associated with changes in E-cadherin protein expression and function. Inactivation of E-cadherin in lobular carcinomas and certain diffuse gastric carcinomas may play a role in the dispersed, discohesive "single cell" growth patterns seen in these tumors. The molecular "signature" of mammary lobular carcinomas is the loss of E-cadherin protein expression as evidenced by immunohistochemistry, whereas ductal carcinomas are typically E-cadherin positive., Patients and Methods: We report on E-cadherin immunostaining patterns in five cases of invasive mammary carcinoma., Results: These were five exceptional instances in which the E-cadherin immunophenotype did not correspond to the apparent histologic classification of the lesion. These cases which are exceedingly rare in our experience are the subject of this report., Conclusion: Findings such as those illustrated in this study occur in virtually all biologic phenomena and they do not invalidate the very high degree of correlation between the expression of E-cadherin and the classification of breast carcinomas as ductal or lobular type on the basis of conventional histologic criteria.

Purpose: Therapy for advanced renal cell carcinoma (RCC) is ineffective in the majority of patients. We have previously reported that retinoid-induced up-regulation of retinoic acid receptor beta (RARbeta) correlated with antitumor effects in RCCs. Recent studies show that there is a reduction in the level of RARbeta2 expression in cancer cells due in part to histone hypoacetylation. Therefore, we tested whether combining histone deacetylase inhibitors with retinoic acid (RA) would restore RARbeta2 receptor expression, leading to increased growth inhibition in RCC cells., Experimental Design: Cell proliferation, Western blot, and reverse transcription-PCR analyses of two RA-resistant RCC cell lines, SK-RC-39 and SK-RC-45, were assessed in the presence of all-trans retinoic acid (ATRA), trichostatin A (TSA), or the combination of ATRA and TSA. Analysis of apoptosis was also done on SK-RC-39 cells treated with these combinations. Additionally, a xenograft tumor model (SK-RC-39) was used in this study to investigate the efficacy of a liposome-encapsulated, i.v. form of ATRA (ATRA-IV) plus TSA combination therapy., Results: Enhanced inhibition of the proliferation of RCC cell lines and of tumor growth in a xenograft model was observed with the combination of ATRA plus TSA. Reactivation of RARbeta2 mRNA expression was observed in SK-RC-39 and SK-RC-45 cells treated with TSA alone or TSA in combination with ATRA. A partial G0-G1 arrest and increased apoptosis were observed with SK-RC-39 cells on treatment with ATRA and TSA., Conclusions: The combination of ATRA and the histone deacetylase inhibitor TSA elicits an additive inhibition of cell proliferation in RCC cell lines. These results indicate that ATRA and histone deacetylase inhibitor therapies should be explored for the treatment of advanced RCC.

Purpose: Retinoids, which include vitamin A (retinol; ROL) and its derivatives, have been investigated in the treatment of bladder cancer. We have shown that expression of the enzyme lecithin:ROL acyltransferase (LRAT), which converts ROL to retinyl esters, is reduced in several human cancers. Here we evaluated expression of LRAT protein and mRNA in normal and malignant bladder tissue specimens from human patients. We also examined the effect of retinoids on LRAT expression in bladder cancer cell lines., Experimental Design: We evaluated 49 bladder cancer specimens for LRAT protein expression using immunohistochemistry with affinity-purified antibodies to human LRAT. LRAT mRNA expression was assessed using reverse transcription-PCR in bladder specimens from an additional 16 patients. We examined the effect of retinoic acid and ROL on LRAT mRNA expression in five human bladder cancer cell lines., Results: LRAT protein was detected throughout the nonneoplastic bladder epithelium in all of the specimens. In bladder tumors, LRAT protein expression was reduced compared with the nonneoplastic epithelium or was completely absent in 7 of 32 (21.9%) superficial tumors versus 16 of 17 (94.1%) invasive tumors (P < 0.001). All of the non-neoplastic bladder specimens tested (11 of 11) showed LRAT mRNA expression, compared with 5 of 8 (62%) superficial tumors and 0 of 5 (0%) invasive tumors (P = 0.001). Three of five human bladder cancer cell lines expressed LRAT mRNA independent of retinoid exposure, whereas in two cell lines LRAT mRNA expression was induced by retinoid treatment., Conclusions: We report a significant reduction in LRAT expression in bladder cancer. Moreover, we demonstrate an inverse correlation of LRAT mRNA and protein expression with increasing tumor stage. These data suggest that loss of LRAT expression is associated with invasive bladder cancer.

Purpose: Retinoids, a group of compounds, including vitamin A (retinol), and related metabolites, have been shown to regulate the growth and differentiation of many types of cells. IFN-alpha and either 13-cis-retinoic acid or liposomal all-trans retinoic acid have been used in the treatment of patients with metastatic renal cell carcinoma. We knew that samples from renal cell carcinomas contained greatly reduced levels of retinol and retinyl esters relative to samples from normal human kidney. This prompted us to examine the levels of LRAT (lecithin:retinol acyltransferase) protein in various subtypes of human kidney cancers relative to normal human kidney by immunohistochemistry., Experimental Design: We examined 31 partial or radical nephrectomy specimens diagnosed with kidney tumors between 1997 and 1998. Representative paraffin-embedded tissue blocks from each tumor, with each containing adjacent nonneoplastic renal parenchyma, were used for immunohistochemical analysis with affinity purified antibodies to human LRAT protein., Results: LRAT protein was detected at high levels in the epithelial cells in the tubules and the lining of Bowman's capsule in the glomeruli of normal, nonneoplastic kidney sections. Among the 31 tumors, there were 13 cases of conventional (clear cell) renal cell carcinoma (RCC; including 2 multilocular cystic RCCs), 7 papillary RCC, 6 chromophobe RCC, 1 RCC, unclassified, and 4 renal oncocytoma. All tumors showed diffuse immunoreactivity for LRAT. In each case, the staining was uniform throughout the tumor, with only minimal variation in the staining intensity between different areas. All 4 renal oncocytomas, 2 of 6 chromophobe RCCs, 1 conventional (clear cell) carcinoma, 1 RCC, unclassified, and 2 conventional RCCs, which were of the multilocular cystic-type stained strongly (3+) for LRAT. In contrast, the remaining conventional RCCs and the papillary RCC samples stained much less intensely for LRAT. Of the 10 tumors that stained 3+ for LRAT in the study, 9 were either benign tumors or tumors with low malignant potential., Conclusions: These data show that LRAT expression is higher in renal tumors with an indolent biological behavior. Additional studies will ascertain if LRAT possesses any prognostic or therapeutic role in renal cancer.

Context: Only limited information exists on the pathologic aspects of thyroid carcinomas with bone metastases, most large studies having concentrated mainly on their clinical features., Objective: To study in detail the morphologic features of thyroid carcinomas with skeletal metastases., Design: Seventy-nine cases of thyroid carcinoma with bone metastases treated at Memorial Sloan-Kettering Cancer Center, New York, NY, between 1964 and 1998 were investigated, with emphasis on the pathology of the primary and/or metastatic tumors and comparison of the morphologic features of the tumors at both the sites, wherever possible. The tumors were also compared for various clinical parameters., Results: The cohort consisted of 22 papillary, 17 follicular, 16 insular, 10 anaplastic, 9 Hürthle cell, and 5 medullary carcinomas. Of these cases, 68% had poorly differentiated or undifferentiated features in the primary and/or metastatic tumors. The metastatic tumors were better differentiated than the primary in one third of the cases (6 of 18). Only one case showed a less differentiated metastasis. The overall 5- and 10-year survival probabilities after the bone metastases were 29% and 13%, respectively (Kaplan-Meier method). Although both the tumor type and differentiation seemed to affect survivals after bone metastasis (P =.007 and.012, respectively) (log-rank test), this was primarily due to the much worse prognosis in the cases of anaplastic and medullary carcinoma. Cases of Hürthle cell carcinoma showed the longest median survival. There was no significant difference in survival among patients up to or older than 45 years at the time of metastases (P =.31)., Conclusions: Most thyroid carcinomas with bone metastases are of papillary type, and most have poorly differentiated or undifferentiated features. The influence of the microscopic tumor type and tumor differentiation on survival after bone metastasis primarily appears to be due to the much worse prognosis among anaplastic and medullary carcinomas. Age at diagnosis of bone metastases does not influence survivals.

Poorly differentiated thyroid cancer lesions often lose the ability to concentrate radioactive [131I]iodine (RAI) and exhibit increased metabolic activity, as evidenced by enhanced glucose uptake. We incorporated [18F]fluorodeoxyglucose (FDG) positron emission tomography (PET) scanning into the routine follow-up of a cohort of thyroid cancer patients undergoing annual evaluations. One hundred and twenty-five patients who had previous thyroidectomies were included. They had diagnostic RAI whole body scans, serum thyroglobulin measurements, and additional imaging studies as clinically indicated. During 41 months of follow-up, 14 patients died. Univariate analysis demonstrated that survival was reduced in those with age over 45 yr, distant metastases, PET positivity, high rates of FDG uptake, and high volume of the FDG-avid disease (>125 mL). Survival did not correlate with gender, RAI uptake, initial histology, or grade. Multivariate analysis demonstrated that the single strongest predictor of survival was the volume of FDG-avid disease. The 3-yr survival probability of patients with FDG volumes of 125 mL or less was 0.96 (95% confidence interval, 0.91, 1.0) compared with 0.18 (95% confidence interval, 0.04, 0.85) in patients with FDG volume greater than 125 mL. Only 1 death (of leukemia) occurred in the PET-negative group (n = 66). Of the 10 patients with distant metastases and negative PET scans, all were alive and well. Patients over 45 yr with distant metastases that concentrate FDG are at the highest risk. Once distant metastases are discovered in patients with differentiated thyroid carcinoma, FDG-PET can identify high and low risk subsets. Subjects with a FDG volume greater than 125 mL have significantly reduced short term survival.

Extra-adrenal myelolipomas are rare; approximately 36 cases have been reported to date. We document a case of myelolipoma presenting as a localized mass in the renal sinus of a 66-year-old man. The chief clinical and radiologic differential diagnostic considerations in this case included a malignant renal tumor arising in the hilum. The patient was being investigated for recurrent urinary tract infections and vague abdominal pains. Histologically, the lesion showed features characteristic of a myelolipoma. There was also marked chronic inflammation in and around the mass. The uneventful follow-up of 62 months is in keeping with the benign nature of this lesion. This report expands the possibilities of the differential diagnoses of renal hilar neoplasms, particularly in view of the increased use of imaging techniques that are bound to detect many incidental lesions in this region.