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Chronic kidney disease and upper tract urothelial carcinomas display a bidirectional relationship. Review of the literature indicates that early diagnosis and correct localization of upper tract urothelial carcinomas in dialysis patients and kidney transplant recipients are important but problematic. Urine cytology and cystoscopy have limited sensitivity for the diagnosis of upper tract urothelial carcinomas in dialysis patients. Enhanced computed tomography and magnetic resonance imaging could prove useful for the detection and staging of upper tract urothelial carcinomas in dialysis patients. Renal ultrasound can detect hydronephrosis caused by upper tract urothelial carcinomas in kidney transplant recipients but cannot visualize the carcinomas themselves. High detection rates for upper tract urothelial carcinomas in kidney transplant recipients have recently been demonstrated using computed tomography urography, which appears to be a promising tool. To detect carcinomas in dialysis patients and kidney transplant recipients as early as possible, regular screening in asymptomatic patients and diagnostic work-up in symptomatic patients should be performed using a combination of urological and imaging methods. Careful assessment of subsequent recurrence within the contralateral upper urinary tract and the urinary bladder is necessary for dialysis patients and kidney transplant recipients with upper tract urothelial carcinomas.

The impact of different carcinogenic exposures on the specific patterns of somatic mutation in human tumors remains unclear. To address this issue, we profiled 209 cholangiocarcinomas (CCAs) from Asia and Europe, including 108 cases caused by infection with the liver fluke Opisthorchis viverrini and 101 cases caused by non-O. viverrini-related etiologies. Whole-exome sequencing (n = 15) and prevalence screening (n = 194) identified recurrent somatic mutations in BAP1 and ARID1A, neither of which, to our knowledge, has previously been reported to be mutated in CCA. Comparisons between intrahepatic O. viverrini-related and non-O. viverrini-related CCAs demonstrated statistically significant differences in mutation patterns: BAP1, IDH1 and IDH2 were more frequently mutated in non-O. viverrini CCAs, whereas TP53 mutations showed the reciprocal pattern. Functional studies demonstrated tumor suppressive functions for BAP1 and ARID1A, establishing the role of chromatin modulators in CCA pathogenesis. These findings indicate that different causative etiologies may induce distinct somatic alterations, even within the same tumor type.

Background: Bladder cancer exists as several distinct subtypes, including urothelial carcinoma (UCa), squamous cell carcinoma (SCCa), adenocarcinoma and small cell carcinoma. These entities, despite showing distinct morphology and clinical behavior, arise from the urothelial lining and are often accompanied by similar precursor/in situ findings. The relationship between these subtypes has not been explored in detail., Methods: We compared gene expression analysis of the two most common subtypes of bladder cancer, UCa (n = 10) and SCCa (n = 9), with an additional comparison to normal urothelium from non-cancer patients (n = 8) using Affymetrix GeneChip Human genome arrays (Affymetrix, Santa Clara, CA). The results were stratified by supervised and unsupervised clustering analysis, as well as by overall fold change in gene expression., Results: When compared to normal urothelium, UCa showed differential expression of 155 genes using a 5-fold cut-off. Examples of differentially regulated genes included topoisomerases, cancer-related transcription factors and cell cycle mediators. A second comparison of normal urothelium to SCCa showed differential expression of 503 genes, many of which were related to squamous-specific morphology (desmosomal complex, intermediate filaments present within squamous epithelium, squamous cornifying proteins, and molecules upregulated in squamous carcinomas from other anatomic sites). When compared, 137 genes were commonly dysregulated in both UCa and SCCa as compared to normal urothelium. All dysregulated genes in UCa were shared in common with SCCa, with the exception of only 18 genes. Supervised clustering analysis yielded correct classification of lesions in 26/27 (96%) of cases and unsupervised clustering analysis yielded correct classification in 25/27 (92.6%) of cases., Conclusions: The results from this analysis suggest that bladder SCCa shares a significant number of gene expression changes with conventional UCa, but also demonstrates an additional set of alterations that is unique to this entity that defines the squamous phenotype. The similarity in deregulated gene products suggests that SCCa may be a much more closely related entity at the molecular level to conventional UCa than previously hypothesized.

Platinum-based concurrent chemoradiotherapy is considered a standard treatment approach for locoregionally advanced nasopharyngeal carcinoma. However, only a minority of patients benefit from this treatment regimen compared with radiotherapy alone. Identification of a set of molecular markers predicting sensitivity of platinum-based chemotherapy may contribute to personalized treatment of patients with nasopharyngeal carcinoma for better clinical outcome with less toxicity. Previously, we generated a cisplatin-sensitive nasopharyngeal carcinoma cell line, S16, by clonal selection from CNE-2 cells and found that eIF3a is upregulated and contributes to cisplatin sensitivity by downregulating the synthesis of nucleotide excision repair proteins. In this study, we conducted a gene expression profiling analysis and found three other genes, asparagine synthetase (ASNS), choriogonadotropin α subunit (CGA), and matrix metalloproteinase 19 (MMP19), that are upregulated in the cisplatin-sensitive S16 cells compared with the CNE-2 cells. However, only ASNS and MMP19, but not CGA, contributes to cisplatin sensitivity by potentiating cisplatin-induced DNA damage and apoptosis. Thus, ASNS and MMP19, along with eIF3a, are the sensitivity factors for cisplatin treatment and may serve as potential candidate molecular markers for predicting cisplatin sensitivity of advanced nasopharyngeal carcinoma., (©2013 AACR.)

As whole-genome sequencing technology rapidly advances, the insights gained from deciphering cancer genomes are shifting the paradigm in the diagnosis and treatment of cancer with the promise of individualized treatment for each patient. Information gained in this way is extensive for certain cancers, but fairly limited in renal cell carcinomas and urothelial carcinoma. Mutations in multiple, potentially druggable genes have been identified in urothelial carcinomas; however, the association between molecular alterations and clinical outcome has not yet been robustly demonstrated. Data in this area are emerging in renal cell carcinoma, leading to the development of targeted agents that have improved overall survival. Unfortunately, these treatments rarely yield complete responses, are not curative, and development of resistance ensues. This Review will focus on the biology of non-hormonally driven urological cancers. We discuss how approaches using whole-genome sequencing can facilitate the discovery of biomarkers of drug sensitivity in both renal cell carcinomas and urothelial carcinomas. For renal cell carcinomas, we will describe how genomic and epigenomic mining has uncovered novel genes and pathways involved in tumorigenesis, tumour classification and mechanisms of resistance in the various subsets of this disease and the potential for exploiting these discoveries in the clinic.

Background & Aims: Almost all gastric cancers are adenocarcinomas, which have considerable heterogeneity among patients. We sought to identify subtypes of gastric adenocarcinomas with particular biological properties and responses to chemotherapy and targeted agents., Methods: We compared gene expression patterns among 248 gastric tumors; using a robust method of unsupervised clustering, consensus hierarchical clustering with iterative feature selection, we identified 3 major subtypes. We developed a classifier for these subtypes and validated it in 70 tumors from a different population. We identified distinct genomic and epigenomic properties of the subtypes. We determined drug sensitivities of the subtypes in primary tumors using clinical survival data, and in cell lines through high-throughput drug screening., Results: We identified 3 subtypes of gastric adenocarcinoma: proliferative, metabolic, and mesenchymal. Tumors of the proliferative subtype had high levels of genomic instability, TP53 mutations, and DNA hypomethylation. Cancer cells of the metabolic subtype were more sensitive to 5-fluorouracil than the other subtypes. Furthermore, in 2 independent groups of patients, those with tumors of the metabolic subtype appeared to have greater benefits with 5-fluorouracil treatment. Tumors of the mesenchymal subtype contain cells with features of cancer stem cells, and cell lines of this subtype are particularly sensitive to phosphatidylinositol 3-kinase-AKT-mTOR inhibitors in vitro., Conclusions: Based on gene expression patterns, we classified gastric cancers into 3 subtypes, and validated these in an independent set of tumors. The subgroups have differences in molecular and genetic features and response to therapy; this information might be used to select specific treatment approaches for patients with gastric cancer., (Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Aristolochic acid (AA), a natural product of Aristolochia plants found in herbal remedies and health supplements, is a group 1 carcinogen that can cause nephrotoxicity and upper urinary tract urothelial cell carcinoma (UTUC). Whole-genome and exome analysis of nine AA-associated UTUCs revealed a strikingly high somatic mutation rate (150 mutations/Mb), exceeding smoking-associated lung cancer (8 mutations/Mb) and ultraviolet radiation-associated melanoma (111 mutations/Mb). The AA-UTUC mutational signature was characterized by A:T to T:A transversions at the sequence motif A[C|T]AGG, located primarily on nontranscribed strands. AA-induced mutations were also significantly enriched at splice sites, suggesting a role for splice-site mutations in UTUC pathogenesis. RNA sequencing of AA-UTUC confirmed a general up-regulation of nonsense-mediated decay machinery components and aberrant splicing events associated with splice-site mutations. We observed a high frequency of somatic mutations in chromatin modifiers, particularly KDM6A, in AA-UTUC, demonstrated the sufficiency of AA to induce renal dysplasia in mice, and reproduced the AA mutational signature in experimentally treated human renal tubular cells. Finally, exploring other malignancies that were not known to be associated with AA, we screened 93 hepatocellular carcinoma genomes/exomes and identified AA-like mutational signatures in 11. Our study highlights an unusual genome-wide AA mutational signature and the potential use of mutation signatures as "molecular fingerprints" for interrogating high-throughput cancer genome data to infer previous carcinogen exposures.

In this multicentre study, we examined 60 cases of Type II enteropathy-associated T-cell lymphoma (EATL) from the Asia-Pacific region by histological review, immunohistochemistry and molecular techniques. Patients were mostly adult males (median age: 58 years, male:female 2.6:1), presenting with abdominal pain (60%), intestinal perforation (40%) and weight loss (28%). None had a history of coeliac disease and the median survival was only 7 months. Histologically, these tumours could be divided into (i) central tumour zone comprising a monotonous population of neoplastic lymphocytes, (ii) peripheral zone featuring stunted villi and morphologically atypical lymphocytes showing epitheliotropism, and (iii) distant mucosa with normal villous architecture and cytologically normal intra-epithelial lymphocytes (IELs). Characterized by extensive nuclear expression of Megakaryocyte-associated tyrosine kinase (MATK) (87%) and usually a CD8(+)CD56(+) (88%) cytotoxic phenotype, there was frequent aberrant expression of CD20 (24%). T-cell receptor (TCR) expression was silent or not evaluable in 40% but of the remainder, there was predominant expression of TCRαβ over TCRγδ (1.6:1). In keeping with the normal ratio of IEL subsets, CD8(+) cases showed predominant CD8αα homodimer expression (77%), regardless of TCR lineage. These tumours constitute a distinct entity from classical EATL, and the pathology may reflect tumour progression from IEL precursors, remnants of which are often seen in the distant mucosa.

The Birt-Hogg-Dube disease occurs as a result of germline mutations in the human Folliculin gene (FLCN), and is characterized by clinical features including fibrofolliculomas, lung cysts and multifocal renal neoplasia. Clinical and genetic evidence suggest that FLCN acts as a tumor suppressor gene. The human cell line UOK257, derived from the renal cell carcinoma of a patient with a germline mutation in the FLCN gene, harbors a truncated version of the FLCN protein. Reconstitution of the wild type FLCN protein into UOK257 cells delays cell cycle progression, due to a slower progression through the late S and G2/M-phases. Similarly, Flcn (-/-) mouse embryonic fibroblasts progress more rapidly through the cell cycle than wild type controls (Flcn (flox/flox)). The reintroduction of tumor-associated FLCN mutants (FLCN ΔF157, FLCN 1-469 or FLCN K508R) fails to delay cell cycle progression in UOK257 cells. Additionally, FLCN phosphorylation (on Serines 62 and 73) fluctuates throughout the cell cycle and peaks during the G2/M phase in cells treated with nocodazole. In keeping with this observation, the reintroduction of a FLCN phosphomimetic mutant into the UOK257 cell line results in faster progression through the cell cycle compared to those expressing the wild type FLCN protein. These findings suggest that the tumor suppression function of FLCN may be linked to its impact on the cell cycle and that FLCN phosphorylation is important for this activity. Additionally, these observations describe a novel in vitro assay for testing the functional significance of FLCN mutations and/or genetic polymorphisms.

MicroRNA, an endogenous noncoding RNA modulating gene expression, is a key molecule that by its dysregulation plays roles in inflammatory-driven carcinogenesis. This study aimed to investigate the role of oncomiR miR-21 and its target, the programmed cell death 4 (PDCD4) in tumor growth and metastasis of the liver fluke Opisthorchis viverrini-associated cholangiocarcinoma (CCA). The expression levels of miR-21 and PDCD4 were analyzed using the TaqMan miRNA expression assay and immunohistochemistry in liver tissues of both O. viverrini plus N-nitrosodimethylamine (NDMA)-treated hamsters and human CCA samples (n=23 cases). The functional assay for miR-21 was performed in CCA cell lines by the anti-miR-21 and pre-miR-21 transfection procedures. The peak of miR-21 levels were reached at 2 (hyperplastic lesions) and 6 (CCA) months of the O. viverrini plus NDMA-induced group and had a reverse response with its target PDCD4 proteins. In human CCA, miR-21 was overexpressed in tumor tissues when compared with nontumor tissues (P=0.0034) and had a negative correlation with PDCD4 protein (P=0.026). It was also found that high expression of miR-21 was significantly correlated with shorter survival (P<0.05) and lymph node metastasis (P=0.037) of CCA patients. Transient transfection of pre-miR-21 reduced the PDCD4 level and resulted in an increase of M213 CCA cell growth and wound-induced migration ability. These results indicated that miR-21 plays a role in the carcinogenesis and metastasis of O. viverrini-associated CCA by suppressing the function of PDCD4. Modulation of aberrantly expressed miR-21 may be a useful strategy to inhibit tumor cell phenotypes or improve response to chemotherapy.

Purpose: Despite more than 70,000 new cases of bladder cancer in the United States annually, patients with advanced disease have a poor prognosis due to limited treatment modalities. We evaluated Aurora kinase A, identified as an upregulated candidate molecule in bladder cancer, as a potential therapeutic target., Experimental Design: Gene expression in human bladder cancer samples was evaluated using RNA microarray and quantitative reverse transcriptase PCR. Effects of the Aurora kinase A inhibitor MLN8237 (Millennium) on cell dynamics in malignant T24 and UM-UC-3 and papilloma-derived RT4 bladder cells were evaluated in vitro and in vivo in a mouse xenograft model., Results: A set of 13 genes involved in the mitotic spindle checkpoint, including Aurora kinases A and B, were upregulated in human urothelial carcinoma compared with normal urothelium. The Aurora kinase A inhibitor MLN8237 induced cell-cycle arrest, aneuploidy, mitotic spindle failure, and apoptosis in the human bladder cancer cell lines T24 and UM-UC-3. MLN8237 also arrested tumor growth when administered orally over 4 weeks in a mouse bladder cancer xenograft model. Finally, in vitro sequential administration of MLN8237 with either paclitaxel or gemcitabine resulted in synergistic cytotoxic effects in T24 cells., Conclusions: Mitotic spindle checkpoint dysfunction is a common characteristic of human urothelial carcinoma and can be exploited with pharmacologic Aurora A inhibition. Given our demonstration of the ability of the Aurora A inhibitor MLN8237 to inhibit growth of bladder cancer in vitro and in vivo, we conclude that Aurora kinase inhibitors warrant further therapeutic investigation in bladder cancer., (©2013 AACR.)

Sustained activation of the stress-regulated transcription factor NRF2 (NFE2L2) is a prominent feature of many types of cancer, implying that mutations driving NRF2 may be important to tumor progression. In hereditary type 2 papillary renal cell carcinoma (PRCC2, also known as hereditary leiomyomatosis and renal cell cancer), NRF2 activation is a direct consequence of the accumulation of intracellular fumarate, a result of fumarate hydratase (FH) inactivation, but it is not clear how NRF2 may be activated in sporadic forms of PRCC2. Here we show that somatic mutations in NRF2, CUL3, and SIRT1 are responsible for driving the NRF2 activation phenotype in sporadic PRCC2. Transcriptome sequencing revealed the expression pattern of mutant alleles of NRF2, CUL3, and SIRT1 and also confirmed NRF2 activation in clinical specimens. Our results show a convergence in somatic mutations in sporadic PRCC2 with FH mutation in hereditary PRCC2., (©2013 AACR.)

Background: The objective of this study was to evaluate the effect of pericyte coverage (PC) of differentiated tumor microvessels on the prognosis of patients with clear cell renal cell carcinoma (CCRCC)., Methods: Samples from 2 cohorts of patients with CCRCC (101 Asian patients and 524 US patients) were prepared using 2 different histologic approaches: routine sectioning versus tissue microarray. Then, the samples were immunohistochemically doubled-stained for a pericyte marker (alpha smooth muscle actin [α-SMA]) and a differentiated vessel marker (cluster of differentiation 34 [CD34]), followed by multispectral image capturing and computerized image analyses to quantify the microvessel density (MVD) and the PC of differentiated vessels. The correlations of PC and the MVD:PC ratio with clinicopathologic characteristics were analyzed., Results: There was an inverse correlation between differentiated MVD and PC. Higher PC correlated with more aggressive clinicopathologic characteristics of CCRCC in both cohorts, including more advanced T-classification, higher pathologic grades, and the occurrence of tumor necrosis. The MVD:PC ratio was an independent favorable prognostic factor for overall and recurrence-free survival in the Asian cohort and for recurrence-free survival in the US cohort. PC also was an independent prognostic factor, with higher PC predicting a poorer outcome. The combination of PC and MVD was better at distinguishing the outcome of patients with CCRCC. PC combined with differentiated MVD or with the MVD:PC ratio provided additional, independent prognostic information to the Leibovich risk model, and that information was used to generate improved risk models., Conclusions: The authors consistently observed that higher PC was correlated with more aggressive clinicopathologic characteristics. PC was an independent unfavorable prognostic factor. The authors concluded that pericytes should be considered for therapeutic targeting., (Copyright © 2012 American Cancer Society.)

Objective: Cisplatin is a widely used gastric cancer (GC) chemotherapy; however, genetic factors regulating GC responses to cisplatin remain obscure. Identifying genes regulating cisplatin resistance could aid clinicians in tailoring treatments, by distinguishing cisplatin sensitive patients from those who might benefit from alternative platinum therapies, and highlight novel targeted strategies for overcoming cisplatin resistance. Here integrated epigenomics is applied to identify genes associated with GC cisplatin resistance., Design: 20 GC cell lines were subjected to gene expression profiling, DNA methylation profiling and drug response assays. The molecular data were integrated to identify genes highly expressed and unmethylated specifically in cisplatin-resistant lines. Candidate genes were functionally tested by several in vitro and in vivo assays. Clinical impact of candidate genes was also assessed in a cohort of 197 GC patients., Results: Epigenomic analysis identified bone morphogenetic protein 4 (BMP4) as an epigenetically regulated gene highly expressed in cisplatin-resistant lines. Functional assays confirmed that BMP4 is necessary and sufficient for the expression of several prooncogenic traits, likely mediated through stimulation of the epithelial-mesenchymal transition. In primary tumours, BMP4 promoter methylation levels were inversely correlated with BMP4 expression, and patients with high BMP4-expressing tumours exhibited significantly worse prognosis. Therapeutically, targeted genetic inhibition of BMP4 caused significant sensitisation of GC cells to cisplatin. Notably, BMP4-expressing GCs also did not exhibit cross resistance to oxaliplatin., Conclusions: BMP4 epigenetic and expression status may represent promising biomarkers for GC cisplatin resistance. Targeting BMP4 may sensitise GC cells to cisplatin. Oxaliplatin, a clinically acceptable cisplatin alternative, may represent a potential therapeutic option for BMP4-positive GCs.

Inherited mutations in the folliculin (FLCN) gene cause the Birt-Hogg-Dubé syndrome of familial hair follicle tumours (fibrofolliculomas), lung cysts and kidney tumours. Though folliculin has features of a tumour suppressor, the precise function of the FLCN gene product is not well characterized. We identified plakophilin-4 (p0071) as a potential novel folliculin interacting protein by yeast two-hybrid analysis. We confirmed the interaction of folliculin with p0071 by co-immunoprecipitation studies and, in view of previous studies linking p0071 to the regulation of rho-signalling, cytokinesis and intercellular junction formation, we investigated the effect of cell folliculin status on p0071-related functions. Folliculin and p0071 partially co-localized at cell junctions and in mitotic cells, at the midbody during cytokinesis. Previously, p0071 has been reported to regulate RhoA signalling during cytokinesis and we found that folliculin deficiency was associated with increased expression and activity of RhoA and evidence of disordered cytokinesis. Treatment of folliculin-deficient cells with a downstream inhibitor of RhoA signalling (the ROCK inhibitor Y-27632) reversed the increased cell migration phenotype observed in folliculin-deficient cells. Deficiency of folliculin and of p0071 resulted in tight junction defects and mislocalization of E-cadherin in mouse inner medullary collecting duct-3 renal tubular cells. These findings suggest that aspects of folliculin tumour suppressor function are linked to interaction with p0071 and the regulation of RhoA signalling.

Aurora kinases have been shown to be involved in the regulation of the cell cycle and are related to tumor progression. This suggests the possibility that they can serve as new anticancer targets for tumor treatment. However, the important roles that Aurora kinases and their signaling pathway play in renal cell carcinoma (RCC) are not fully understood and addressed to date. In this study, we aimed to address these questions. We observed that downregulation of Aurora kinases induced by AurA miRNA, AurB miRNA or VX680 could inhibit proliferation and metastasis, induce G2/M phase arrest in clear cell renal cell carcinoma cells and exert antitumor activity in an SN12C xenograft model. We also show that either silencing of Aurora kinases or treating the cells with VX680 could downregulate the expression of cdc25c and cyclin B/cdc2, upregulate the expression of p-cdc2 (Tyr15) via blocking the activity of ERK. All these changes may contribute to inhibition of proliferation, metastasis and G2/M arrest in ccRCC. In summary, we proved that both Aurora kinases A and B are key elements of tumor growth regulation, and inhibition of Aurora kinases may contribute to blocking ccRCC progression. We conclude that Aurora kinases could be potential therapeutic targets in the management of renal cell carcinoma.

Treatment of renal-cell carcinoma has progressed over the past decade, in terms of surgical and systemic therapy. Current treatment guidelines are based on clinical evidence, but do not take into account resource limitations among different countries. These limitations, which include financial and logistical challenges and lack of skilled health-care professionals, have the greatest effect in low-income countries. This consolidated statement gives treatment recommendations for renal-cell carcinoma that are based on clinical evidence and stratified according to extent of resource availability. The statement was formulated by a panel of urologists, medical oncologists, and clinical oncologists from Asian countries, at a consensus session on kidney cancer that was held as part of the 2012 Asian Oncology Summit in Singapore. Resource levels are defined according to a four-tier system (basic, limited, enhanced, and maximum), and treatment recommendations are specified based on availability of financial, skill, and logistical resources., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Epigenetic alterations are fundamental hallmarks of cancer genomes. We surveyed the landscape of DNA methylation alterations in gastric cancer by analyzing genome-wide CG dinucleotide (CpG) methylation profiles of 240 gastric cancers (203 tumors and 37 cell lines) and 94 matched normal gastric tissues. Cancer-specific epigenetic alterations were observed in 44% of CpGs, comprising both tumor hyper- and hypomethylation. Twenty-five percent of the methylation alterations were significantly associated with changes in tumor gene expression. Whereas most methylation-expression correlations were negative, several positively correlated methylation-expression interactions were also observed, associated with CpG sites exhibiting atypical transcription start site distances and gene body localization. Methylation clustering of the tumors revealed a CpG island methylator phenotype (CIMP) subgroup associated with widespread hypermethylation, young patient age, and adverse patient outcome in a disease stage-independent manner. CIMP cell lines displayed sensitivity to 5-aza-2'-deoxycytidine, a clinically approved demethylating drug. We also identified long-range regions of epigenetic silencing (LRESs) in CIMP tumors. Combined analysis of the methylation, gene expression, and drug treatment data suggests that certain LRESs may silence specific genes within the region, rather than all genes. Finally, we discovered regions of long-range tumor hypomethylation, associated with increased chromosomal instability. Our results provide insights into the epigenetic impact of environmental and biological agents on gastric epithelial cells, which may contribute to cancer.

The pituitary tumor transforming gene (PTTG1) is a recently discovered oncogene implicated in malignant progression of both endocrine and nonendocrine malignancies. Clear cell renal cell carcinoma (ccRCC) is cytogenetically characterized by chromosome 3p deletions that harbor the ccRCC-related von Hippel-Lindau, PBRM1, BAP1, and SETD2 tumor suppressor genes, along with chromosome 5q amplifications where the significance has been unclear. PTTG1 localizes to the chromosome 5q region where amplifications occur in ccRCC. In this study, we report a functional role for PTTG1 in ccRCC tumorigenesis. PTTG1 was amplified in ccRCC, overexpressed in tumor tissue, and associated with high-grade tumor cells and poor patient prognosis. In preclinical models, PTTG1 ablation reduced tumorigenesis and invasion. An analysis of gene expression affected by PTTG1 indicated an association with invasive and metastatic disease. PTTG1-dependent expression of the RhoGEF proto-oncogene ECT2 was observed in a number of ccRCC cell lines. Moreover, ECT2 expression correlated with PTTG1 expression and poor clinical features. Together, our findings reveal features of PTTG1 that are consistent with its identification of an oncogene amplified on chromsome 5q in ccRCC, where it may offer a novel therapeutic target of pathologic significance in this disease., (©2012 AACR.)

The processes by which cells sense and respond to ambient oxygen concentration are fundamental to cell survival and function, and they commonly target gene regulatory events. To date, however, little is known about the link between the microRNA pathway and hypoxia signaling. Here, we show in vitro and in vivo that chronic hypoxia impairs Dicer (DICER1) expression and activity, resulting in global consequences on microRNA biogenesis. We show that von Hippel-Lindau-dependent down-regulation of Dicer is key to the expression and function of hypoxia-inducible factor α (HIF-α) subunits. Specifically, we show that EPAS1/HIF-2α is regulated by the Dicer-dependent microRNA miR-185, which is down-regulated by hypoxia. Full expression of hypoxia-responsive/HIF target genes in chronic hypoxia (e.g. VEGFA, FLT1/VEGFR1, KDR/VEGFR2, BNIP3L, and SLC2A1/GLUT1), the function of which is to regulate various adaptive responses to compromised oxygen availability, is also dependent on hypoxia-mediated down-regulation of Dicer function and changes in post-transcriptional gene regulation. Therefore, functional deficiency of Dicer in chronic hypoxia is relevant to both HIF-α isoforms and hypoxia-responsive/HIF target genes, especially in the vascular endothelium. These findings have relevance to emerging therapies given that we show that the efficacy of RNA interference under chronic hypoxia, but not normal oxygen availability, is Dicer-dependent. Collectively, these findings show that the down-regulation of Dicer under chronic hypoxia is an adaptive mechanism that serves to maintain the cellular hypoxic response through HIF-α- and microRNA-dependent mechanisms, thereby providing an essential mechanistic insight into the oxygen-dependent microRNA regulatory pathway.

Unlabelled: The molecular pathogenesis of natural killer/T-cell lymphoma (NKTCL) is not well understood. We conducted whole-exome sequencing and identified Janus kinase 3 (JAK3) somatic-activating mutations (A572V and A573V) in 2 of 4 patients with NKTCLs. Further validation of the prevalence of JAK3 mutations was determined by Sanger sequencing and high-resolution melt (HRM) analysis in an additional 61 cases. In total, 23 of 65 (35.4%) cases harbored JAK3 mutations. Functional characterization of the JAK3 mutations support its involvement in cytokine-independent JAK/STAT constitutive activation leading to increased cell growth. Moreover, treatment of both JAK3-mutant and wild-type NKTCL cell lines with a novel pan-JAK inhibitor, CP-690550, resulted in dose-dependent reduction of phosphorylated STAT5, reduced cell viability, and increased apoptosis. Hence, targeting the deregulated JAK/STAT pathway could be a promising therapy for patients with NKTCLs., Significance: Gene mutations causing NKTCL have not been fully identified. Through exome sequencing, we identified activating mutations of JAK3 that may play a significant role in the pathogenesis of NKTCLs. Our findings have important implications for the management of patients with NKTCLs.

Nasopharyngeal carcinoma (NPC) has the highest metastatic potential among head and neck cancers. Distant metastasis is the major cause of treatment failure. The role of interleukin-8 (IL-8) in NPC progression remains unknown. Our multivariate survival analyses of 255 patients with NPC revealed that higher IL-8 expression in primary NPC tissue was an independent prognostic factor for overall survival, disease-free survival, and distant metastasis-free survival of the patients. In vitro study revealed that IL-8 was highly expressed in the established high-metastasis NPC clone S18 relative to the low-metastasis cells. Suppression of IL-8 by short-hairpin RNA reduced the expression of IL-8 in S18 cells and subsequently inhibited migration, invasion, and hepatic metastasis of the cells without influencing cellular growth. Overexpression of IL-8 in S26 cells resulted in increased migration, invasion, and metastasis capabilities of the cells without affecting cellular growth. Exogenous IL-8 enhanced the migration and invasion of low-metastasis CNE-2 cells in a dose-dependent manner. An epithelial-mesenchymal transition (EMT) could be induced by IL-8 in various NPC cell lines. The high level of phosphorylated AKT in S18 cells could be suppressed by knocking down IL-8 expression. Further, IL-8-promoted migration and invasion could be abolished by either the application of the phosphoinositide-3-kinase inhibitor LY294002 or the knock down of AKT expression by using small-interfering RNA. In summary, IL-8 serves as an independent prognostic indicator of overall survival, disease-free survival, and metastasis-free survival for patients with NPC. IL-8 promotes NPC metastasis via autocrine and paracrine means, involving activation of AKT signaling and inducing EMT in NPC cells.

Renal cell carcinoma (RCC) is the most common type of renal cancer in adults. RCC is notoriously resistant to current therapies suggesting the need to improve our knowledge and create more effective therapies. The molecular genetic defects that occur in RCC are extensive and complex ranging from single DNA changes, to large chromosomal defects, to signature disruptions in the transcription of hundreds of genes. These changes are often shared within each histological RCC subtype, illustrating their significance to the disease phenotype. This review presents an overview of the genetic abnormalities that occur within the most common subtypes of RCC. We discuss the recent molecular findings that have advanced our understanding of the somatic architecture of renal tumors and their impact on disease therapeutics.

Opisthorchis viverrini-related cholangiocarcinoma (CCA), a fatal bile duct cancer, is a major public health concern in areas endemic for this parasite. We report here whole-exome sequencing of eight O. viverrini-related tumors and matched normal tissue. We identified and validated 206 somatic mutations in 187 genes using Sanger sequencing and selected 15 genes for mutation prevalence screening in an additional 46 individuals with CCA (cases). In addition to the known cancer-related genes TP53 (mutated in 44.4% of cases), KRAS (16.7%) and SMAD4 (16.7%), we identified somatic mutations in 10 newly implicated genes in 14.8-3.7% of cases. These included inactivating mutations in MLL3 (in 14.8% of cases), ROBO2 (9.3%), RNF43 (9.3%) and PEG3 (5.6%), and activating mutations in the GNAS oncogene (9.3%). These genes have functions that can be broadly grouped into three biological classes: (i) deactivation of histone modifiers, (ii) activation of G protein signaling and (iii) loss of genome stability. This study provides insight into the mutational landscape contributing to O. viverrini-related CCA.

Multiple endocrine neoplasia 1 (MEN1) is an autosomal dominant syndrome characterized by a triad of endocrine (parathyroid, enteropancreatic and pituitary) tumors. Familial MEN1 is defined by one first-degree relative having at least one of these 3 main tumors, and is associated with germline mutations in the MEN1 gene on 11q13 in a large proportion of cases. MEN1 patients may also develop non-endocrine tumors, notably thymic carcinoid. These are rare tumors found predominantly in men, and are a major cause of death in MEN1 due to their insidious nature, lack of effective treatment and unpredictable recurrence. Prophylactic thymectomy has been advocated for prevention but continued surveillance for recurrence is necessary. Although genotype-phenotype correlation in MEN1-related thymic carcinoid is inconsistent, there is a high prevalence of truncating mutations in this condition. We describe a father and son with MEN1, associated with thymic carcinoid (father) and the truncating mutation R29X (son), which was not previously reported in MEN1-related thymic carcinoid, and review the literature about thymic carcinoids in MEN1. Our cases illustrate the importance of a high index of suspicion for early diagnosis and lifelong surveillance in MEN1, and the utility of genetic analysis in defining surveillance for MEN1-related thymic carcinoid., (© J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York.)

Gastric cancer is a major cause of global cancer mortality. We surveyed the spectrum of somatic alterations in gastric cancer by sequencing the exomes of 15 gastric adenocarcinomas and their matched normal DNAs. Frequently mutated genes in the adenocarcinomas included TP53 (11/15 tumors), PIK3CA (3/15) and ARID1A (3/15). Cell adhesion was the most enriched biological pathway among the frequently mutated genes. A prevalence screening confirmed mutations in FAT4, a cadherin family gene, in 5% of gastric cancers (6/110) and FAT4 genomic deletions in 4% (3/83) of gastric tumors. Frequent mutations in chromatin remodeling genes (ARID1A, MLL3 and MLL) also occurred in 47% of the gastric cancers. We detected ARID1A mutations in 8% of tumors (9/110), which were associated with concurrent PIK3CA mutations and microsatellite instability. In functional assays, we observed both FAT4 and ARID1A to exert tumor-suppressor activity. Somatic inactivation of FAT4 and ARID1A may thus be key tumorigenic events in a subset of gastric cancers.

Caveolin-1 (CAV1) is an essential structural constituent of caveolae, specialized lipid raft microdomains on the cell membrane involved in endocytosis and signal transduction, which are inexplicably deregulated and are associated with aggressiveness in numerous cancers. Here we identify CAV1 as a direct transcriptional target of oxygen-labile hypoxia-inducible factor 1 and 2 that accentuates the formation of caveolae, leading to increased dimerization of EGF receptor within the confined surface area of caveolae and its subsequent phosphorylation in the absence of ligand. Hypoxia-inducible factor-dependent up-regulation of CAV1 enhanced the oncogenic potential of tumor cells by increasing the cell proliferative, migratory, and invasive capacities. These results support a concept in which a crisis in oxygen availability or a tumor exhibiting hypoxic signature triggers caveolae formation that bypasses the requirement for ligand engagement to initiate receptor activation and the critical downstream adaptive signaling during a period when ligands required to activate these receptors are limited or are not yet available.

Breast phyllodes tumors are rare neoplasms which present challenges for histological classification. Microscopic features are not always predictive of clinical behavior, and scarce data exist on the prognostic role of biological markers. Our study evaluated a series of 145 phyllodes tumors diagnosed at the Department of Pathology, Singapore General Hospital between 2006 and 2009, incorporating 91 (62.8%) benign, 40 (27.6%) borderline, and 14 (9.7%) malignant phyllodes tumors. Antibodies to keratin 15 (KRT15), transcobalamin I (TCN1), and homeobox gene Hox-B13 (HOXB13) were applied to sections cut from tissue microarray blocks. KRT15 and TCN1 positivity was defined when there was reactivity of 1% or more stromal cells, while HOXB13 positivity was defined using a H-score of 100 and above. Positive immunohistochemical expression for KRT15, TCN1, and HOXB13 was seen in 21 (14.5%), 96 (66.2%), and 66 (45.5%) of tumors, respectively. Stromal expression of KRT15, TCN1, and HOXB13 was significantly correlated with tumor grade (P < 0.001, P < 0.001, P = 0.012), stromal hypercellularity (P = 0.005, P < 0.001, P = 0.023), mitotic activity (P < 0.001), and microscopic borders (P = 0.006, P < 0.001, P = 0.011). Co-expression of TCN1 and HOXB13 was seen in 21 of 91 (23.1%) benign, 18 of 40 (45.0%) borderline, and 11 of 14 (78.6%) malignant tumors, suggesting that the dual-marker panels of TCN1 and HOXB13 might be helpful in classifying borderline and malignant tumors. Although expression of TCN1 alone was present in all malignant and 34 of 40 (85.0%) borderline tumors, a combined panel with HOXB13 excluded some benign cases and was a better discriminant for a significant proportion of borderline and malignant tumors.

Chronic lymphocytic leukemia (CLL) cells express the receptor for Epstein-Barr virus (EBV) and can be infected in vitro. Infected cells do not express the growth-promoting set of EBV-encoded genes and therefore they do not yield LCLs, in most experiments. With exceptional clones, lines were obtained however. We describe a new line, HG3, established by in vitro EBV-infection from an IGHV1-2 unmutated CLL patient clone. All cells expressed EBNA-2 and LMP-1, the EBV-encoded genes pivotal for transformation. The karyotype, FISH cytogenetics and SNP-array profile of the line and the patient's ex vivo clone showed biallelic 13q14 deletions with genomic loss of DLEU7, miR15a/miR16-1, the two micro-RNAs that are deleted in 50% of CLL cases. Further features of CLL cells were: expression of CD5/CD20/CD27/CD43 and release of IgM natural antibodies reacting with oxLDL-like epitopes on apoptotic cells (cf. stereotyped subset-1). Comparison with two LCLs established from normal B cells showed 32 genes expressed at higher levels (> 2-fold). Among these were LHX2 and LILRA. These genes may play a role in the development of the disease. LHX2 expression was shown in self-renewing multipotent hematopoietic stem cells, and LILRA4 codes for a receptor for bone marrow stromal cell antigen-2 that contributes to B cell development. Twenty-four genes were expressed at lower levels, among these PARD3 that is essential for asymmetric cell division. These genes may contribute to establish precursors of CLL clones by regulation of cellular phenotype in the hematopoietic compartment. Expression of CD5/CD20/CD27/CD43 and spontaneous production of natural antibodies may identify the CLL cell as a self-renewing B1 lymphocyte.

Introduction: The extent of lymph nodes (LNs) metastasis is a major determinant for the staging and the most reliable adverse prognostic factor. Primary tumours can induce lymphatics and vasculature reorganisations within sentinel LN before the arrival of cancer cells and these key blood vessels are identified as high endothelial venules (HEV). The alterations of HEV in the presence of cancer, coupled with the increased proliferation rate of the endothelial cells, results in a functional shift of HEV from immune response mediator to blood flow carrier. We aim to evaluate tumour-induced vascularisation in regional LN of cancer patients by studying the morphological and functional alterations of HEV and its correlation to clinico-pathological features., Materials and Methods: This multi-centre study with a prospective database identified 65 consecutive patients with tongue squamous cell carcinoma (SCC) who underwent primary surgical treatment from 2001 to 2005. Immunohistochemical staining for HEV and image analysis were performed and analysed with correlation to the patients' clinico-pathological features., Results: The total number of HEV is significantly associated to disease-free interval when controlling for the group (P = 0.022) as well as combining both groups as one cohort (P = 0.023). There is also a similar association comparing the HEV parameters to overall survival., Conclusion: Our results suggest that HEV possibly plays a key role in the pathogenesis of lymphatic and subsequent distant metastases and may provide the missing link in cancer metastasis. Confirmation of this hypothesis would offer a novel therapeutic approach to preventing metastasis by blocking the remodeling processes of HEV in LN.

Translational control at the initiation step has been recognized as a major and important regulatory mechanism of gene expression. Eukaryotic initiation factor-3a (eIF3a), a putative subunit of the eIF3 complex, has recently been shown to have an important role in regulating the translation of a subset of mRNAs and is found to correlate with the prognosis of cancers. In this study, using nasopharyngeal carcinoma (NPC) cells as a model system, we tested the hypothesis that eIF3a negatively regulates the synthesis of nucleotide excision repair (NER) proteins, and, in turn, cellular response to treatments with DNA-damaging agents such as cisplatin (cis-dichlorodiammine platinum(II) (CDDP)). We found that a CDDP-sensitive sub-clone S16 isolated through limited dilution from an NPC cell line CNE-2 has increased eIF3a expression. Knocking down its expression in S16 cells increased cellular resistance to CDDP, NER activity and synthesis of the NER proteins XPA, XPC, RAD23B and RPA32. Altering eIF3a expression also changed the cellular response to CDDP and UV treatment in other NPC cell lines. Taken together, we conclude that eIF3a has an important role in the CDDP response and in NER activity of NPCs by suppressing the synthesis of NER proteins.

So far, no common environmental and/or phenotypic factor has been associated with melanoma and renal cell carcinoma (RCC). The known risk factors for melanoma include sun exposure, pigmentation and nevus phenotypes; risk factors associated with RCC include smoking, obesity and hypertension. A recent study of coexisting melanoma and RCC in the same patients supports a genetic predisposition underlying the association between these two cancers. The microphthalmia-associated transcription factor (MITF) has been proposed to act as a melanoma oncogene; it also stimulates the transcription of hypoxia inducible factor (HIF1A), the pathway of which is targeted by kidney cancer susceptibility genes. We therefore proposed that MITF might have a role in conferring a genetic predisposition to co-occurring melanoma and RCC. Here we identify a germline missense substitution in MITF (Mi-E318K) that occurred at a significantly higher frequency in genetically enriched patients affected with melanoma, RCC or both cancers, when compared with controls. Overall, Mi-E318K carriers had a higher than fivefold increased risk of developing melanoma, RCC or both cancers. Codon 318 is located in a small-ubiquitin-like modifier (SUMO) consensus site (ΨKXE) and Mi-E318K severely impaired SUMOylation of MITF. Mi-E318K enhanced MITF protein binding to the HIF1A promoter and increased its transcriptional activity compared to wild-type MITF. Further, we observed a global increase in Mi-E318K-occupied loci. In an RCC cell line, gene expression profiling identified a Mi-E318K signature related to cell growth, proliferation and inflammation. Lastly, the mutant protein enhanced melanocytic and renal cell clonogenicity, migration and invasion, consistent with a gain-of-function role in tumorigenesis. Our data provide insights into the link between SUMOylation, transcription and cancer.

Fumarate hydratase (FH) mutation causes hereditary type 2 papillary renal cell carcinoma (PRCC2). The main effect of FH mutation is fumarate accumulation. The current paradigm posits that the main consequence of fumarate accumulation is HIF-α stabilization. Paradoxically, FH mutation differs from other HIF-α stabilizing mutations, such as VHL and SDH mutations, in its associated tumor types. We identified that fumarate can directly up-regulate antioxidant response element (ARE)-controlled genes. We demonstrated that aldo-keto reductase family 1 member B10 (AKR1B10) is an ARE-controlled gene and is up-regulated upon FH knockdown as well as in FH null cell lines. AKR1B10 overexpression is also a prominent feature in both hereditary and sporadic PRCC2. This phenotype better explains the similarities between hereditary and sporadic PRCC2., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Well differentiated papillary mesothelioma of the peritoneum (WDPMP) is a rare variant of epithelial mesothelioma of low malignancy potential, usually found in women with no history of asbestos exposure. In this study, we perform the first exome sequencing of WDPMP., Results: WDPMP exome sequencing reveals the first somatic mutation of E2F1, R166H, to be identified in human cancer. The location is in the evolutionarily conserved DNA binding domain and computationally predicted to be mutated in the critical contact point between E2F1 and its DNA target. We show that the R166H mutation abrogates E2F1's DNA binding ability and is associated with reduced activation of E2F1 downstream target genes. Mutant E2F1 proteins are also observed in higher quantities when compared with wild-type E2F1 protein levels and the mutant protein's resistance to degradation was found to be the cause of its accumulation within mutant over-expressing cells. Cells over-expressing wild-type E2F1 show decreased proliferation compared to mutant over-expressing cells, but cell proliferation rates of mutant over-expressing cells were comparable to cells over-expressing the empty vector., Conclusions: The R166H mutation in E2F1 is shown to have a deleterious effect on its DNA binding ability as well as increasing its stability and subsequent accumulation in R166H mutant cells. Based on the results, two compatible theories can be formed: R166H mutation appears to allow for protein over-expression while minimizing the apoptotic consequence and the R166H mutation may behave similarly to SV40 large T antigen, inhibiting tumor suppressive functions of retinoblastoma protein 1.

Phyllodes tumors of the breast are rare fibroepithelial neoplasms with a potential for recurrence. Current histological classification is not always predictive of clinical behavior. The aim of this study was to identify genetic changes associated with the development of borderline and malignant phyllodes tumors in an Asian population, and to assess if genetic data supported the categorization of these tumors into the existing three grades of benign, borderline, and malignant. Expression profiling of 21 phyllodes tumors (6 benign, 10 borderline, 5 malignant) was performed using Affymetrix U133Plus 2.0 GeneChips(®). Gene expression among benign, borderline, and malignant tumors was compared and a 29 gene list was able to classify them according to their histologic grade. Among these 29 genes are those responsible for matrix formation, cell adhesion, epidermis formation, and cell proliferation. Comparative genomic microarray analysis showed that the most common chromosomal alteration associated with borderline and malignant tumors was 1q gain, and an increasing number of chromosomal changes was noted with increasing histological grade. Upregulation of HOXB13 was seen in malignant relative to borderline phyllodes tumors and further investigated by immunohistochemistry in a corresponding set of formalin-fixed, paraffin-embedded tumors. HOXB13 protein overexpression was found to be correlated with stromal hypercellularity and atypia (P = 0.03, P = 0.039, respectively) and may be implicated in the development of malignant phyllodes tumors.

In recent years, several molecularly targeted therapies have been approved for clear cell renal cell carcinoma (ccRCC), a highly aggressive cancer. Although these therapies significantly extend overall survival, nearly all patients with advanced ccRCC eventually succumb to the disease. To identify other molecular targets, we profiled gene expression in 90 ccRCC patient specimens for which tumor grade information was available. Gene set enrichment analysis indicated that cell-cycle-related genes, in particular, Polo-like kinase 1 (PLK1), were associated with disease aggressiveness. We also carried out RNAi screening to identify kinases and phosphatases that when inhibited could prevent cell proliferation. As expected, RNAi-mediated knockdown of PLK1 and other cell-cycle kinases was sufficient to suppress ccRCC cell proliferation. The association of PLK1 in both disease aggression and in vitro growth prompted us to examine the effects of a small-molecule inhibitor of PLK1, BI 2536, in ccRCC cell lines. BI 2536 inhibited the proliferation of ccRCC cell lines at concentrations required to inhibit PLK1 kinase activity, and sustained inhibition of PLK1 by BI 2536 led to dramatic regression of ccRCC xenograft tumors in vivo. Taken together, these findings highlight PLK1 as a rational therapeutic target for ccRCC.

Parathyroid carcinoma is associated with mutations in the HRPT2/CDC73 gene and with decreased parafibromin and calcium-sensing receptor (CASR) expression, but in some cases establishing an unequivocal diagnosis remains a challenge. The aim of our study was to evaluate the prognostic value of CASR and parafibromin expression and of HRPT2/CDC73 mutations in patients with an established diagnosis of parathyroid carcinoma. Data on survival and disease-free survival were obtained from hospital records of 23 patients with an established diagnosis of parathyroid carcinoma in whom CASR and parafibromin expression and HRPT2/CDC73 mutation analyses were available from paraffin-embedded pathological specimens. Kaplan-Meier curves were used for survival analysis. Downregulation of CASR expression, global loss of parafibromin staining and a HRPT2/CDC73 mutation were, respectively, found in 7 (30%), 13 (59%) and 4 (17%) patients, and were associated with, respectively, 16-fold, 4-fold and 7-fold increased risk of developing local or distant metastasis. These findings suggest that although downregulation of CASR expression, global loss of parafibromin staining and mutations in the HRPT2/CDC73 gene are tools of proven value to assist in establishing a diagnosis of parathyroid carcinoma, their absence does not exclude it. Notwithstanding, we demonstrate a significant added value of these markers as strong determinants of increased malignant potential and thus as negative prognostic markers in this malignancy.

Nasopharyngeal carcinoma (NPC) is known for its high-metastatic potential. Here we report the identification of the proteoglycan serglycin as a functionally significant regulator of metastasis in this setting. Comparative genomic expression profiling of NPC cell line clones with high- and low-metastatic potential revealed the serglycin gene (SRGN) as one of the most upregulated genes in highly metastatic cells. RNAi-mediated inhibition of serglycin expression blocked serglycin secretion and the invasive motility of highly metastatic cells, reducing metastatic capacity in vivo. Conversely, serglycin overexpression in poorly metastatic cells increased their motile behavior and metastatic capacity in vivo. Growth rate was not influenced by serglycin in either highly or poorly metastatic cells. Secreted but not bacterial recombinant serglycin promoted motile behavior, suggesting a critical role for glycosylation in serglycin activity. Serglycin inhibition was associated with reduced expression of vimentin but not other epithelial-mesenchymal transition proteins. In clinical specimens, serglycin expression was elevated significantly in liver metastases from NPC relative to primary NPC tumors. We evaluated the prognostic value of serglycin by immunohistochemical staining of tissue microarrays from 263 NPC patients followed by multivariate analyses. High serglycin expression in primary NPC was found to be an unfavorable independent indicator of distant metastasis-free and disease-free survival. Our findings establish that glycosylated serglycin regulates NPC metastasis via autocrine and paracrine routes, and that it serves as an independent prognostic indicator of metastasis-free survival and disease-free survival in NPC patients., (©2011 AACR.)

Background: Hereditary leiomyomatosis and renal cell cancer (HLRCC) is an autosomal dominant disorder predisposing humans to cutaneous and uterine leiomyomas; in 20% of affected families, type 2 papillary renal cell cancers (PRCCII) also occur with aggressive course and poor prognosis. HLRCC results from heterozygous germline mutations in the tumour suppressor fumarate hydratase (FH) gene., Methods: As part of the French National Cancer Institute (INCa) 'Inherited predispositions to kidney cancer' network, sequence analysis and a functional study of FH were preformed in 56 families with clinically proven or suspected HLRCC and in 23 patients with isolated PRCCII (5 familial and 18 sporadic)., Results: The study identified 32 different germline FH mutations (15 missense, 6 frameshifts, 4 nonsense, 1 deletion/insertion, 5 splice site, and 1 complete deletion) in 40/56 (71.4%) families with proven or suspected HLRCC and in 4/23 (17.4%) probands with PRCCII alone, including 2 sporadic cases. 21 of these were novel and all were demonstrated as deleterious by significant reduction of FH enzymatic activity. In addition, 5 asymptomatic parents in 3 families were confirmed as carrying disease-causing mutations., Conclusions: This study identified and characterised 21 novel FH mutations and demonstrated that PRCCII can be the only one manifestation of HLRCC. Due to the incomplete penetrance of HLRCC, the authors propose to extend the FH mutation analysis to every patient with PRCCII occurring before 40 years of age or when renal tumour harbours characteristic histologic features, in order to discover previously ignored HLRCC affected families.

Next generation sequencing technology has revolutionized the study of cancers. Through matched normal-tumor pairs, it is now possible to identify genome-wide germline and somatic mutations. The generation and analysis of the data requires rigorous quality checks and filtering, and the current analytical pipeline is constantly undergoing improvements. We noted however that in analyzing matched pairs, there is an implicit assumption that the sequenced data are matched, without any quality check such as those implemented in association studies. There are serious implications in this assumption as identification of germline and rare somatic variants depend on the normal sample being the matched pair. Using a genetics concept on measuring relatedness between individuals, we demonstrate that the matchedness of tumor pairs can be quantified and should be included as part of a quality protocol in analysis of sequenced data. Despite the mutation changes in cancer samples, matched tumor-normal pairs are still relatively similar in sequence compared to non-matched pairs. We demonstrate that the approach can be used to assess the mutation landscape between individuals.

Objective: Urothelial carcinoma (UC) of the kidney is a relatively rare but aggressive form of kidney cancer. Differential diagnosis of renal UC from renal cell carcinoma (RCC) can be difficult, but is critical for correct patient management. We aimed to use global gene expression profiling to identify genes specifically expressed in urothelial carcinoma (UC) of the kidney, with purpose of finding new biomarkers for differential diagnosis of UC of both upper and lower tract from normal tissues., Materials and Methods: Microarray gene expression profiling was performed on a variety of human kidney tumor samples, including clear cell, papillary, chromophobe, oncocytoma, renal UC and normal kidney controls. Differentially expressed mRNAs in various kidney tumor subtypes were thus identified. Protein expression in human UC tumor samples from both upper and lower urinary tract was evaluated by immunohistochemistry., Results: FXYD3 (MAT-8) mRNA was specifically expressed in UC of the kidney and not in normal kidney tissue or in any RCC tumor subtypes. FXYD3 mRNA levels displayed equal or better prediction rate for the detection of renal UC than the mRNA levels of selected known UC markers as p63, vimentin, S100P, KRT20 and KRT7. Finally, immunohistochemical staining of clinical UC samples showed that FXYD3 protein is overexpressed in majority of UC of the upper genitourinary tract (encompassing the kidney, ∼90%) and in majority of high grade bladder UC (∼84%, it's <40% in low grade tumors, P < 0.001) compared to normal kidney and bladder tissues., Conclusion: FXYD3 may be a promising novel biomarker for the differential diagnosis of renal UC and a promising prognosis marker of UC from bladder. Because it was identified genome-widely, FXYD3 may have important biological ramifications for the genetic study of UC.

Molecular pathways associated with pathogenesis of sporadic papillary renal cell carcinoma (PRCC), the second most common form of kidney cancer, are poorly understood. We analyzed primary tumor specimens from 35 PRCC patients treated by nephrectomy via gene expression analysis and tissue microarrays constructed from an additional 57 paraffin-embedded PRCC samples via immunohistochemistry. Gene products were validated and further studied by Western blot analyses using primary PRCC tumor samples and established renal cell carcinoma cell lines, and potential associations with pathologic variables and survival in 27 patients with follow-up information were determined. We show that the expression of E2-EPF ubiquitin carrier protein, which targets the principal negative regulator of hypoxia-inducible factor (HIF), von Hippel-Lindau protein, for proteasome-dependent degradation, is markedly elevated in the majority of PRCC tumors exhibiting increased HIF1α expression, and is associated with poor prognosis. In addition, we identified multiple hypoxia-responsive elements within the E2-EPF promoter, and for the first time we demonstrated that E2-EPF is a hypoxia-inducible gene directly regulated via HIF1. These findings reveal deregulation of the oxygen-sensing pathway impinging on the positive feedback mechanism of HIF1-mediated regulation of E2-EPF in PRCC., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The genetics of renal cancer is dominated by inactivation of the VHL tumour suppressor gene in clear cell carcinoma (ccRCC), the commonest histological subtype. A recent large-scale screen of ∼3,500 genes by PCR-based exon re-sequencing identified several new cancer genes in ccRCC including UTX (also known as KDM6A), JARID1C (also known as KDM5C) and SETD2 (ref. 2). These genes encode enzymes that demethylate (UTX, JARID1C) or methylate (SETD2) key lysine residues of histone H3. Modification of the methylation state of these lysine residues of histone H3 regulates chromatin structure and is implicated in transcriptional control. However, together these mutations are present in fewer than 15% of ccRCC, suggesting the existence of additional, currently unidentified cancer genes. Here, we have sequenced the protein coding exome in a series of primary ccRCC and report the identification of the SWI/SNF chromatin remodelling complex gene PBRM1 (ref. 4) as a second major ccRCC cancer gene, with truncating mutations in 41% (92/227) of cases. These data further elucidate the somatic genetic architecture of ccRCC and emphasize the marked contribution of aberrant chromatin biology.

The receptor tyrosine kinase MET is frequently amplified in human tumors, resulting in high cell surface densities and constitutive activation even in the absence of growth factor stimulation by its endogenous ligand, hepatocyte growth factor (HGF). We sought to identify mechanisms of signaling crosstalk that promote MET activation by searching for kinases that are coordinately dysregulated with wild-type MET in human tumors. Our bioinformatic analysis identified leucine-rich repeat kinase-2 (LRRK2), which is amplified and overexpressed in papillary renal and thyroid carcinomas. Down-regulation of LRRK2 in cultured tumor cells compromises MET activation and selectively reduces downstream MET signaling to mTOR and STAT3. Loss of these critical mitogenic pathways induces cell cycle arrest and cell death due to loss of ATP production, indicating that MET and LRRK2 cooperate to promote efficient tumor cell growth and survival in these cancers.

Genetic alterations in kinases have been linked to multiple human pathologies. To explore the landscape of kinase genetic variation in gastric cancer (GC), we used targeted, paired-end deep sequencing to analyze 532 protein and phosphoinositide kinases in 14 GC cell lines. We identified 10,604 single-nucleotide variants (SNV) in kinase exons including greater than 300 novel nonsynonymous SNVs. Family-wise analysis of the nonsynonymous SNVs revealed a significant enrichment in mitogen-activated protein kinase (MAPK)-related genes (P < 0.01), suggesting a preferential involvement of this kinase family in GC. A potential antioncogenic role for MAP2K4, a gene exhibiting recurrent alterations in 2 lines, was functionally supported by siRNA knockdown and overexpression studies in wild-type and MAP2K4 variant lines. The deep sequencing data also revealed novel, large-scale structural rearrangement events involving kinases including gene fusions involving CDK12 and the ERBB2 receptor tyrosine kinase in MKN7 cells. Integrating SNVs and copy number alterations, we identified Hs746T as a cell line exhibiting both splice-site mutations and genomic amplification of MET, resulting in MET protein overexpression. When applied to primary GCs, we identified somatic mutations in 8 kinases, 4 of which were recurrently altered in both primary tumors and cell lines (MAP3K6, STK31, FER, and CDKL5). These results demonstrate that how targeted deep sequencing approaches can deliver unprecedented multilevel characterization of a medically and pharmacologically relevant gene family. The catalog of kinome genetic variants assembled here may broaden our knowledge on kinases and provide useful information on genetic alterations in GC., (© 2011 AACR.)

Background: Germline mutations in the folliculin (FLCN) gene are associated with the development of Birt-Hogg-Dubé syndrome (BHDS), a disease characterized by papular skin lesions, a high occurrence of spontaneous pneumothorax, and the development of renal neoplasias. The majority of renal tumors that arise in BHDS-affected individuals are histologically similar to sporadic chromophobe renal cell carcinoma (RCC) and sporadic renal oncocytoma. However, most sporadic tumors lack FLCN mutations and the extent to which the BHDS-derived renal tumors share genetic defects associated with the sporadic tumors has not been well studied., Methods: BHDS individuals were identified symptomatically and FLCN mutations were confirmed by DNA sequencing. Comparative gene expression profiling analyses were carried out on renal tumors isolated from individuals afflicted with BHDS and a panel of sporadic renal tumors of different subtypes using discriminate and clustering approaches. qRT-PCR was used to confirm selected results of the gene expression analyses. We further analyzed differentially expressed genes using gene set enrichment analysis and pathway analysis approaches. Pathway analysis results were confirmed by generation of independent pathway signatures and application to additional datasets., Results: Renal tumors isolated from individuals with BHDS showed distinct gene expression and cytogenetic characteristics from sporadic renal oncocytoma and chromophobe RCC. The most prominent molecular feature of BHDS-derived kidney tumors was high expression of mitochondria-and oxidative phosphorylation (OXPHOS)-associated genes. This mitochondria expression phenotype was associated with deregulation of the PGC-1α-TFAM signaling axis. Loss of FLCN expression across various tumor types is also associated with increased nuclear mitochondrial gene expression., Conclusions: Our results support a genetic distinction between BHDS-associated tumors and other renal neoplasias. In addition, deregulation of the PGC-1α-TFAM signaling axis is most pronounced in renal tumors that harbor FLCN mutations and in tumors from other organs that have relatively low expression of FLCN. These results are consistent with the recently discovered interaction between FLCN and AMPK and support a model in which FLCN is a regulator of mitochondrial function.

Background: Urothelial carcinoma (UC) can arise at any location along the urothelial tract, including the urethra, bladder, ureter, or renal pelvis. Although tumors arising in these various locations have similar morphology, it is unclear whether the gene expression profiles are similar between the upper-tract (ureter and renal pelvis) and lower-tract (bladder and urethra) carcinomas. Because differences may facilitate different screening and treatment modalities, we sought to examine the relationship between urothelial carcinoma of the renal pelvis (rUC) and urothelial carcinoma of the bladder (bUC)., Methods: Fresh tumor tissue was collected from patients with bUC (n = 10) and benign mucosa from the bladder of individuals undergoing resection for non-UC conditions (n = 7). Gene expression profiles from these samples were determined using high-throughput Affymetrix gene expression microarray chips. Bioinformatic approaches were used to compare the gene expression profiles of these samples with those of rUC samples and normal kidney samples that had been described previously., Results: Using unsupervised analytic approaches, rUC and bUC were indistinguishable. Yet when a supervised analytic approach was used, a small number of differentially expressed genes were identified; these differences were most likely limited to a single pathway--the chloride ion binding activity pathway--which was more frequently activated in rUC than in bUC., Conclusions: We found that the gene expression profiles of UCs from the upper and lower tract were extremely similar, suggesting that similar pathogenic mechanisms likely function in the development of these tumors. The differential expression of genes in the identified pathway may represent a new avenue for detection of upper-tract tumors.

Purpose: Sunitinib is an approved treatment for metastatic renal cell carcinoma. We performed a prospective clinical trial to evaluate the safety and clinical response to sunitinib administered before nephrectomy in patients with localized or metastatic clear cell renal cell carcinoma., Materials and Methods: Patients with biopsy proven clear cell renal cell carcinoma were enrolled in the study and treated with 37.5 mg sunitinib malate daily for 3 months before nephrectomy. The primary end point was safety., Results: In an 18-month period 20 patients were enrolled. The most common toxicities were gastrointestinal symptoms and hematological effects. Grade 3 toxicity developed in 6 patients (30%). No surgical complications were attributable to sunitinib treatment. Of the 20 patients 17 (85%) experienced reduced tumor diameter (mean change -11.8%, range -27% to 11%) and cross-sectional area (mean change -27.9%, range -43% to 23%). Enhancement on contrast enhanced computerized tomography decreased in 15 patients (mean HU change -22%, range -74% to 29%). After tumor reduction 8 patients with cT1b disease underwent laparoscopic partial nephrectomy. Surgical parameters, such as blood loss, transfusion rate, operative time and complications, were similar to those in patients who underwent surgery during the study period and were not enrolled in the trial., Conclusions: Preoperative treatment with sunitinib is safe. Sunitinib decreased the size of primary renal cell carcinoma in 17 of 20 patients. Future trials can be considered to evaluate neoadjuvant sunitinib to maximize nephron sparing and decrease the recurrence of high risk, localized renal cell carcinoma., (2010 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)