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1.  TMPRSS2:ERG Gene Fusion Predicts Subsequent Detection of Prostate Cancer in Patients With High-Grade Prostatic Intraepithelial Neoplasia 
Journal of Clinical Oncology  2013;32(3):206-211.
Purpose
High-grade prostatic intraepithelial neoplasia (HGPIN) is considered a precursor lesion of prostate cancer (PCa). The predictive value of ERG gene fusion in HGPIN for PCa was interrogated as a post hoc analysis in the context of a randomized clinical trial.
Patients and Methods
The GTx Protocol G300104 randomly assigned 1,590 men with biopsy-diagnosed HGPIN to receive toremifene or placebo for 3 years or until a diagnosis of PCa was made on prostate biopsy. As part of this phase III clinical trial, a central pathologist evaluated biopsies of patients with isolated HGPIN at baseline and 12, 24, and 36 months of follow-up. ERG immunohistochemistry was performed on biopsies from 461 patients and evaluated for protein overexpression.
Results
ERG expression was detected in 11.1% of patients (51 of 461 patients) with isolated HGPIN. In the first year and during the 3-year clinical trial, 14.7% and 36.9% of 461 patients were diagnosed with PCa, respectively. Patients with ERG expression were more likely to develop PCa, with 27 (53%) of 51 ERG-positive and 143 (35%) of 410 ERG-negative patients experiencing progression to PCa (P = .014, Fisher's exact test). ERG expression was not associated with age, baseline PSA, Gleason score, or tumor volume.
Conclusion
This study underscores the necessity of more stringent follow-up for men with HGPIN that is also positive for ERG overexpression. Clinicians should consider molecular characterization of HGPIN as a means to improve risk stratification.
doi:10.1200/JCO.2013.49.8386
PMCID: PMC3887478  PMID: 24297949
2.  The oestrogen receptor alpha-regulated lncRNA NEAT1 is a critical modulator of prostate cancer 
Nature Communications  2014;5:5383.
The androgen receptor (AR) plays a central role in establishing an oncogenic cascade that drives prostate cancer progression. Some prostate cancers escape androgen dependence and are often associated with an aggressive phenotype. The oestrogen receptor alpha (ERα) is expressed in prostate cancers, independent of AR status. However, the role of ERα remains elusive. Using a combination of chromatin immunoprecipitation (ChIP) and RNA-sequencing data, we identified an ERα-specific non-coding transcriptome signature. Among putatively ERα-regulated intergenic long non-coding RNAs (lncRNAs), we identified nuclear enriched abundant transcript 1 (NEAT1) as the most significantly overexpressed lncRNA in prostate cancer. Analysis of two large clinical cohorts also revealed that NEAT1 expression is associated with prostate cancer progression. Prostate cancer cells expressing high levels of NEAT1 were recalcitrant to androgen or AR antagonists. Finally, we provide evidence that NEAT1 drives oncogenic growth by altering the epigenetic landscape of target gene promoters to favour transcription.
While prostate cancer predominantly exhibits androgen dependence, oestrogen receptor (ER) signalling is also involved. Here, Chakravarty et al. show that ERα regulates the expression of the NEAT1 long non-coding RNA, which in turn promotes tumorigenesis by maintaining an oncogenic programme/cascade.
doi:10.1038/ncomms6383
PMCID: PMC4241506  PMID: 25415230
3.  ERG induces taxane resistance in castration-resistant prostate cancer 
Nature Communications  2014;5:5548.
Taxanes are the only chemotherapies used to treat patients with metastatic castration-resistant prostate cancer (CRPC). Despite the initial efficacy of taxanes in treating CRPC, all patients ultimately fail due to the development of drug resistance. In this study, we show that ERG overexpression in in vitro and in vivo models of CRPC is associated with decreased sensitivity to taxanes. ERG affects several parameters of microtubule dynamics and inhibits effective drug-target engagement of docetaxel or cabazitaxel with tubulin. Finally, analysis of a cohort of 34 men with metastatic CRPC treated with docetaxel chemotherapy reveals that ERG-overexpressing prostate cancers have twice the chance of docetaxel resistance than ERG-negative cancers. Our data suggest that ERG plays a role beyond regulating gene expression and functions outside the nucleus to cooperate with tubulin towards taxane insensitivity. Determining ERG rearrangement status may aid in patient selection for docetaxel or cabazitaxel therapy and/or influence co-targeting approaches.
Metastatic castration-resistant prostate cancer is treated with the microtubule-stabilizing drugs taxanes, but resistance ultimately develops. Here Galletti et al. show that ERG, a transcription factor commonly overexpressed in prostate cancers, confers taxane resistance by binding to soluble tubulin.
doi:10.1038/ncomms6548
PMCID: PMC4244604  PMID: 25420520
4.  Ubiquitylome analysis identifies dysregulation of effector substrates in SPOP-mutant prostate cancer 
Science (New York, N.Y.)  2014;346(6205):85-89.
Cancer genome characterization has revealed driver mutations in genes that govern ubiquitylation; however, the mechanisms by which these alterations promote tumorigenesis remain incompletely characterized. Here, we analyzed changes in the ubiquitin landscape induced by prostate cancer-associated mutations of SPOP, an E3 ubiquitin ligase substrate binding protein. SPOP mutants impaired ubiquitylation of a subset of proteins in a dominant-negative fashion. Of these, DEK and TRIM24 emerged as effector substrates consistently up-regulated by SPOP mutants. We highlight DEK as a SPOP substrate that exhibited decreases in ubiquitylation and proteasomal degradation resulting from heteromeric complexes of wild-type and mutant SPOP protein. DEK stabilization promoted prostate epithelial cell invasion, implicating DEK as an oncogenic effector. More generally, these results provide a framework to decipher tumorigenic mechanisms linked to dysregulated ubiquitylation.
doi:10.1126/science.1250255
PMCID: PMC4257137  PMID: 25278611
5.  Novel MIR143-NOTCH Fusions in Benign and Malignant Glomus Tumors 
Genes, chromosomes & cancer  2013;52(11):1075-1087.
Glomus tumors (GT) have been classified among tumors of perivascular smooth muscle differentiation, together with myopericytoma, myofibroma/tosis, and angioleiomyoma, based on their morphologic overlap. However, no molecular studies have been carried out to date to investigate their genetic phenotype and to confirm their shared pathogenesis. RNA sequencing was performed in three index cases (GT1, malignant GT; GT2, benign GT and M1, multifocal myopericytoma), followed by FusionSeq data analysis, a modular computational tool developed to discover gene fusions from paired-end RNA-seq data. A gene fusion involving MIR143 in band 5q32 was identified in both GTs with either NOTCH2 in 1p13 in GT1 or NOTCH1 in 9q34 in GT2, but none in M1. After being validated by FISH and RT-PCR, these abnormalities were screened on 33 GTs, 6 myopericytomas, 9 myofibroma/toses, 18 angioleiomyomas and in a control group of 5 sino-nasal hemangiopericytomas. Overall NOTCH2 gene rearrangements were identified in 52% of GT, including all malignant cases and one NF1-related GT. No additional cases showed NOTCH1 rearrangement. As NOTCH3 shares similar functions with NOTCH2 in regulating vascular smooth muscle development, the study group was also investigated for abnormalities in this gene by FISH. Indeed, NOTCH3 rearrangements were identified in 9% of GTs, all present in benign soft tissue GT, one case being fused to MIR143. Only 1/18 angioleiomyomas showed NOTCH2 gene rearrangement, while all the myopericytomas and myofibroma/toses were negative. In summary we describe novel NOTCH1-3 rearrangements in benign and malignant, visceral and soft tissue GTs.
doi:10.1002/gcc.22102
PMCID: PMC3889711  PMID: 23999936
NOTCH2; NOTCH3; NOTCH1; miR143; glomus tumor
6.  Antibody-independent targeted quantification of TMPRSS2-ERG fusion protein products in prostate cancer 
Molecular oncology  2014;8(7):1169-1180.
Fusions between the transmembrane protease serine 2 (TMPRSS2) and ETS related gene (ERG) represent one of the most specific biomarkers that define a distinct molecular subtype of prostate cancer. Studies of TMPRSS2-ERG gene fusions have seldom been performed at the protein level, primarily due to the lack of high-quality antibodies suitable for quantitative studies. Herein, we applied a recently developed PRISM (high-pressure high-resolution separations with intelligent selection and multiplexing)-SRM (selected reaction monitoring) strategy for quantifying ERG protein in prostate cancer cell lines and tumors. The highly sensitive PRISM-SRM assays provided confident detection of 6 unique ERG peptides in both TMPRSS2-ERG positive cell lines and tissues, but not in cell lines or tissues lacking the TMPRSS2-ERG rearrangement, clearly indicating that ERG protein expression is significantly increased in the presence of the TMPRSS2-ERG gene fusion. Significantly, our results provide evidence that two distinct ERG protein isoforms are simultaneously expressed in TMPRSS2-ERG positive samples as evidenced by the concomitant detection of two mutually exclusive peptides in two patient tumors and in the VCaP prostate cancer cell line. Three peptides, shared across almost all fusion protein products, were determined to be the most abundant peptides, providing “signature” peptides for detection of ERG over-expression resulting from TMPRSS2-ERG gene fusion. The PRISM-SRM assays provide valuable tools for studying TMPRSS2-ERG gene fusion protein products in prostate cancer.
doi:10.1016/j.molonc.2014.02.004
PMCID: PMC4183720  PMID: 25266362
TMPRSS2-ERG gene fusion; ERG protein isoform; PRISM-SRM; Targeted quantification; Prostate cancer
7.  Common Mutations in BRCA1 and BRCA2 Do Not Contribute to Early Prostate Cancer in Jewish Men 
The Prostate  1999;40(3):172-177.
Background
Families with a high incidence of hereditary breast cancer, and subsequently shown to have terminating mutations in BRCA1 or BRCA2 appear to have a higher incidence of prostate cancer among the male relatives. We aimed to determine whether the common germline mutations of BRCA1 or BRCA2 in Ashkenazi Jewish men predisposed them to prostate cancer.
Methods
We examined genomic DNA from 83 (for BRCA1 185delAG) or 82 (for BRCA2 6174delT) Ashkenazi Jewish prostate cancer patients, most of whom were treated at a relatively young age, for the most common germline mutation in each gene seen in the Ashkenazi population.
Results
Our study should be able to detect a four to five fold increase in the risk of prostate cancer due to mutation of BRCA1 or BRCA2. However, only one (1.15%, 95% CI 0-3.6%) of the patients was heterozygous for the BRCA1 mutant allele, and only two were heterozygous for the BRCA2 mutation (2.4%, 95% CI 0-6.2%).
Conclusions
The incidence of each of the germline mutations in these prostate cancer patients closely matched their incidence (about 1%) in the general Ashkenazi Jewish population. This suggests that unlike the cases of breast and ovarian cancers, mutations in BRCA1 or BRCA2 do not significantly predispose men to prostate cancer.
PMCID: PMC4196372  PMID: 10398279
risk; gene; breast; neoplastic; heterozygote
8.  Unraveling the clonal hierarchy of somatic genomic aberrations 
Genome Biology  2014;15(8):439.
Defining the chronology of molecular alterations may identify milestones in carcinogenesis. To unravel the temporal evolution of aberrations from clinical tumors, we developed CLONET, which upon estimation of tumor admixture and ploidy infers the clonal hierarchy of genomic aberrations. Comparative analysis across 100 sequenced genomes from prostate, melanoma, and lung cancers established diverse evolutionary hierarchies, demonstrating the early disruption of tumor-specific pathways. The analyses highlight the diversity of clonal evolution within and across tumor types that might be informative for risk stratification and patient selection for targeted therapies. CLONET addresses heterogeneous clinical samples seen in the setting of precision medicine.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0439-6) contains supplementary material, which is available to authorized users.
doi:10.1186/s13059-014-0439-6
PMCID: PMC4167267  PMID: 25160065
9.  New Strategies in Prostate Cancer: Translating Genomics into the Clinic 
With the rapidly developing use of Next Generation Sequencing (NGS) technologies, there has been a surge in our knowledge of the genomic landscape of prostate cancer and a movement towards developing a molecular sub-classification system for the disease. With this new understanding comes enormous clinical potential, both for the development of biomarkers and new therapeutic targets. Herein, we highlight the potential clinical utility of recent discoveries and how they fit in to our current paradigm. We describe the challenges that lie ahead as we move genomic sequencing towards routine clinical practice and adopt precision cancer care for prostate cancer patients.
doi:10.1158/1078-0432.CCR-12-1452
PMCID: PMC4123124  PMID: 23248095
11.  Proposed Morphologic Classification of Prostate Cancer With Neuroendocrine Differentiation 
On July 31, 2013, the Prostate Cancer Foundation assembled a working committee on the molecular biology and pathologic classification of neuroendocrine differentiation in prostate cancer. The committee consisted of genitourinary oncologists, urologists, urological surgical pathologists, basic scientists, and translational researchers, with expertise in this field. It was concluded that the proceedings of the meeting should be reported in 2 manuscripts appealing to different target audiences, one to focus on surgical pathology and the other to review the molecular aspects of this disease. New clinical and molecular data emerging from prostate cancers treated by contemporary androgen deprivation therapies, as well as primary lesions, have highlighted the need for refinement of diagnostic terminology to encompass the full spectrum of neuroendocrine differentiation. It is envisioned that specific criteria associated with the refined diagnostic terminology will lead to clinically relevant pathologic diagnoses that will stimulate further clinical and molecular investigation and identification of appropriate targeted therapies.
doi:10.1097/PAS.0000000000000208
PMCID: PMC4112087  PMID: 24705311
small cell carcinoma; Paneth cell like; large cell neuroendocrine carcinoma; carcinoid; prostate adenocarcinonma
12.  Novel YAP1-TFE3 Fusion Defines a Distinct Subset of Epithelioid Hemangioendothelioma 
Genes, chromosomes & cancer  2013;52(8):775-784.
Conventional epithelioid hemangioendotheliomas (EHE) have a distinctive morphologic appearance and are characterized by a recurrent t(1;3) translocation, resulting in a WWTR1-CAMTA1 fusion gene. We have recently encountered a fusion-negative subset characterized by a somewhat different morphology, including focally well-formed vasoformative features, which was further investigated for recurrent genetic abnormalities. Based on a case showing strong TFE3 immunoreactivity, FISH analysis for TFE3 gene rearrangement was applied to the index case as well as to 9 additional cases, selected through negative WWTR1-CAMTA1 screening. A control group, including 18 epithelioid hemangiomas, 9 pseudomyogenic HE and 3 epithelioid angiosarcomas, was also tested. TFE3 gene rearrangement was identified in 10 patients, with equal gender distribution and a mean age of 30 years old. The lesions were located in somatic soft tissue in 6 cases, lung in 3 and one in bone. One case with available frozen tissue was tested by RNA sequencing and FusionSeq data analysis to detect novel fusions. A YAP1-TFE3 fusion was thus detected, which was further validated by FISH and RT-PCR. YAP1 gene rearrangements were then confirmed in 7 of the remaining 9 TFE3-rearranged EHEs by FISH. No TFE3 structural abnormalities were detected in any of the controls. The TFE3-rearranged EHEs showed similar morphologic features with at least focally, well-formed vascular channels, in addition to a variably solid architecture. All tumors expressed endothelial markers, as well as strong nuclear TFE3. In summary we are reporting a novel subset of EHE occurring in young adults, showing a distinct phenotype and YAP1-TFE3 fusions.
doi:10.1002/gcc.22073
PMCID: PMC4089994  PMID: 23737213
TFE3; YAP1; epithelioid hemangioendothelioma; WWTR1
13.  Recurrent Prostate Cancer Genomic Alterations Predict Response to Brachytherapy Treatment 
Background
This study aimed to evaluate the association of recurrent molecular alterations in prostate cancer, such as ERG rearrangements and phosphatase and tensin homolog gene (PTEN) deletions, with oncologic outcomes in patients with prostate cancer treated with brachytherapy.
Methods
Ninety-two men underwent I-125 brachytherapy with a 145 Gy delivered dose between 2000 and 2008. Pretreatment prostate biopsies were analyzed by immunohistochemistry (IHC) and FISH for ERG rearrangement and overexpression, PTEN deletion, and expression loss. Univariable and multivariable Cox-regression analyses evaluated association of ERG and PTEN status with biochemical recurrence (BCR).
Results
Within a median follow-up of 73 months, 11% of patients experienced BCR. Of 80 samples with both IHC and FISH performed for ERG, 46 (57.8%) demonstrated rearrangement by FISH and 45 (56.3%) by IHC. Of 77 samples with both IHC and FISH for PTEN, 14 (18.2%) had PTEN deletion by FISH and 22 (28.6%) by IHC. No significant associations were found between ERG, PTEN status, and clinicopathologic features. Patients with concurrent ERG rearrangement and PTEN deletion demonstrated significantly worse relapse-free survival rates compared with those with ERG or PTEN wild type (P < 0.01). In multivariable Cox regression analysis adjusted for the effects of standard clinicopathologic features, combined ERG rearranged and PTEN deletion was independently associated with BCR (HR = 2.6; P = 0.02).
Conclusions
Concurrent ERG rearrangement and PTEN loss was independently associated with time to BCR in patients undergoing brachytherapy. Future studies are needed to validate prostate cancer molecular subtyping for risk stratification.
Impact
Identifying patients in the ERG-rearranged/PTEN-deleted molecular subclass may improve treatment personalization.
doi:10.1158/1055-9965.EPI-13-1180
PMCID: PMC4083705  PMID: 24515272
14.  Combining Urinary Detection of TMPRSS2:ERG and PCA3 with Serum PSA to Predict Diagnosis of Prostate Cancer 
Urologic oncology  2011;31(5):566-571.
Objectives
We sought to develop a clinical algorithm combining serum PSA with detection of TMPRSS2:ERG fusion and PCA3 in urine collected after digital rectal exam (post-DRE urine) to predict prostate cancer on subsequent biopsy.
Materials and Methods
Post-DRE urine was collected in 48 consecutive patients before prostate biopsy at two centers; qRT-PCR was used to detect PCA3 and TMPRSS2:ERG fusion transcript expression. Serum PSA was measured by clinical assay. The performance of TMPRSS2:ERG fusion, PCA3, and serum PSA as biomarkers predicting prostate cancer at biopsy was measured; a clinically practical algorithm combining serum PSA with TMPRSS2:ERG and PCA3 in post-DRE urine to predict prostate cancer was developed.
Results
Post-DRE urine sediment provided informative RNA in 45 patients; prostate cancer was present on subsequent biopsy in 15. TMPRSS2:ERG in post-DRE urine was associated with prostate cancer (OR = 12.02; p< 0.001). PCA3 had the highest sensitivity in predicting prostate cancer diagnosis (93%), whereas TMPRSS2:ERG had the highest specificity (87%). TMPRSS2:ERG had the greatest discriminatory value in predicting prostate cancer (AUC = 0.77 compared to 0.65 for PCA3 and 0.72 for serum PSA alone). Combining serum PSA, PCA3 and TMPRSS2:ERG in a multivariable algorithm optimized for clinical utility improved cancer prediction (AUC = 0.88; specificity = 90% at 80% sensitivity).
Conclusions
A clinical algorithm specifying biopsy for all patients with PSA ≥10ng/ml, while restricting biopsy among those with PSA <10ng/ml to only those with detectable PCA3 or TMPRSS2:ERG in post-DRE urine, performed better than the individual biomarkers alone in predicting prostate cancer.
doi:10.1016/j.urolonc.2011.04.001
PMCID: PMC3210917  PMID: 21600800
Screening; DRE; Biomarkers; Cancer Detection; Gene Fusion
15.  The Alien Limb Phenomenon 
Journal of neurology  2013;260(7):1880-1888.
Background
Alien limb phenomenon refers to involuntary motor activity of a limb in conjunction with the feeling of estrangement from that limb. Alien limb serves as a diagnostic feature of corticobasal syndrome.
Objective
Our objective was to determine the differential diagnoses of alien limb and to determine the features in a large group of patients with the alien limb with different underlying etiologies.
Methods
We searched the Mayo Clinic Medical Records Linkage system to identify patients with the diagnosis of alien limb seen between January 1, 1996, and July 11, 2011.
Results
One hundred fifty patients with alien limb were identified. Twenty two were followed in the Alzheimer’s Disease Research Center. Etiologies of alien limb included corticobasal syndrome (n=108), stroke (n=14), Creutzfeldt Jacob disease (n=9), Hereditary diffuse leukoencephalopathy with spheroids (n=5), tumor (n=4), progressive multifocal leukoencephalopathy(n=2), demyelinating disease (n=2), progressive dementia not otherwise specified (n=2), posterior reversible encephalopathy syndrome (n=1), corpus callosotomy (n=1), intracerebral hemorrhage (n=1) and thalamic dementia (n=1). Ten of fourteen cerebrovascular cases were right hemisphere in origin. All cases involved the parietal lobe. Of the 44 patients with corticobasal syndrome from the Alzheimer’s Disease Research Center cohort, 22 had alien limb, and 73% had the alien limb affecting the left extremities. Left sided corticobasal syndrome was significantly associated with the presence of alien limb (p=0.004).
Conclusions
These findings support the notion that the alien limb phenomenon is partially related to damage underlying the parietal cortex, especially the right parietal, disconnecting it from other cortical areas.
doi:10.1007/s00415-013-6898-y
PMCID: PMC3914666  PMID: 23572346
Alien limb; corticobasal syndrome
16.  SLC45A3-ELK4 is a Novel and Frequent ETS Fusion Transcript in Prostate Cancer 
Cancer research  2009;69(7):2734-2738.
Chromosomal rearrangements account for all erythroblast transformation specific (ETS) family member gene fusions that have been reported in prostate cancer and have clinical, diagnostic and prognostic implications. Androgen-regulated genes account for the majority of the 5’ genomic regulatory promoter elements fused with ETS genes. TMPRSS2-ERG, TMPRSS2-ETV1 and SLC45A3-ERG rearrangements account for roughly 90% of ETS fusion prostate cancer. ELK4, another ETS family member, is androgen-regulated, involved in promoting cell growth, and highly expressed in a subset of prostate cancer, yet the mechanism of ELK4 over-expression is unknown. In this study, we identified a novel ETS family fusion transcript, SLC45A3-ELK4, and found it to be expressed in both benign prostate tissue and prostate cancer. We found high levels of SLC45A3-ELK4 mRNA restricted to a subset of prostate cancer samples. SLC45A3-ELK4 transcript can be detected at high levels in urine samples from men at risk for prostate cancer. Characterization of the fusion mRNA revealed a major variant in which SLC45A3 exon 1 is fused to ELK4 exon 2. Based on quantitative PCR analyses of DNA, unlike other ETS fusions described in prostate cancer, the expression of SLC45A3-ELK4 mRNA is not exclusive to cases harbouring a chromosomal rearrangement. Treatment of LNCaP cancer cells with a synthetic androgen (R1881) revealed that SLC45A3-ELK4, and not endogenous ELK4, mRNA expression is androgen-regulated. Altogether, our findings show that SLC45A3-ELK4 mRNA expression is heterogeneous, highly induced in a subset of prostate cancers, androgen-regulated, and most commonly occurs through a mechanism other than chromosomal rearrangement (e.g., trans-splicing).
doi:10.1158/0008-5472.CAN-08-4926
PMCID: PMC4063441  PMID: 19293179
prostate cancer; ETS genes; splicing; SLC45A3; ELK4
17.  Recurrent NCOA2 gene rearrangements in congenital/infantile spindle cell rhabdomyosarcoma 
Genes, chromosomes & cancer  2013;52(6):10.1002/gcc.22050.
Spindle cell rhabdomyosarcoma (RMS) is a rare form of RMS with different clinical characteristics and behavior between children and adult patients. Its genetic hallmark remains unknown and it remains debatable if there is pathogenetic relationship between the spindle cell and the so-called sclerosing RMS. We studied two pediatric and one adult spindle cell RMS by next generation RNA sequencing and used FusionSeq for data analysis to detect novel fusions. An SRF-NCOA2 gene fusion was detected in a spindle cell RMS from the posterior neck in a 7 month-old child. The fusion matched the tumor karyotype and was further confirmed by fluorescence in situ hybridization (FISH) and by RT-PCR, which showed fusion of SRF exon 6 to NCOA2 exon 12. Additional 14 spindle cell (from 8 children and 6 adults) and 4 sclerosing (from 2 children and 2 adults) RMS were tested by FISH for the presence of abnormalities in NCOA2, SRF, as well as for PAX3 and NCOA1, identifying NCOA2 rearrangements in two additional spindle cell RMS from a 3 month-old and a 4 week-old child, both arising in the chest wall. In the latter tumor, TEAD1 was identified by rapid amplification of cDNA ends (RACE) to be the NCOA2 gene fusion partner. None of the adult tumors were positive for NCOA2 rearrangement. Despite similar histomorphology in adults and young children, these results suggest that spindle cell RMS is a heterogeneous disease genetically as well as clinically. Our findings also support a relationship between NCOA2-rearranged spindle cell RMS occurring in young childhood and the so-called congenital RMS, which often displays rearrangements at 8q13 locus (NCOA2).
doi:10.1002/gcc.22050
PMCID: PMC3734530  PMID: 23463663
rhabdomyosarcoma; spindle cell; NCOA2; SRF; TEAD1; translocation; infantile
18.  Frequent truncating mutations of STAG2 in bladder cancer 
Nature genetics  2013;45(12):10.1038/ng.2800.
Here we report the discovery of truncating mutations of the gene encoding the cohesin subunit STAG2, which regulates sister chromatid cohesion and segregation, in 36% of papillary non-invasive urothelial carcinomas and 16% of invasive urothelial carcinomas of the bladder. Our studies suggest that STAG2 plays a role in controlling chromosome number but not proliferation of bladder cancer cells. These findings identify STAG2 as among the most commonly mutated genes in bladder cancer discovered to date.
doi:10.1038/ng.2800
PMCID: PMC3875130  PMID: 24121789
19.  Targeted Next-generation Sequencing of Advanced Prostate Cancer Identifies Potential Therapeutic Targets and Disease Heterogeneity 
European urology  2012;63(5):920-926.
Background
Most personalized cancer care strategies involving DNA sequencing are highly reliant on acquiring sufficient fresh or frozen tissue. It has been challenging to comprehensively evaluate the genome of advanced prostate cancer (PCa) because of limited access to metastatic tissue.
Objective
To demonstrate the feasibility of a novel next-generation sequencing (NGS) based platform that can be used with archival formalin-fixed paraffin-embedded (FFPE) biopsy tissue to evaluate the spectrum of DNA alterations seen in advanced PCa.
Design, setting, and participants
FFPE samples (including archival prostatectomies and prostate needle biopsies) were obtained from 45 patients representing the spectrum of disease: localized PCa, metastatic hormone-naive PCa, and metastatic castration-resistant PCa (CRPC). We also assessed paired primaries and metastases to understand disease heterogeneity and disease progression.
Intervention
At least 50 ng of tumor DNA was extracted from FFPE samples and used for hybridization capture and NGS using the Illumina HiSeq 2000 platform.
Outcome measurements and statistical analysis
A total of 3320 exons of 182 cancer-associated genes and 37 introns of 14 commonly rearranged genes were evaluated for genomic alterations.
Results and limitations
We obtained an average sequencing depth of >900X. Overall, 44% of CRPCs harbored genomic alterations involving the androgen receptor gene (AR), including AR copy number gain (24% of CRPCs) or AR point mutation (20% of CRPCs). Other recurrent mutations included transmembrane protease, serine 2 gene (TMPRSS2):v-ets erythroblastosis virus E26 oncogene homolog (avian) gene (ERG) fusion (44%); phosphatase and tensin homolog gene (PTEN) loss (44%); tumor protein p53 gene (TP53) mutation (40%); retinoblastoma gene (RB) loss (28%); v-myc myelocytomatosis viral oncogene homolog (avian) gene (MYC) gain (12%); and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α gene (PIK3CA) mutation (4%). There was a high incidence of genomic alterations involving key genes important for DNA repair, including breast cancer 2, early onset gene (BRCA2) loss (12%) and ataxia telangiectasia mutated gene (ATM) mutations (8%); these alterations are potentially targetable with poly(adenosine diphosphate-ribose)polymerase inhibitors. A novel and actionable rearrangement involving the v-raf murine sarcoma viral oncogene homolog B1 gene (BRAF) was also detected.
Conclusions
This first-in-principle study demonstrates the feasibility of performing in-depth DNA analyses using FFPE tissue and brings new insight toward understanding the genomic landscape within advanced PCa.
doi:10.1016/j.eururo.2012.08.053
PMCID: PMC3615043  PMID: 22981675
Next-generation sequencing; Castration-resistant prostate cancer; Prostate cancer genome
20.  Punctuated Evolution of Prostate Cancer Genomes 
Cell  2013;153(3):666-677.
SUMMARY
The analysis of exonic DNA from prostate cancers has identified recurrently mutated genes, but the spectrum of genome-wide alterations has not been profiled extensively in this disease. We sequenced the genomes of 57 prostate tumors and matched normal tissues to characterize somatic alterations and to study how they accumulate during oncogenesis and progression. By modeling the genesis of genomic rearrangements, we identified abundant DNA translocations and deletions that arise in a highly interdependent manner. This phenomenon, which we term “chromoplexy”, frequently accounts for the dysregulation of prostate cancer genes and appears to disrupt multiple cancer genes coordinately. Our modeling suggests that chromoplexy may induce considerable genomic derangement over relatively few events in prostate cancer and other neoplasms, supporting a model of punctuated cancer evolution. By characterizing the clonal hierarchy of genomic lesions in prostate tumors, we charted a path of oncogenic events along which chromoplexy may drive prostate carcinogenesis.
doi:10.1016/j.cell.2013.03.021
PMCID: PMC3690918  PMID: 23622249
21.  Asymptomatic carotid stenosis: What we can learn from the next generation of randomized clinical trials 
JRSM Cardiovascular Disease  2014;3:2048004014529419.
Stroke remains an exceedingly incident and prevalent public health burden across the globe, with an estimated 16 million new strokes per annum and prevalence over 60 million, and extracranial internal carotid artery atherosclerotic disease is an important risk factor for stroke. Randomized trials of surgical treatment were conducted (North American Symptomatic Carotid Endarterectomy Trial, European Carotid Surgery Trial) and demonstrated efficacy of carotid endarterectomy for secondary prevention of stroke in patients with cerebrovascular events (e.g. ipsilateral stroke, transient ischemic attack, and/or amaurosis fugax) attributable to a diseased artery with 50–99% stenosis. Therapeutic clarity, however, proved elusive with asymptomatic carotid artery disease. Asymptomatic Carotid Atherosclerosis Study (ACAS), Asymptomatic Carotid Surgery Trial, and Veterans Affairs Cooperative Study (VACS) suggested only modest benefit from surgical intervention for primary stroke prevention and the best medical therapy at the time of these trials is not comparable to modern medical therapy. ACT-1, Asymptomatic Carotid Surgery Trial-2, Stent-Protected Angioplasty in asymptomatic Carotid artery stenosis versus Endarterectomy Trial-2, European Carotid Surgery Trial-2, Carotid Revascularization Endarterectomy Versus Stenting Trial-2 are trials that are recent, ongoing, or in development that include diverse populations across Europe and North America, complementary trial designs, and a collaborative spirit that should provide clinicians with evidence that informs best clinical practice for asymptomatic carotid artery disease.
doi:10.1177/2048004014529419
PMCID: PMC4157468  PMID: 25247072
Carotid stenosis; primary and secondary stroke prevention; cardiology; carotid endarterectomy; angioplasty and stenting
22.  Lenalidomide in combination with gemcitabine as first-line treatment for patients with metastatic carcinoma of the pancreas 
Cancer Biology & Therapy  2013;14(4):340-346.
Objectives: To evaluate the 6-mo overall survival, safety and tolerability of lenalidomide in combination with standard gemcitabine as first-line treatment for patients with metastatic pancreatic cancer. Methods: Eligibility included: previously untreated metastatic adenocarcinoma of the pancreas with metastases incurable by surgery/radiation therapy; ECOG PS 0–2; adequate organ function; prophylactic anticoagulation for venous thromboembolic events (VTEs). Patients received lenalidomide 25 mg PO (days 1–21) and gemcitabine 1,000 mg/m2 IV (days 1, 8 and 15) each 28-day cycle, with response evaluations every eight weeks. Results: Between 5/2009–4/2010, 72 patients (median age 64 years; 68% male; 42% ECOG PS 0) were enrolled in this multicenter, community-based study. Six-month OS was 37% (95% CI 26–48%). Median PFS and OS were 2.3 (95% CI 1.9–3.5) and 4.7 (95% CI 3.4–5.7) months, respectively. Eight partial responses (11%) were documented. Thirty-nine patients (54%) experienced thrombocytopenia (2 patients, 3% grade 4). Hematologic toxicities resulted in dose modifications for the majority of patients. Twenty patients (28%) developed VTEs during treatment. Conclusions: The observed 6-month OS (37%) of lenalidomide with gemcitabine does not suggest improvement compared with historical results with gemcitabine alone. Toxicities and dose modifications likely limited dose intensity. Further development of this regimen in pancreas cancer is not recommended.
doi:10.4161/cbt.23625
PMCID: PMC3667874  PMID: 23358470
pancreas cancer; lenalidomide; phase II; gemcitabine combination
23.  Integrative Annotation of Variants from 1092 Humans: Application to Cancer Genomics 
Science (New York, N.Y.)  2013;342(6154):1235587.
Interpreting variants, especially noncoding ones, in the increasing number of personal genomes is challenging. We used patterns of polymorphisms in functionally annotated regions in 1092 humans to identify deleterious variants; then we experimentally validated candidates. We analyzed both coding and noncoding regions, with the former corroborating the latter. We found regions particularly sensitive to mutations (“ultrasensitive”) and variants that are disruptive because of mechanistic effects on transcription-factor binding (that is, “motif-breakers”). We also found variants in regions with higher network centrality tend to be deleterious. Insertions and deletions followed a similar pattern to single-nucleotide variants, with some notable exceptions (e.g., certain deletions and enhancers). On the basis of these patterns, we developed a computational tool (FunSeq), whose application to ~90 cancer genomes reveals nearly a hundred candidate noncoding drivers.
doi:10.1126/science.1235587
PMCID: PMC3947637  PMID: 24092746
24.  The Phosphoinositide Kinase PIKfyve Mediates EGF Receptor Trafficking to the Nucleus 
Cancer research  2007;67(19):9229-9237.
ErbB receptor tyrosine kinases can transit to nuclei in tumor cells, where they have been shown to regulate gene expression as components of transcriptional complexes. Quantitative analysis of a human bladder cancer tissue microarray identified nuclear EGFR in tumor cells and also showed an increased frequency of this histologic feature in cancer relative to normal tissues. This observation suggests a potential role for nuclear EGFR in bladder cancer. We confirmed that EGFR could be induced to transit to nuclei in cultured human bladder cancer cells in response to the urothelial cell growth factor and EGFR ligand, HB-EGF. Mass spectrometric analysis of EGFR immune complexes from a transitional carcinoma cell line (TCCSUP) identified the phosphoinositide kinase, PIKfyve, as a potential component of the EGFR trafficking mechanism. RNA silencing indicated that PIKfyve is a mediator of HB-EGF-stimulated EGFR nuclear trafficking, EGFR binding to the cyclin D1 promoter, and cell cycle progression. These results identify a novel mediator of the EGFR transcription function and further suggest that nuclear EGFR and the lipid kinase PIKfyve may play a role in bladder oncogenesis.
doi:10.1158/0008-5472.CAN-07-1333
PMCID: PMC3934554  PMID: 17909029
nuclear EGFR; bladder cancer; PIKfyve; trafficking
25.  Molecular Archeology: Unearthing Androgen Induced Structural Rearrangements in Prostate Cancer Genomes 
Cancer cell  2013;23(2):133-135.
Summary
In this issue of Cancer Cell, Weischenfeldt et al. report on whole genome sequencing of 11 early onset prostate cancers. Compared to elderly onset prostate cancer, these tumors demonstrate enrichment for androgen driven structural rearrangements involving ETS family genes. This study confirms observations that prostate cancer manifests discrete genomic subclasses.
doi:10.1016/j.ccr.2013.01.019
PMCID: PMC3630373  PMID: 23410968

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