Specific Fusion Genes Research Articles

Abstract 101: Identification of pancreatobilliary cancer specific fusion genes in patient tissues

Abstract Gene fusion occurs when a part of one gene fuses with or attaches to a part of another gene by genome rearrangement and the result in gene fusion may possess oncogenic properties; fused gene may be translated into a unique protein that may promotes cancer properties. Pancreatic cancer and bile duct cancer are one of the most lethal cancer types with low 5-year survival rates, but lack of proper diagnostic or prognostic markers. Our aim was to investigate pancreatobilliary- specific fusion genes found in patient's specimens. We used TRIzol to extract total RNA from seven pancreatic cancer tissues and normal tissues from the same patients, and five bile duct cancer tissues and normal tissues from the same patients. Total RNA was assessed for RNA sequencing and the result data was analyzed using ChimeraScan to detect gene fusion. To identify the cancer somatic fusion gene, the fusion occurred in normal tissues and previously reported fusion genes found in normal tissues were excluded and the fusion occurred in coding region was included. As a result, we found 87 pancreatic cancer tissue specific- and 52 bile duct cancer tissue specific- fusion genes. We then selected fusion genes that either one of the gene has been reported to express in cancer tissues. We confirmed the expression of selected fusion genes by RT-PCR in cancer and normal tissues as well as in cancer cell lines. As a result we observed novel pancreatobilliary cancer specific fusion genes. These findings indicate the presence of novel fusion genes as well as its possible application for the early diagnosis or prognosis of pancreatic and bile duct cancer. Citation Format: Kahee Kim, Dawoon E. Jung, Mi-kyoung Seo, Sang Woo Kim, Si Young Song. Identification of pancreatobilliary cancer specific fusion genes in patient tissues. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 101.

RNA-Seq methods for transcriptome analysis.

Deep sequencing has been revolutionizing biology and medicine in recent years, providing single base-level precision for our understanding of nucleic acid sequences in high throughput fashion. Sequencing of RNA, or RNA-Seq, is now a common method to analyze gene expression and to uncover novel RNA species. Aspects of RNA biogenesis and metabolism can be interrogated with specialized methods for cDNA library preparation. In this study, we review current RNA-Seq methods for general analysis of gene expression and several specific applications, including isoform and gene fusion detection, digital gene expression profiling, targeted sequencing and single-cell analysis. In addition, we discuss approaches to examine aspects of RNA in the cell, technical challenges of existing RNA-Seq methods, and future directions. WIREs RNA 2017, 8:e1364. doi: 10.1002/wrna.1364 For further resources related to this article, please visit the WIREs website.

TFE3-Fusion Variant Analysis Defines Specific Clinicopathologic Associations Among Xp11 Translocation Cancers.

Xp11 translocation cancers include Xp11 translocation renal cell carcinoma (RCC), Xp11 translocation perivascular epithelioid cell tumor (PEComa), and melanotic Xp11 translocation renal cancer. In Xp11 translocation cancers, oncogenic activation of TFE3 is driven by the fusion of TFE3 with a number of different gene partners; however, the impact of individual fusion variant on specific clinicopathologic features of Xp11 translocation cancers has not been well defined. In this study, we analyze 60 Xp11 translocation cancers by fluorescence in situ hybridization using custom bacterial artificial chromosome probes to establish their TFE3 fusion gene partner. In 5 cases RNA sequencing was also used to further characterize the fusion transcripts. The 60 Xp11 translocation cancers included 47 Xp11 translocation RCC, 8 Xp11 translocation PEComas, and 5 melanotic Xp11 translocation renal cancers. A fusion partner was identified in 53/60 (88%) cases, including 18 SFPQ (PSF), 16 PRCC, 12 ASPSCR1 (ASPL), 6 NONO, and 1 DVL2. We provide the first morphologic description of the NONO-TFE3 RCC, which frequently demonstrates subnuclear vacuoles leading to distinctive suprabasal nuclear palisading. Similar subnuclear vacuolization was also characteristic of SFPQ-TFE3 RCC, creating overlapping features with clear cell papillary RCC. We also describe the first RCC with a DVL2-TFE3 gene fusion, in addition to an extrarenal pigmented PEComa with a NONO-TFE3 gene fusion. Furthermore, among neoplasms with the SFPQ-TFE3, NONO-TFE3, DVL2-TFE3, and ASPL-TFE3 gene fusions, the RCCs are almost always PAX8 positive, cathepsin K negative by immunohistochemistry, whereas the mesenchymal counterparts (Xp11 translocation PEComas, melanotic Xp11 translocation renal cancers, and alveolar soft part sarcoma) are PAX8 negative, cathepsin K positive. These findings support the concept that despite an identical gene fusion, the RCCs are distinct from the corresponding mesenchymal neoplasms, perhaps due to the cellular context in which the translocation occurs. We corroborate prior data showing that the PRCC-TFE3 RCCs are the only known Xp11 translocation RCC molecular subtype that are consistently cathepsin K positive. In summary, our data expand further the clinicopathologic features of cancers with specific TFE3 gene fusions and should allow for more meaningful clinicopathologic associations to be drawn.

Analysis of gene expression and DNA methylation patterns in childhood acute lymphoblastic leukemia

The 5-year survival rate of patients with childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) now exceeds 90%, though there are still patients who fail to achieve remission or relapse early. To improve the outcomes of these cases, new diagnostic markers for stratification of those with unfavorable outcomes and novel targets for treatment have been investigated based on data from the OMICs analysis. We performed gene expression analysis of leukemic cells from 91 near-diploid BCP-ALL cases without specific fusion genes enrolled in Tokyo Children's Cancer Study Group (TCCSG)-L0416 & L0616 clinical trials employing the Affymetrix Human Genome U133 Plus 2.0 Array. Among them, DNA methylation status was analyzed in 24 cases by using the Infinium HumanMethylation450 BeadChip. Herein, initially, the current situations of gene expression analysis and DNA methylation analysis of childhood BCP-ALL are reviewed. Then, our analyses of gene expressions and DNA methylation related to the prognosis of childhood ALL without fusion genes are presented.

Abstract A75: Pan-cancer identification of fusion genes as therapeutic targets in cancer

Abstract Fusion genes are a well established category of oncogenic mutations, in which a chromosomal rearrangement brings together parts of two different genes to create a novel gene with oncogenic potential. Originally identified mostly in hematologic malignancies (e.g. BCR-ABL1 in CML), over the last years fusion genes have been shown to occur in practically all types of cancers examined. Some of the best known examples of these are ETS-family gene fusions (e.g. TMPRSS2-ERG) in prostate cancer and EML4-ALK fusions in non-small cell lung cancer. Although the prevalence of a specific gene fusion in a single cancer is often low, the example of treating e.g. EML4-ALK fusion positive NSCLC cancers with ALK inhibitors, such as crizotinib and ceritinib, has shown gene fusions to have significant value as targets of drug development. In this study, we describe a comprehensive survey of gene fusions in 7428 cancer samples from 27 different cancer types, using paired-end RNA-seq data created by the TCGA project. At least one fusion gene was identified in 3801/7428 samples, with over 9000 genes taking part in a total of ∼11000 fusion gene events. The number of gene fusions per tumor is highly variable, with the largest number of fusions typically identified in tumors with high level amplifications. This suggests many of the fusion genes identified here may arise as byproducts of amplicon formation, making them likely passenger events. Several potentially targetable genes take part in gene fusions across different cancer types. For instance, we identified a total of 12 gene fusions involving ALK in 5 different cancer types. Strikingly, only 5 of these involve EML4 as 5' partner gene. NTRK fusions were identified in 24 samples from 9 cancer types, FGFR fusions in 39 samples from 14 cancer types and MAST family kinase fusions in 14 samples from 7 cancer types. In summary, our analysis of fusion gene occurrence across a wide variety of cancer types identifies several interesting and potentially targetable gene fusions. Furthermore, it illustrates that many of these are found across several different cancer types, as well as involve a large number of different fusion partner genes, suggesting this may need to be taken into account in diagnostics of fusion genes as well as selection of patients for clinical trials. Citation Format: Henrik Edgren, Kalle Ojala, Anja Ruusulehto. Pan-cancer identification of fusion genes as therapeutic targets in cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A75.

The Molecular Taxonomy of Primary Prostate Cancer

There is substantial heterogeneity among primary prostate cancers, evident in the spectrum of molecular abnormalities and its variable clinical course. As part of The Cancer Genome Atlas (TCGA), we present a comprehensive molecular analysis of 333 primary prostate carcinomas. Our results revealed a molecular taxonomy in which 74% of these tumors fell into one of seven subtypes defined by specific gene fusions (ERG, ETV1/4, and FLI1) or mutations (SPOP, FOXA1, and IDH1). Epigenetic profiles showed substantial heterogeneity, including an IDH1 mutant subset with a methylator phenotype. Androgen receptor (AR) activity varied widely and in a subtype-specific manner, with SPOP and FOXA1 mutant tumors having the highest levels of AR-induced transcripts. 25% of the prostate cancers had a presumed actionable lesion in the PI3K or MAPK signaling pathways, and DNA repair genes were inactivated in 19%. Our analysis reveals molecular heterogeneity among primary prostate cancers, as well as potentially actionable molecular defects.

Intraparenchymal mesenchymal chondrosarcoma of the frontal lobe – a case report and molecular detection of specific gene fusions from archival FFPE sample

Mesenchymal chondrosarcoma is a rare tumor of cartilaginous origin characterized by its bimorphic pattern composed of highly undifferentiated small round cells separated by islands of well-differentiated hyaline cartilage. It exhibits higher malignancy and earlier occurrence in comparison to classic chondrosarcomas. Recently identified HEY1-NCOA2 and IRF2BP2-CDX1 gene fusions confirm their distinct molecular origin and pose a promising diagnostic marker. The majority of cases arise from craniofacial bones. In this study, we present a rare case of mesenchymal chondrosarcoma encompassed within the brain parenchyma of the frontal lobe without any dural or bone attachment. We demonstrate histopathological findings and confirm the HEY1-NCOA2 gene fusion in a formalin-fixed paraffin-embedded archival sample using simple reverse transcription polymerase chain reaction (RT-PCR) method. IRF2BP2-CDX1 gene fusion was absent in the analyzed sample. The clinical follow-up is also presented with a review of treatment modalities for this entity.

Abstract 4859: Development of a cancer transcriptome analysis toolkit: identification of gene fusions in chronic lymphocytic leukemia

Abstract RNA-Seq has become an invaluable tool for improving the understanding of the biological mechanisms at work in various cancers, with tantalizing prospects for application in the area of clinical diagnostics and for the design and monitoring of therapy. Transcriptome data uniquely provides information on gene expression, and the presence of somatic expressed splice variants, point mutations, CNVs and gene fusions. Although diverse algorithms have been developed to identify specific classes of somatic alterations, an approach for streamlined, comprehensive and large-scale processing and mining of RNA-Seq data remains a formidable challenge. To address this challenge, we have developed the Cancer Transcriptome Analysis Toolkit (CTAT).The CTAT is an amalgamation of both existing and novel tools for RNAseq data analysis, wrapped in Galaxy as a user-friendly bioinformatics pipeline. We have tested CTAT on a RNA-seq dataset generated from 138 patients with chronic lymphocytic leukemia (CLL), whose malignant cells have been already extensively characterized at the DNA level by analysis of whole-exome sequencing (WES) and SNP array data. In addition, we have also analyzed RNA-seq data from CD19+ B cells isolated from 19 healthy adult volunteers. As expected, CTAT could detect the established features of CLL. We could reliably detect at the RNA-level previously identified mutated CLL driver genes as per WES analysis. Overexpression of CLL-associated genes (i.e. ZAP70, and CD38) was also detected. CTAT also could detect novel features of CLL. Focusing on gene fusions, an important class of genetic alteration across blood malignancies but which have not been well-characterized in CLL, we detected a total of 509 gene fusion events across the trio of fusion detection algorithms used, and 25 were detected unanimously. From an arbitrary subset of 40 fusions, we validated 38 by RT-PCR (95% validation rate). 450 of 509 (88%) of gene fusions involved intrachromosomal partners. After adjusting for gene density, CTAT detected the highest incidence of high confidence gene fusions on chr13 (30%). GSEA revealed that gene fusions appeared to cluster to known driver pathways of CLL, such as the BCR, NOTCH, NFKB, RNA processing and Apoptosis pathways. Specific gene fusions in our CLL patient cohort appeared to be predominantly private events and with variable predicted oncogenic potential. However, several potential oncogenic drivers (NCOR2–UBC, PHF3–PTP4A1, CYTIP–ERMN, G3BP2–CCNG2) each appeared in 5-10% of patients, targeting cell cycle control, NOTCH signaling and cell adhesion pathways, and their biological significance can now be investigated. Citation Format: Nikolaus D. Obholzer, Brian J. Haas, Dan-Avi Landau, Nathalie Pochet, Aviv Regev, Catherine Wu. Development of a cancer transcriptome analysis toolkit: identification of gene fusions in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4859. doi:10.1158/1538-7445.AM2015-4859

Abstract 1818: Analytical platform evaluation for quantification of ERG oncoprotein in prostate cancer using protein and mRNA detection methods

Abstract Current molecular tests that aid in the diagnosis of prostate cancer (PCa) are based on the detection of PSA and PSA isoforms (blood), overexpression of PCA3 (urine), and overexpression of AMACR (tissue) as biomarkers. The discovery of cancer specific fusion transcripts, which are detected in over 50% of prostate cancers, revolutionized the field. As such, the cancer specific TMPRSS2-ERG gene fusion drives the overexpression of the ERG oncoprotein. Based on this, we hypothesized that ERG protein may serve as a valuable biomarker and for which quantification methods can be applied for the diagnosis of prostate cancer. An antibody-free assay for ERG protein detection was developed based on PRISM (high-pressure high-resolution separations with intelligent selection and multiplexing)-SRM (selected reaction monitoring) mass spectrometry. We utilized TMPRSS2-ERG positive VCaP and TMPRSS2-ERG negative LNCaP cells to simulate three different sample types (cells, tissue, and post-DRE urine sediment) for evaluation. Recombinant ERG protein spiked into LNCaP cell lysates could be detected at levels as low as 20 pg by PRISM-SRM analysis. The sensitivity of the PRISM-SRM assay was approximately 10,000 VCaP cells in a mixed cell population model of VCaP and LNCaP cells. Interestingly, ERG protein could be detected in as few as 600 VCaP cells spiked into female urine, presumably due to more effective loading of ERG protein and the reduced sample complexity. In comparison the detection limit of the in-house enzyme-linked immunosorbent assay was 30 pg of recombinant ERG protein and 10,000 VCaP cells, while Western blot analysis demonstrated the detection of 195 pg of recombinant ERG protein and 10,000 VCaP cells in these two sample types, respectively. On the other hand, qRT-PCR exhibited a higher sensitivity, as TMPRSS2-ERG transcripts were detected in as few as 250 VCaP cells spiked into female urine, in comparison to NanoString methodologies which detected 10,000 VCaP cells in the same sample. In summary the data presented here suggest that qRT-PCR and PRISM-SRM platforms are highly sensitive in detecting TMPRSS2-ERG transcripts and proteins, respectively. Compared to other RNA (without preamplifcation step) and protein detection technologies, the PRISM-SRM assay has several significant advantages: it is not affected by RNA stability, it does not rely on a specific antibody, and it is ideal for isoform-specific detection with high multiplexing capability. Therefore, PRISM-SRM assays can be adapted to detect ERG protein in cells present in clinical specimens (e.g., tissue and urine), and cell-free ERG protein present in the blood sera, providing an opportunity for their use in the clinical setting for detection of ERG in prostate cancer patients and for defining appropriate treatment strategies. Citation Format: Jintang He, Athena Schepmoes, Anshu Rastogi, Shyh-Han Tan, Wusheng Yan, Wei Huang, Sreedatta Banerjee, Tujin Shi, Chaochao Wu, Thomas Fillmore, Yuqian Gao, Jacob Kagan, Sudhir Srivastava, Richard Smith, Wei-Jun Qian, David McLeod, Gyorgy Petrovics, Albert Dobi, Alagarsamy Srinivasan, Shiv Srivastava, Karin Rodland, Tao Liu, David Camp. Analytical platform evaluation for quantification of ERG oncoprotein in prostate cancer using protein and mRNA detection methods. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1818. doi:10.1158/1538-7445.AM2015-1818

Diagnosis and treatment of dermatofibrosarcoma protuberans. European consensus-based interdisciplinary guideline

Dermatofibrosarcoma protuberans (DFSP) is a skin fibroblastic tumour that is locally aggressive, with a tendency for local recurrence, but rarely metastasizes. A unique collaboration of multi-disciplinary experts from the European Dermatology Forum (EDF), the European Association of Dermato-Oncology (EADO) and the European Organization of Research and Treatment of Cancer (EORTC) was formed to make recommendations on DFSP diagnosis and treatment, based on systematic literature reviews and the experts’ experience. Diagnosis is suspected clinically and confirmed by pathology. Analysis by fluorescence in situ hybridisation (FISH) or multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) to detect specific chromosomal translocations and fusion gene transcripts is useful to confirm a difficult DFSP diagnosis. Treatment is mainly surgical, with the aim to achieve complete resection of the tumour. In order to reduce the recurrence rate, the treatment of choice of DFSP seems to be Mohs’ micrographic surgery (MMS) and related variants. In hospitals where only standard histopathological procedures are available, standard excision with lateral safety margin of 3cm is advisable. Imatinib (Glivec®) is approved in Europe for the treatment of inoperable primary tumours, locally inoperable recurrent disease, and metastatic DFSP. Imatinib has also been given to patients with extensive, difficult-to-operate tumours for preoperative reduction of tumour size, but the usefulness of this attitude should be confirmed by clinical trials. Therapeutic decisions for patients with fibrosarcomatous DFSP should be primarily made by an interdisciplinary oncology team (‘tumour board’).

Prenatal imaging and immunohistochemical analysis of congenital peribronchial myofibroblastic tumor

A 32-year-old pregnant woman, gravida 4 para 1, was referred to us at 27 weeks' gestation because of fetal hydrops. Ultrasound examination revealed a male fetus with a large homogeneous mass occupying the whole mediastinum with encasement of the great vessels and bronchus (Figure 1 and Videoclip S1). The tumor had a spiculated margin and infiltrated deep into adjacent tissues, suggestive of a sarcoma or other malignancy (Videoclip S2). Subsequent karyotyping following amniocentesis provided a normal result. Maternal blood tests were negative for active infection of adenovirus, toxoplasma and cytomegalovirus. Fetal magnetic resonance imaging revealed a large mediastinal tumor of 6 cm in longitudinal diameter with encasement of adjacent mediastinal structures (Figure 2). At 29 weeks' gestation, the neonate was delivered by elective Cesarean section and intubated by ex-utero intrapartum treatment owing to fetal distress. Apgar scores were 0, 1 and 4 at 1, 5 and 10 min, respectively. The newborn was put on immediate ventilator support for severe hypoxemia, and bilateral chest tubes were inserted to drain the pleural effusions. Transthoracic sonography showed a large extracardiac mediastinal mass with compression of the left atrium. Despite the advanced medical support, the newborn developed refractory hypotension and died 2 days later. Autopsy revealed multiple cystic changes in the periventricular area of the brain (Figure S1). The mediastinal tumor was brown and soft and weighed 26.2 g with dimensions of 69 × 37 mm (Figure 3). It was found to encompass the esophagus, trachea, pulmonary trunk, aortic arch and other great vessels. Microscopic examination of the mass revealed interlacing fascicles of uniform spindle cells, with focal nuclear atypia (Figure S2). These tumor cells were diffusely positive for vimentin, focally positive for smooth muscle actin and CD68, but negative for CD31, CD34, S-100 and desmin. Tumor complementary DNA of the fusion gene transcript ETV6-NTRK3 was amplified specifically and no specific fusion gene transcripts were detected. Fluorescence in-situ hybridization revealed a wild-type EWSR1, encoding the Ewing sarcoma fusion protein, with no splitting of the alleles. The diagnosis of congenital peribronchial myofibroblastic tumor (CPMT) was made based on the myofibroblastic immunophenotype and genetic investigations (Table 1 and Table S1)123. CPMT was first defined in 1993 by McGinnis et al.4 to describe a neoplasm that develops from the embryonic pluripotent mesenchyme found surrounding large bronchi. In the past it was recognized as a malignancy or low-grade sarcoma, but it has now been categorized as a benign mesenchymal tumor with different extents of nuclear atypia. Fewer than 30 cases have been reported in the English medical literature12345. CPMT presents typically as a solid lung mass in the perinatal period, but has not been seen previously as a mediastinal tumor. Heart failure is considered the result of increased mediastinal pressure caused by encasement of the great vessels and direct cardiac compression. The clinical behaviors of CPMT are diverse, and survival rate is only 36% after excluding cases of elective termination123 5. However, patients with CPMT may have a good outcome if they can survive long enough for surgical resection5. In conclusion, when non-immune hydrops fetalis is encountered, the mediastinum should be examined carefully when other etiologies are unlikely to be the cause. Subsequent counseling and management pose more challenges than the detection of the fetal neoplasm itself. Despite its benign histopathology, CPMT in the mediastinum can have a devastating effect on the fetus because of its location and rapid growth. Y.-A. Tu†, W.-C. Lin‡, H.-J. Chen‡ and J.-C. Shih*† †Department of Obstetrics and Gynecology, National Taiwan University Hospital and National Taiwan University College of Medicine, 15F, No. 8, Chung-Shan South Road, 10002, Taipei, Taiwan; ‡Department of Pathology, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan *Correspondence. (e-mail: jcshih@ntu.edu.tw) Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

PD44-07 EFFECTIVENESS OF SERUM PSA, URINE PCA3 AND URINE TMPRSS2:ERG FUSION AS A NOVEL URINARY BIOMARKER PANEL IN CLINICAL PRACTICE

You have accessJournal of UrologyProstate Cancer: Detection and Screening IV1 Apr 2015PD44-07 EFFECTIVENESS OF SERUM PSA, URINE PCA3 AND URINE TMPRSS2:ERG FUSION AS A NOVEL URINARY BIOMARKER PANEL IN CLINICAL PRACTICE John Wei, Javed Siddiqui, Rabia Siddiqui, Arul M. Chinnaiyan, Lakshmi P. Kunju, Rohit Mehra, Debbie Snyder, and Scott A. Tomlins John WeiJohn Wei More articles by this author , Javed SiddiquiJaved Siddiqui More articles by this author , Rabia SiddiquiRabia Siddiqui More articles by this author , Arul M. ChinnaiyanArul M. Chinnaiyan More articles by this author , Lakshmi P. KunjuLakshmi P. Kunju More articles by this author , Rohit MehraRohit Mehra More articles by this author , Debbie SnyderDebbie Snyder More articles by this author , and Scott A. TomlinsScott A. Tomlins More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2553AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES When new technology enters clinical practice, effectiveness deviates from efficacy due to external patient, provider, and systems related factors that may moderate its effect. PCA3, a prostate-specific noncoding gene that is significantly over-expressed in cancer and TMPRSS2(T2):ERG, a prostate cancer specific gene fusion, are both quantifiable by urinary assays that along with serum PSA comprise the testing panel Michigan Prostate Score (MiPS). This novel panel reports individual patient risk estimates for biopsy detection of any prostate cancer (MiPS) and high grade (Gleason score >6) cancer (HG MiPS). We examined the impact of this new test panel on clinical decision-making in men without cancer who were presenting for a prostate biopsy. METHODS During the past year, men referred for prostate biopsy at a single institution were offered MiPS as an alternative to proceeding directly to a biopsy. Patient characteristics, PSA, PCA3, T2:ERG, and biopsy pathology were analyzed to see how MiPS and HG MiPS affected the decision for prostate biopsy and pathology at biopsy. One-way ANOVA was used to compare PCA3, T2:ERG and risk levels. RESULTS 121 men (21.5% with prior biopsy) underwent MiPS testing with a mean age of 64 years, median PSA of 7 ng/ml, PCA3 score of 22, T2:ERG score of 2. Based on MiPS, the average predicted risk for detection of any and high grade cancer were 42.4% and 26.6%, respectively. Men who then chose to have a biopsy (n=58/47.9%) had higher MiPS (54.1% vs 31.6%, p<0.05) and HG MiPS scores (35.3% vs 18.5%, p<0.05) than those who did not. Among those biopsied, MiPS, HG MiPS, PCA3, T2:ERG were higher in those with high grade cancer found on biopsy but only T2:ERG was significantly higher (178.2 vs 44.5, p<0.05). CONCLUSIONS The combination of PSA, PCA3 and T2:ERG as a test panel for prostate cancer reduced the use of prostate biopsy in half. T2:ERG was strongly associated with detection of high grade cancer. These findings support the use of MiPS for assessing prostate cancer risk. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e899-e900 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information John Wei More articles by this author Javed Siddiqui More articles by this author Rabia Siddiqui More articles by this author Arul M. Chinnaiyan More articles by this author Lakshmi P. Kunju More articles by this author Rohit Mehra More articles by this author Debbie Snyder More articles by this author Scott A. Tomlins More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MiT family translocation renal cell carcinoma.

The MiT subfamily of transcription factors includes TFE3, TFEB, TFC, and MiTF. Gene fusions involving two of these transcription factors have been identified in renal cell carcinoma (RCC). The Xp11 translocation RCCs were first officially recognized in the 2004 WHO renal tumor classification, and harbor gene fusions involving TFE3. The t(6;11) RCCs harbor a specific Alpha-TFEB gene fusion and were first officially recognized in the 2013 International Society of Urologic Pathology (ISUP) Vancouver classification of renal neoplasia. These two subtypes of translocation RCC have many similarities. Both were initially described in and disproportionately involve young patients, though adult translocation RCC may overall outnumber pediatric cases. Both often have unusual and distinctive morphologies; the Xp11 translocation RCCs frequently have clear cells with papillary architecture and abundant psammomatous bodies, while the t(6;11) RCCs frequently have a biphasic appearance with both large and small epithelioid cells and nodules of basement membrane material. However, the morphology of these two neoplasms can overlap, with one mimicking the other. Both of these RCCs underexpress epithelial immunohistochemical markers like cytokeratin and epithelial membrane antigen (EMA) relative to most other RCCs. Unlike other RCCs, both frequently express the cysteine protease cathepsin k and often express melanocytic markers like HMB45 and Melan A. Finally, TFE3 and TFEB have overlapping functional activity as these two transcription factors frequently heterodimerize and bind to the same targets. Therefore, on the basis of clinical, morphologic, immunohistochemical, and genetic similarities, the 2013 ISUP Vancouver classification of renal neoplasia grouped these two neoplasms together under the heading of "MiT family translocation RCC." This review summarizes our current knowledge of these recently described RCCs.

Clinical significance of ERG rearrangement subtype and its association with increased p53 expression in Japanese and German prostate cancer.

This study investigated differences in prevalence of the androgen-regulated transmembrane protease serine 2 (TMPRSS2) and ETS transcription factor family member, v-ets erythroblastosis virus E26 oncogene homolog (ERG) fusion gene (TMPRSS2-ERG fusions) in clinically localized prostate cancer Japanese and German patients. Atotal of 105 specimens, including 69 Japanese and 36 German patients, were collected. The status of TMPRSS2-ERG fusion was determined by fluorescence in situ hybridization, and correlations of the TMPRSS2-ERG fusion with clinicopathological characteristics and immunohistochemistry were studied. Gene fusions were identified in 20% (14/69) of Japanese and 53% (19/36) of German patients (P < 0.001). The difference in the type of gene fusion between the two ethnic groups was statistically significant (P=0.024). Overexpression of ERG protein was significantly associated with gene fusion. Biochemical recurrence was significantly higher in patients with ERG overexpression than in those without, and not related to TMPRSS2-ERG fusion status. Interestingly, two types of gene fusions (deletion and increase of copy number) were significantly associated with increased p53 expression (P = 0.005). Association of specific gene fusions harboring higher genomic alterations with p53 expression levels suggests that p53 mutation might drive more aggressive arrangements of TMPRSS2-ERG fusion in prostate cancer. ERG, p53, prostate cancer, TMPRSS2-ERG fusion.

A minimal dose of electrically induced muscle activity regulates distinct gene signaling pathways in humans with spinal cord injury.

Paralysis after a spinal cord injury (SCI) induces physiological adaptations that compromise the musculoskeletal and metabolic systems. Unlike non-SCI individuals, people with spinal cord injury experience minimal muscle activity which compromises optimal glucose utilization and metabolic control. Acute or chronic muscle activity, induced through electrical stimulation, may regulate key genes that enhance oxidative metabolism in paralyzed muscle. We investigated the short and long term effects of electrically induced exercise on mRNA expression of human paralyzed muscle. We developed an exercise dose that activated the muscle for only 0.6% of the day. The short term effects were assessed 3 hours after a single dose of exercise, while the long term effects were assessed after training 5 days per week for at least one year (adherence 81%). We found a single dose of exercise regulated 117 biological pathways as compared to 35 pathways after one year of training. A single dose of electrical stimulation increased the mRNA expression of transcriptional, translational, and enzyme regulators of metabolism important to shift muscle toward an oxidative phenotype (PGC-1α, NR4A3, IFRD1, ABRA, PDK4). However, chronic training increased the mRNA expression of specific metabolic pathway genes (BRP44, BRP44L, SDHB, ACADVL), mitochondrial fission and fusion genes (MFF, MFN1, MFN2), and slow muscle fiber genes (MYH6, MYH7, MYL3, MYL2). These findings support that a dose of electrical stimulation (∼10 minutes/day) regulates metabolic gene signaling pathways in human paralyzed muscle. Regulating these pathways early after SCI may contribute to reducing diabetes in people with longstanding paralysis from SCI.

Recurrent LRP1-SNRNP25 and KCNMB4-CCND3 fusion genes promote tumor cell motility in human osteosarcoma.

BackgroundThe identification of fusion genes such as SYT-SSX1/SSX2, PAX3-FOXO1, TPM3/TPM4-ALK and EWS-FLI1 in human sarcomas has provided important insight into the diagnosis and targeted therapy of sarcomas. No recurrent fusion has been reported in human osteosarcoma.MethodsTranscriptome sequencing was used to characterize the gene fusions and mutations in 11 human osteosarcomas.ResultsNine of 11 samples were found to harbor genetic inactivating alterations in the TP53 pathway. Two recurrent fusion genes associated with the 12q locus, LRP1-SNRNP25 and KCNMB4-CCND3, were identified and validated by RT-PCR, Sanger sequencing and fluorescence in situ hybridization, and were found to be osteosarcoma specific in a validation cohort of 240 other sarcomas. Expression of LRP1-SNRNP25 fusion gene promoted SAOS-2 osteosarcoma cell migration and invasion. Expression of KCNMB4-CCND3 fusion gene promoted SAOS-2 cell migration.ConclusionsOur study represents the first whole transcriptome analysis of untreated human osteosarcoma. Our discovery of two osteosarcoma specific fusion genes associated with osteosarcoma cellular motility highlights the heterogeneity of osteosarcoma and provides opportunities for new treatment modalities.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-014-0076-2) contains supplementary material, which is available to authorized users.

Abstract 1539: Fusion events in NSCLC cell lines identified by whole transcriptome analysis

Abstract Since the discovery of Philadelphia chromosome, human chromosomal rearrangements consequence the great deal within the distinct area of cancer biomarker discovery and targeted therapy. These events are commonly generated by breaking and re-joining the segments on either inter- or intra-chromosomal locations, creating an error within DNA repair mechanism responsible for oncogenic properties and, one of the reasons, causing cancer. Also, alteration in the programing of gene regulation changes the transcriptome structure resulting pathological conditions. Recent advances in the cancer epidemiology predicted the highest death rates within Europe and Korea due to lung cancer representing the leading cause of death within cancer patients. Therefore, we have sequenced the whole transcriptome following the highly sensitive path of RNA-seq with the combination of bioinformatics tools and experimental confirmation. The mRNA was subjected on Illumina HiSeq2000 for paired-end (PE) sequencing and bioinformatics analyses were conducted on Linux based software tools. The PE reads were aligned with Ensemble reference human genome version hg19 using TopHat2 and bowtie1. The fusion events were predicted using two different software packages, deFuse and Tophat-Fusion, and expression analysis was conducted using Cufflinks and R-package cumeRbund. The predicted fusion break-points were confirmed by target specific sanger-sequencing. Through the RNA-seq analysis, we identified a number of fusion events shared by oncogenes in non-small cell lung cancer (NSCLC) cell-lines including EML4:ALK fusion identified in recently established gefitinib resistant SNU2292 cell-line and some novel ones. Moreover, a significant increase in the expression of cell-line specific fusion genes was also observed. Our result will be helpful not only for discovering novel biomarkers but also for understanding the tumorigenesis of NSCLC. Note: This abstract was not presented at the meeting. Citation Format: Seung-Hyun Jung, Yeun-Jun Chung. Fusion events in NSCLC cell lines identified by whole transcriptome analysis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1539. doi:10.1158/1538-7445.AM2014-1539

ERG monoclonal antibody in the diagnosis and biological stratification of prostate cancer: delineation of minimal epitope, critical residues for binding, and molecular basis of specificity.

Recently we reported the development of a highly specific murine monoclonal antibody (ERG MAb 9FY) against the ERG oncoprotein. ERG is expressed in over half of all prostate cancers (CaP) as a result of specific gene fusions involving ERG and the androgen regulated TMPRSS2 promoter. ERG MAb 9FY has been extensively used in the evaluations of CaP. Increasing use of ERG MAb in CaP has prompted us to characterize the precise ERG epitope it binds to and to define the molecular basis of its specificity to ERG. The 9FY antibody binds to an epitope formed by amino acid residues 42-66 of the ERG protein. To determine the key residues involved in 9FY binding, experiments were carried out using a combination of approaches including overlapping peptides, alanine scanning mutagenesis, ELISA, and immunoblot assays. Analysis of both overlapping and variant peptides harboring truncations of amino acids revealed that a minimal epitope of eight residues (RVPQQDWL) is sufficient for binding to the 9FY antibody. In order to further identify key residues that mediate the binding of the antibody to ERG protein, a 14-residue peptide (P23) with optimal reactivity was subjected to alanine scanning mutagenesis. Alterations to residues QQDW were found to eliminate binding to the antibody, while residues (R50 and L57) were found to contribute to the binding of the antibody. Further experiments showed that peptide P23 competed effectively with ERG protein for binding 9FY. On the other hand, peptides with alanine substitutions for residues Q53 and W56 (P27 and P30, respectively) failed to interfere with binding. These data provide new information about a minimal epitope (RVPQQDWL) within amino acid residues 42-66 of the ERG protein that is recognized by MAb 9FY, which may aid in the diagnosis and also development of antibody based therapeutics against prostate and other cancers showing ERG overexpression.

VCL-ALK Renal Cell Carcinoma in Children With Sickle-cell Trait

We report the third case of a renal cell carcinoma bearing a fusion of the vinculin (VCL) and anaplastic lymphoma kinase (ALK) genes. Like the 2 other reported cases, this neoplasm occurred in a young patient (6 y old) with sickle-cell trait and demonstrated distinctive morphologic features including medullary epicenter, discohesive polygonal or spindle-shaped cells with prominent cytoplasmic vacuoles, and prominent lymphocytic infiltrate. The neoplastic cells demonstrated focal membranous labeling for ALK protein by immunohistochemistry, ALK gene rearrangement by fluorescence in situ hybridization, and a specific VCL-ALK gene fusion by reverse transcriptase polymerase chain reaction. VCL-ALK renal cell carcinoma may represent the eighth sickle-cell nephropathy.

A near complete snapshot of the Zea mays seedling transcriptome revealed from ultra-deep sequencing

RNA-sequencing (RNA-seq) enables in-depth exploration of transcriptomes, but typical sequencing depth often limits its comprehensiveness. In this study, we generated nearly 3 billion RNA-Seq reads, totaling 341 Gb of sequence, from a Zea mays seedling sample. At this depth, a near complete snapshot of the transcriptome was observed consisting of over 90% of the annotated transcripts, including lowly expressed transcription factors. A novel hybrid strategy combining de novo and reference-based assemblies yielded a transcriptome consisting of 126,708 transcripts with 88% of expressed known genes assembled to full-length. We improved current annotations by adding 4,842 previously unannotated transcript variants and many new features, including 212 maize transcripts, 201 genes, 10 genes with undocumented potential roles in seedlings as well as maize lineage specific gene fusion events. We demonstrated the power of deep sequencing for large transcriptome studies by generating a high quality transcriptome, which provides a rich resource for the research community.