Regional perturbation of gene transcription is associated with intrachromosomal rearrangements and gene fusion transcripts in high grade ovarian cancer.

Genomic rearrangements are a hallmark of cancer biology and progression, allowing cells to rapidly transform through alterations in regulatory structures, changes in expression patterns, reprogramming of signaling pathways, and creation of novel transcripts via gene fusion events. Though functional gene fusions encoding oncogenic proteins are the most dramatic outcomes of genomic rearrangements, we investigated the relationship between rearrangements evidenced by fusion transcripts and local expression changes in cancer using transcriptome data alone. 9,953 gene fusion predictions from 418 primary serious ovarian cancer tumors were analyzed, identifying depletions of gene fusion breakpoints within coding regions of fused genes as well as an N-terminal enrichment of breakpoints within fused genes. We identified 48 genes with significant fusion-associated upregulation and furthermore demonstrate that significant regional overexpression of intact genes in patient transcriptomes occurs within 1 megabase of 78 novel gene fusions that function as central markers of these regions. We reveal that cancer transcriptomes select for gene fusions that preserve protein and protein domain coding potential. The association of gene fusion transcripts with neighboring gene overexpression supports rearrangements as mechanism through which cancer cells remodel their transcriptomes and identifies a new way to utilize gene fusions as indicators of regional expression changes in diseased cells with only transcriptomic data

Massive NGS data analysis reveals hundreds of potential novel gene fusions in human cell lines

Background: Gene fusions derive from chromosomal rearrangements and the resulting chimeric transcripts are often endowed with oncogenic potential. Furthermore, they serve as diagnostic tools for the clinical classification of cancer subgroups with different prognosis and, in some cases, they can provide specific drug targets. So far, many efforts have been carried out to study gene fusion events occurring in tumor samples. In recent years, the availability of a comprehensive Next Generation Sequencing dataset for all the existing human tumor cell lines has provided the opportunity to further investigate these data in order to identify novel and still uncharacterized gene fusion events. Results: In our work, we have extensively reanalyzed 935 paired-end RNA-seq experiments downloaded from "The Cancer Cell Line Encyclopedia" repository, aiming at addressing novel putative cell-line specific gene fusion events in human malignancies. The bioinformatics analysis has been performed by the execution of four different gene fusion detection algorithms. The results have been further prioritized by running a bayesian classifier which makes an in silico validation. The collection of fusion events supported by all of the predictive softwares results in a robust set of ∼ 1,700 in-silico predicted novel candidates suitable for downstream analyses. Given the huge amount of data and information produced, computational results have been systematized in a database named LiGeA. The database can be browsed through a dynamical and interactive web portal, further integrated with validated data from other well known repositories. Taking advantage of the intuitive query forms, the users can easily access, navigate, filter and select the putative gene fusions for further validations and studies. They can also find suitable experimental models for a given fusion of interest. Conclusions: We believe that the LiGeA resource can represent not only the first compendium of both known and putative novel gene fusion events in the catalog of all of the human malignant cell lines, but it can also become a handy starting point for wet-lab biologists who wish to investigate novel cancer biomarkers and specific drug targets

INTEGRATE-Neo: A pipeline for personalized gene fusion neoantigen discovery

Abstract Motivation While high-throughput sequencing (HTS) has been used successfully to discover tumor-specific mutant peptides (neoantigens) from somatic missense mutations, the field currently lacks a method for identifying which gene fusions may generate neoantigens. Results We demonstrate the application of our gene fusion neoantigen discovery pipeline, called INTEGRATE-Neo, by identifying gene fusions in prostate cancers that may produce neoantigens. Availability and Implementation INTEGRATE-Neo is implemented in C ++ and Python. Full source code and installation instructions are freely available from https://github.com/ChrisMaherLab/INTEGRATE-Neo. Supplementary information Supplementary data are available at Bioinformatics online. </jats:sec

Gene Fusion Markup Language: a prototype for exchanging gene fusion data

Abstract Background An avalanche of next generation sequencing (NGS) studies has generated an unprecedented amount of genomic structural variation data. These studies have also identified many novel gene fusion candidates with more detailed resolution than previously achieved. However, in the excitement and necessity of publishing the observations from this recently developed cutting-edge technology, no community standardization approach has arisen to organize and represent the data with the essential attributes in an interchangeable manner. As transcriptome studies have been widely used for gene fusion discoveries, the current non-standard mode of data representation could potentially impede data accessibility, critical analyses, and further discoveries in the near future. Results Here we propose a prototype, Gene Fusion Markup Language (GFML) as an initiative to provide a standard format for organizing and representing the significant features of gene fusion data. GFML will offer the advantage of representing the data in a machine-readable format to enable data exchange, automated analysis interpretation, and independent verification. As this database-independent exchange initiative evolves it will further facilitate the formation of related databases, repositories, and analysis tools. The GFML prototype is made available at http://code.google.com/p/gfml-prototype/ . Conclusion The Gene Fusion Markup Language (GFML) presented here could facilitate the development of a standard format for organizing, integrating and representing the significant features of gene fusion data in an inter-operable and query-able fashion that will enable biologically intuitive access to gene fusion findings and expedite functional characterization. A similar model is envisaged for other NGS data analyses.http://deepblue.lib.umich.edu/bitstream/2027.42/112901/1/12859_2011_Article_5754.pd

Diagnostic yield of NanoString nCounter FusionPlex profiling in soft tissue tumors

Diagnostic histopathology of soft tissue tumors can be troublesome as many entities are quite rare and have overlapping morphologic features. Many soft tissue tumors harbor tumor-defining gene translocations, which may provide an important ancillary tool for tumor diagnosis. The NanoString nCounter platform enables multiplex detection of pre-defined gene fusion transcripts in formalin-fixed and paraffin-embedded tissue. A cohort of 104 soft tissue tumors representing 20 different histological types was analyzed for the expression of 174 unique gene fusion transcripts. A tumor-defining gene fusion transcript was detected in 60 cases (58%). Sensitivity and specificity of the NanoString assay calculated against the result of an alternative molecular method were 85% and 100%, respectively. Highest diagnostic coverage was obtained for Ewing sarcoma, synovial sarcoma, myxoid liposarcoma, alveolar rhabdomyosarcoma, and desmoplastic small round cell tumor. For these tumor types, the NanoString assay is a rapid, cost-effective, sensitive, and specific ancillary screening tool for molecular diagnosis. For other sarcomas, additional molecular testing may be required when a translocation transcript is not identified with the current 174 gene fusion panel

Solitary fibrous tumour of the genitourinary tract: a clinicopathological study of 11 cases and their association with the NAB2-STAT6 fusion gene

Aims To characterise clinicopathological features and clinical outcomes of the genitourinary tract solitary fibrous tumours, incorporating NAB2-STAT6 gene fusion status. Methods The presence of the molecular hallmark NAB2-STAT6 gene fusion and for the defining fusion partner product STAT6 was assessed in 11 cases of the genitourinary tract solitary fibrous tumours. NAB2-STAT6 gene fusion analysis was performed using a break-apart fluorescence in situ hybridisation (FISH) probe using a probe cocktail with Bacterial artificial chromosome (BAC) clones for STAT6 and NAB2. Results Eleven solitary fibrous tumours were diagnosed in eight male patients and three female patients with a mean age of 46 years (range: 11–64 years). Four of the tumours had malignant histological features, and three were considered moderate risk for metastasis. With a mean follow-up time of 61 months, 1 recurred locally and 2 presented at distant metastatic sites. Using a break-apart FISH probe cocktail, we found the NAB2-STAT6 gene fusion and nuclear STAT6 expression in 58% and 91% of cases, respectively. However, the NAB2-STAT6 fusion status was not correlated with STAT6 expression or useful in discriminating between malignant histological features or subsequent clinical outcomes in the genitourinary solitary fibrous tumours. Conclusions A subset of solitary fibrous tumours of the genitourinary tract behaved aggressively. Using a break-apart FISH probe cocktail, we found the NAB2-STAT6 gene fusion in 64% of cases. However, the NAB2-STAT6 fusion status was not correlated with STAT6 expression or useful in discriminating between low-risk or high-risk tumours and subsequent clinical outcomes

Genetic analyses of undifferentiated small round cell sarcoma identifies a novel sarcoma subtype with a recurrent CRTC1-SS18 gene fusion

In recent years, undifferentiated small round cell sarcomas (USRCSs) have been divided into a variety of new, rare, sarcoma subtypes, including the group of Ewing-like sarcomas, which have the morphological appearance of Ewing sarcomas, but carry CIC-DUX4, BCOR-CCNB3 and other gene fusions different from the classic EWSR1-ETS gene fusion. Using high-throughput RNA-sequencing (RNA-seq) analyses, we identified a novel recurrent gene fusion, CRTC1-SS18, in two cases of USRCS that lacked any known translocation. RNA-seq results were confirmed by reverse transcription polymerase chain reaction, long-range polymerase chain reaction, and fluorescence in situ hybridization. In vitro, we showed that the cells expressing the gene fusion were morphologically distinct and had enhanced oncogenic potential as compared with control cells. Expression profile comparisons with tumours of other sarcoma subtypes demonstrated that both cases clustered close to EWSR1-CREB1-positive tumours. Moreover, these analyses indicated enhanced NTRK1 expression in CRTC1-SS18-positive tumours. We conclude that the novel gene fusion identified in this study adds a new subtype to the USRCSs with unique gene signatures, and may be of therapeutic relevance. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd

Evaluation of NAB2-STAT6 Fusion Variants and Other Molecular Alterations as Prognostic Biomarkers in a Case Series of 83 Solitary Fibrous Tumors

Risk stratification of solitary fibrous tumor (SFT) patients based on clinicopathological features has limited efficacy, especially in predicting late relapse or metastasis. The hallmark alteration of SFT is the gene fusion NAB2-STAT6, whose prognostic value remains controversial. As biological knowledge of this entity has increased in recent years, new molecular alterations have emerged that could be helpful to refine current risk models. Here, we evaluated NAB2-STAT6 fusion variants and other molecular alterations in a series of 83 SFTs that are enriched in progressing cases. Gene fusion variants were identified by targeted RNA-seq in the whole series, whereas TERT promoter (pTERT) mutations were inspected by Sanger sequencing in a subset of 18 cases. Immunohistochemical assays were performed to assess BCOR and NTRK expression as well as P53 mutation status in 45, 44, and 44 cases, respectively. While confirming the associations of gene fusion variants with clinicopathological parameters, our results do not prove their prognostic value. Pan-TRK immunoexpresion correlated with recurrence/progression, P53 staining associated with higher mitotic counts, and pTERT mutations were enriched in cases with fatal outcome. An intriguing correlation was found for BCOR protein expression with gene fusion variants, size, and tumor location

A rare case of paediatric astroblastoma with concomitant MN1-GTSE1 and EWSR1-PATZ1 gene fusions altering management

In a case of astroblastoma, methylation analysis was uninformative, with no clustering with known CNS-HGNET-MN1 cases. Whole genome sequencing however identified a novel MN1-GTSE1 gene fusion (image), confirming the diagnosis of astroblastoma, as well as an EWSR1-PATZ1 gene fusion. Whole genome sequencing, alongside methylation profiling and conventional neuropathology, will continue to lead to improved diagnostics and prognostication for children with brain tumours

Exosomes: A Novel Zika Virus Vaccine Candidate

With the recent emergence of Zika virus (ZIKV) diseases, increasing global concern has driven the demand for a vaccine. One promising vaccine platform has presented itself in the form of exosomes: a subgroup of extracellular vesicles released by many human cell types that facilitate intercellular communication. The objective of this study is to engineer exosomes that incorporate ZIKV structural proteins into its phospholipid bilayer. Previous studies indicate that CD9 and CD63 proteins are highly enriched in exosomal membranes. From this, it was hypothesized that attaching ZIKV genes to CD9 or CD63 to produce a gene fusion may enable exosomes to act as antigen-presenting vesicles. These engineered exosomes may potentially stimulate T-cells to mount a strong immune response. The cDNA of the CD9, CD63, and the highly immunogenic ZIKV genes (envelope, precursor membrane, and NS1) were generated using RT-PCR. These products were used as a template for regular PCR, and cloned into pcDNA3.1/V5 vector. The chimeric gene fusion was assembled using the Gibson assembly kit, and transfected into human embryonic kidney epithelial (HEK293T) cells for expression. The exosomes were purified from the supernatant and subjected to immunoblotting and immunofluorescence assays to confirm the presence of ZIKV proteins. The results of this study are pending at the time of this abstract submission. A future study will be conducted using an in vitro activation assay to determine if the engineered exosomes induce T-cell activation. The potential candidates will be used in an animal study for immunity against ZIKV infection