BACKGROUND
cancers result from the accumulation of genomic mutations and epigenetic alterations that change gene expression and function.
in particular, gene fusions have been recognized as an associated and significant feature of cancer since the characterization of the philadelphia chromosome  <cit> .
the occurrence of gene fusions in solid tumors has long been noted, but their importance has been appreciated only recently, largely due to high throughput technologies such as transcriptome sequencing  .
rna-seq permits genome-wide transcription analysis for novel transcript discovery.
rna-seq has spurred important gene fusion discoveries for a number of different cancers, including lung  <cit> , prostate  <cit> , breast , brain  <cit> , thyroid  <cit>  and bladder carcinomas  <cit> .
one obvious benefit from gene fusion discovery is the potential to develop novel treatments that target these genetic abnormalities.
the eml4-alk translocation fusion is an example in which the fusion causes constitutive kinase activity.
mouse fibroblasts transfected with eml4-alk formed tumors when this fusion was injected into nude mice, thus demonstrating the oncogenic activity of the resultant protein  <cit> .
crizotinib, a competitive inhibitor of alk, has recently been granted fda approval for the treatment of specific late-stage, non-small cell lung cancers, and presently there are two phase  <dig> trials in progress  <cit> .
glioblastoma multiforme , a grade iv astrocytoma, is the most common form of primary brain cancer, with a median survival of approximately 1 year after multi-modal treatments  <cit> .
recent studies suggest that nearly 80% of all malignant brain tumors are accounted for by the broad category of gliomas, and 54% of all malignant brain tumors are gbm  <cit> .
the first fusion protein discovered in glioblastoma was the fig-ros <dig> fusion, in which an intra-chromosomal deletion of 240 kb leads to a constitutively active kinase, suggesting oncogenic activity  <cit> .
two more studies reported fusions of pdgfra-kdr  <cit>  and leo1-slc12a <dig>  <cit> , each in a single patient sample.
the fgfr-tacc fusion is one of the recurrent fusions in gbm and it has been reported in three studies  <cit> .
the oral administration of an fgfr inhibitor has been shown to prolong the survival of mice harboring intracranial fgfr-tacc-initiated glioma  <cit> .
egfr-sept <dig> fusions present in about 4% of gbms were shown to be functional and sensitive to egfr inhibition in a recent study  <cit> .
in this study, we focus on identification of gene fusion events from gbm transcriptome data.
using our in-house pipeline, we identify and validate  <dig> fusion events in  <dig> gbm samples by analyzing solid single-end 50 bp data.
we also identify  <dig> high-confidence gene fusion events in  <dig> gbm samples by analyzing tcga illumina hiseq paired-end 75 bp transcriptome data.
we integrate gene fusion data with copy number data to elucidate fusion mechanisms in gbm.
RESULTS
gene fusion discovery pipeline for solid single-end 50 bp data
we profiled the transcriptome of  <dig> gbm samples and  <dig> non-tumor samples using the solid sequencer.
we generated 50 bp single-end rna-seq reads with sequencing depths ranging from  <dig> to  <dig> million reads .
a variety of software packages are available for gene fusion discovery for illumina paired-end, illumina single-end and solid paired-end data .
we developed an in-house gene fusion discovery pipeline, as no software package was available for single-end solid data .
first, we aligned all the reads and calculated reads per kilobase per million  for each exon using bioscope  <dig>  software package  <cit> .
gene annotations were combined from three databases: ensembl gene annotation version  <dig>  ucsc and refseq genes .
cancer outlier profile analysis   <cit>  was performed for each exon to identify exons with substantially higher expression in a small set of samples.
we evaluated the expression variation at 5′ and 3′ exons of all genes that had at least one exon with outlier expression levels.
we extracted reads that partially mapped to the junction where there was a significant change in expression levels for a given gene.
we then constructed a consensus sequence from the partially extracted reads.
after converting the consensus sequence from color space to base space format, we used the ucsc blat tool  <cit>  to map the consensus sequence to the human genome .
if part of the consensus sequence mapped to the original gene and the rest mapped uniquely to another region in the genome, then the sequence was considered a fusion sequence.
we identified  <dig> such sequences  in eight samples.
we were able to validate all of the  <dig> fusion transcripts using fusion qpcr followed by sanger sequencing.
figure  <dig> illustrates the mon2-mars gene fusion as one example of a fusion transcript.
the outlier expression of the mon <dig> and mars exons can be observed with a z-score >  <dig>  panel a shows the rna-seq read distribution across all exons for both genes.
the mon <dig> read distribution shows higher 5′ expression relative to its 3′ expression, and the mars read distribution shows higher 3′ expression relative to its 5′ expression.
partially mapped reads at exon  <dig> of mon <dig> and exon  <dig> of mars map to the mon2-mars fusion sequence.
panel b shows the gel image for fusion qpcr.
the product can be observed in sample sn <dig>  but not in non-tumor brain and mon2-mars fusion negative gbm samples.
panel c shows the sanger sequencing trace of the fusion pcr product.
detailed images for the other  <dig> fusion sequences are available in additional file  <dig> figure  <dig>
gene fusion discovery pipeline for the ivy center solid single-end data.
reads were aligned to the hg <dig> assembly using bioscope- <dig>  software by life technologies.
rpkm values were calculated for each exon, followed by modified cancer outlier profile analysis .
if any of the exons of a gene displayed outlier expression in a sample, then the read distribution across that gene was evaluated for that sample.
if either the 3′ or 5′ end of the gene had a considerably lower rpkm value compared to the other, the gene was further evaluated for fusion.
all partially mapped sequences to a potential fusion breakpoint were extracted.
one or more consensus sequence was generated and translated to base space format from the color space format.
the consensus sequences were then aligned to the hg <dig> human genome using ucsc blat.
if part of the consensus sequence mapped to the known exon and the other part uniquely mapped to the genome, the sequence was considered a potential fusion sequence.
all potential fusion sequences were validated with fusion qpcr followed by sanger sequencing.
ivy center fusions
fusions identified in the ivy center solid single-end dataset.
example of a gene fusion identified in the ivy center solid single-end data.
this figure illustrates the mon2-mars gene fusion.
a. outlier expression of the mon <dig> and mars exons can be observed with a z-score >  <dig>  mon <dig> read distribution shows a drop after exon  <dig>  and mars read distribution shows a rise at exon  <dig>  partially mapped reads at exon  <dig> of mon <dig> and exon  <dig> of mars map to the mon2-mars fusion sequence.
the sequence is represented both in color space and base space format.
the purple color indicates a mismatch in the color space format.
b. gel images showing the fusion qpcr result.
the product can be observed in sample sn <dig>  but not in the non-tumor brain and mon2-mars fusion negative gbm samples.
c. trace from sanger sequencing of the fusion pcr product.
gene fusions in the ivy center solid dataset
we identified  <dig> fusion transcripts in eight out of the  <dig> gbm samples .
two samples, sn <dig> and sn <dig>  harbored multiple fusions.
both fusion partners in eight of the fusion transcripts are well annotated genes.
five  out of these eight transcripts are predicted to be in-frame fusions coding for a chimeric protein product.
transcript creb1 → pard3b results in a c-terminal truncation of the 5′ fusion gene partner due to a frameshift.
in transcript plekha6 → pik3c2b, the entire pik3c2b coding sequence is preserved, but the fusion junction is at a novel 5′ utr exon for plekha <dig>  in transcript lancl2 → rp11-745c <dig>  the 5′ partner gene fuses with a non-coding rna resulting in c-terminal truncation.
for the other five fusion transcripts, the 5′ partner gene fuses with genomic sequence without gene annotation, denoted as “uar” in table  <dig>  three of these transcripts  result in c-terminal truncation of the 5′ partner genes.
one transcript  is predicted to result in a shorter isoform with an extended 3′ utr, and one transcript does not have any predicted protein product .
we had tissue available from surgery at recurrence for patient sn <dig>  and we were able to validate the presence of sec61g → uar at recurrence.
predicted protein sequences are provided in additional file  <dig>  all fusions are intra-chromosomal in our cohort, and fusion partners are in close proximity, ranging from a distance of  <dig>  million base pairs to  <dig> kilo base pairs between the two partners.
although there are no recurrent fusions in our small cohort, there are multiple genes, slc35e <dig>  pik3c2b and znf <dig>  that occurred in more than one fusion transcript.
an fgfr3 → tacc <dig> fusion was recently reported by three independent studies as a recurrent gene fusion  <cit> .
all our fusion transcripts are highly overexpressed compared to their wildtype gene partners, as is evident in the third column in table  <dig>  which shows a much higher number of reads spanning the fusion junction compared to the number of reads spanning the known wildtype junctions.
one of the fusions, slc35e3 → uar, has two isoforms.
gene fusions in the illumina hiseq tcga dataset
we downloaded rna-seq data for  <dig> tcga samples from cghub  <cit>  to explore the gene fusion landscape of gbm beyond our cohort.
the tcga transcriptome data are 75 bp paired-end reads generated using illumina hiseq with sequencing depths ranging from  <dig> to  <dig> million reads per sample.
we used the tophat-fusion  <cit>  and snowshoes-ftd  <cit>  software packages to identify fusions because both packages are expected to have a very low false-positive rate.
there were  <dig> and  <dig> fusion sequences identified by tophat-fusion and snowshoes ftd suggesting a large number of false positives .
the number of fusion sequences could be reduced by increasing the threshold for the minimum number of fusion spanning reads, but this modification can lead to the failure to identify some truly important fusions, such as cep85l → ros <dig>  in our solid dataset, the fgfr3 → tacc <dig> fusion has the second lowest number of junction spanning reads.
because our method resulted in a 100% validation rate, we applied filtering steps based on our method to the fusion sequences identified by both packages.
we required that at least one of the breakpoints must be a known exon boundary.
to reduce the likelihood of identifying passenger fusions  <cit> , we required that at least one of the fusion spanning reads must have a ratio of greater than two compared with its corresponding wild-type exon-exon spanning reads.
we discarded gene fusions involving adjacent genes.
exact details are provided in the methods section.
after curating fusion sequences from both packages, we obtained a set of  <dig> high-confidence fusion sequences, which was referred to as the curated set.
curated fusions were present in 53%  of patients, and 22%  of these patients harbored more than one fusion.
the curated fusion set is available in additional file  <dig>
gene fusions and copy number changes
the circos plot of all curated fusions  shows specific genomic hotspots where fusions occur in gbm.
two major genomic hotspots are on chromosomes  <dig>  and  <dig> .
in our solid dataset,  <dig> of  <dig> validated fusions were located on 7p <dig> and 12q14- <dig>  other regions with higher frequency of fusions are on chromosomes  <dig>   <dig>   <dig> and  <dig>  these genomic hotspots for fusions are the regions that are frequently amplified in gbm, as observed in figure  <dig> <cit> .
because affymetrix snp array data were available for all but two tcga samples, we looked for associations between fusion points and copy number data.
we downloaded level  <dig> segmented copy number data from tcga  <cit> .
the start and end points of each segment were considered to be the genomic breakpoints.
for the curated set, copy number data were available for  <dig> fusion sequences, out of which at least one of the partner genes harbored a genomic breakpoint in  <dig> cases .
we also predicted the fusion mechanism for each of the fusion sequences based on the copy number data.
figure  <dig> shows the distribution of different fusion mechanisms for all curated fusions.
we binned fusion mechanisms into six types:figure  <dig>
gene fusions identified in the tcga illumina hiseq paired-end data and the ivy center solid single-end data.
this circos plot shows all the curated fusions identified in tcga dataset.
fusions are represented by arcs.
the thickness of the arc represents the number of fusion-spanning reads.
the colors of the arc represent the likely mechanism of the fusion formation.
green arcs represent fusions formed by focal amplifications, cyan arcs represent fusions formed by tandem duplications, magenta arcs represent deletion-based fusions, dark orange arcs represent fusions formed by complex genomic rearrangements and blue arcs show the fusions formed by inter-chromosomal rearrangements.
the outer ring shows the fusion breakpoint density histogram at a given genomic location.
yellow triangles represent the fusions detected in the ivy center samples.
the size of the triangle indicates the number of breakpoints in that location.
the two rings outside of the ideogram show frequency of the copy number gain and loss in tcga samples.
two major genomic hotspots for fusions can be observed on chromosomes  <dig>  and  <dig> .
these regions also show frequent focal gains.
association between gene fusions and copy number changes in the tcga dataset.
a. distribution of each fusion mechanism over three chromosomes  with most fusions.
majority of fusions due to focal amplifications  are found on chromosome  <dig>  fusions formed due to complex genomic rearrangements are mostly present on chromosomes  <dig> and  <dig>  b. fusion without associated copy number changes.
c. fusion points within a genomic amplicon.
d. fusions due to tandem duplications.
e. black arc showing fusion due to interstitial genomic deletion.
f. fusions due to complex genomic rearrangements.
g. inter-chromosomal fusion with at least one fusion point near a genomic breakpoint.
h. distribution of fusion mechanisms for  <dig> fusion sequences.
eight percent of the fusion sequences do not have associated copy number changes.
forty percent of fusion sequences are formed by complex genomic rearrangements.
fusions in the proneural subtype are mostly formed by complex genomic rearrangements.
no copy number changes - there are no genomic breakpoints around fusion points.
these could be either inter- or intra-chromosomal fusions, see figure 4b.
focal amplifications - fusion points are within a genomic amplicon, see figure 4c.
tandem duplications - fusion points are around the start and end of an amplified genomic segment, see figure 4d.
deletion-based - fusion points are around the start and end of a relatively deleted genomic segment, see figure 4e.
complex genomic rearrangements - both fusion points are around genomic breakpoints with multiple segments between the two breakpoints, see figure 4f.
inter-chromosomal - fusion partners are located on different chromosomes with at least one fusion point near a genomic breakpoint, see figure 4g.
only 8% of the fusions are without accompanying copy number changes suggesting that the majority of the fusions in gbm are associated with unbalanced genomic rearrangements.
majority of the fusions in focal amplicons are present on chromosome  <dig> and restricted to the egfr locus, see figure  <dig> and figure 4a.
approximately 40% of all the fusions in gbm result from complex genomic rearrangements , see figure 4h.
some of the inter-chromosomal rearrangements also display complex fusion mechanisms, see figure 4g.
a recent study analyzed whole genome sequencing data and showed a high incidence of cgr in gbm resulting from chromothripsis—39% in gbm compared to 9% in other tumor types  <cit> .
fusions generated through cgrs are largely present on chromosomes  <dig> and  <dig>  see figure 4a.
the distribution of cgr based fusions on chromosome  <dig> is largely restricted to 12q14- <dig> amplicon, see figure  <dig>  even though partners of fusion sequences formed due to cgrs belong to different copy number segments, they have highly correlated copy number value, see figure 4f.
this suggests co-amplification of segments involving fusion.
gene fusions and molecular features
we checked to see if samples with at least one curated fusion were enriched in any clinically associated molecular features.
we did not find any association with presence of egfr viii, mutation/homozygous deletion of pten or tp <dig>  mutation of idh <dig> or g-cimp status .
amplifications of egfr were more prevalent in samples with at least one fusion compared to samples with no fusions .
we observed that the samples with classical subtype were more likely to have fusions  and that samples with mesenchymal subtype were less likely to have fusions , see figure 5a.
this result can be explained by the association of genomic fusion hotspots with subtypes.
almost all of the samples with a classical subtype have focal amplification of the egfr locus, and samples with a mesenchymal subtype have a much lower incidence of focal amplifications on chromosomes  <dig> and  <dig>  <cit> .
figure 5b shows the chromosomal distribution of fusion breakpoints for each subtype.
the majority of the fusions in the classical subtype are located on chromosomes  <dig>   <dig> and  <dig>  fusions in the mesenchymal subtype are mostly present on chromosomes  <dig>   <dig>   <dig> and  <dig>  whereas chromosomes  <dig> and  <dig> harbor the majority of the fusions with the proneural subtype.
proneural subtype shows enrichment of fusions formed by complex genomic rearrangements, see figure 4h.
gene fusions in samples with the neural subtype have a broader chromosomal distribution, with the majority of breakpoints on chromosomes  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> figure  <dig>
association between gene fusions and molecular subtype in the tcga dataset.
a. seventy-two percent of the samples with a classical subtype had at least one fusion event compared to 39% of samples with a mesenchymal subtype .
b. chromosomal distribution of fusion events for different molecular subtypes.
predicted structure of the curated fusions set
we predicted the amino acid sequence of all curated fusions based on their chimeric nucleotide sequence.
a significant portion of the fusions  were predicted to be in-frame fusions with amino acid sequences present from both fusion partner genes.
another 18% were predicted to have c-terminal truncation due to either the out of frame fusion with another gene or fusion with an unannotated region.
approximately 8% of fusions are predicted to result in the same protein product as its 3′ partner gene by borrowing only the promoter from the 5′ partner.
in approximately 10% of the fusions, the 5′ gene partner is predicted to contribute only the promoter, but the n-terminal of the 3′ gene is truncated.
we also observed another novel class of fusions that involve non-coding rna genes.
in approximately 14%  of the fusions, the 5′ partner gene is predicted to have a c-terminal truncation due to fusion with a non-coding rna.
these fusions also result in the expression of non-coding rnas that are not expressed in other samples.
another important set of fusions involve tyrosine kinases.
in  <dig> cases, the fusion sequences retained the tyrosine kinase domain.
egfr , fgfr <dig>  and ntrk <dig>  were recurrently fused.
other kinase genes included ephb <dig>  flt <dig> and ros <dig>
recurrent gene fusions
although more than half of gbms showed evidence of gene fusions, there were very few fusions that were present in more than one sample.
one of those fusions is the already reported fgfr3 → tacc <dig> fusion, which was found in two patients in the tcga cohort and in one patient in the ivy center cohort.
egfr → sept <dig>  an in-frame fusion with c-terminal deletion of egfr, was found in three patients in the tcga cohort.
lancl2 → sept <dig>  an out of frame fusion that leads to the c-terminal truncation of lancl <dig>  was found in two tcga patients.
two additional patients, one tcga and one ivy center, had fusions of lancl <dig> with non-coding rna rp11-745c <dig> , which also resulted in the c-terminal truncation of lancl <dig>  the same non-coding rna rp11-745c <dig>  fused with egfr in two tcga patients, resulting in the c-terminal truncation of egfr.
there are  <dig> genes that are fusion partners in more than one patient sample .
the majority of these genes  are on genomic fusion hotspots located on chromosomes  <dig> and  <dig> table  <dig>
fusions involving genes that partner in more than one fusion
discussion
our study highlights the prevalence of gene fusions as one of the major genomic abnormalities in gbm.
fusions occur in approximately 30-50% of gbm patient samples.
in the ivy center cohort of  <dig> patients, 33% of samples harbored fusions that were validated by qpcr and sanger sequencing.
we were able to identify high-confidence gene fusions from rna-seq data in 53% of samples in a tcga cohort of  <dig> patients.
we identified  <dig> cases  with fusions retaining the tyrosine kinase domain in the tcga cohort and one case in the ivy center cohort.
recent advances in the development of tyrosine kinase inhibitors  have demonstrated that these drugs can provide significant benefit to patients whose tumors have a specific genetic abnormality.
we also identified a novel class of fusions  that result in the c-terminal truncation of its 5′ partner due to fusion with non-coding rna genes.
one such case was also present in the ivy center cohort.
this study reveals the diversity of gene fusions in gbm samples.
the majority of the fusions are private fusions occurring in one patient.
there are a few fusions that recur at low frequency in gbm.
our study is the first to provide a comprehensive view of the gene fusion landscape in gbm by examining sequences from  <dig> patients from two independent cohorts.
we successfully utilized our in-house pipeline for fusion discovery using solid single-end, 50 bp rna-seq data with a 100% validation rate.
for the tcga cohort, we used two different gene fusion detection software packages to comprehensively identify fusions from illumina paired-end, 75 bp rna-seq data.
ours is the first study to describe recurrent fusions involving non-coding genes.
we combined copy number data with gene fusion discovery to elucidate mechanisms of the formation of gene fusions in gbm.
all of the fusions detected in this study can be further visualized and analyzed on our website .
we were able to validate all of the fusions in our solid single-end rna-seq data by using strict filtering criteria.
it is likely that we may have underestimated fusions for ivy center data.
due to lack of access to the tissue samples, we could not determine the validation rate for our set of curated fusions in the tcga cohort.
the curated fusion set did have a significantly higher percentage of fusions associated with copy number changes relative to the low-confidence set.
we applied filters to discard likely passenger fusions  <cit> , but the functional significance of these fusions still needs to be evaluated.
singh et al.
was the first study to describe multiple fusions of fgfr-tacc in gbm, reporting this phenomenon in  <dig> of the  <dig> tumors examined.
they showed that the fusion protein has oncogenic activity when introduced into astrocytes and oral administration of an fgfr inhibitor prolongs the survival of mice harboring intracranial fgfr-tacc-initiated glioma  <cit> .
a second study by parker et al.
showed that the fusion gene is overexpressed by escaping mir-99a regulation due to loss of the 3′ utr of fgfr <dig>  <cit> .
in their cohort,  <dig> out of  <dig> samples harbored the fgfr3 → tacc <dig> fusion.
in our ivy center cohort, the fgfr3 → tacc <dig> fusion was detected in one out of  <dig> samples.
we tested for this fusion in an additional  <dig> samples in addition to the  <dig> rna-seq samples, but did not detect any fusion events.
in the tcga cohort,  <dig> of  <dig> samples harbored the fgfr3 → tacc <dig> fusion.
fusions of fgfr genes are identified in other cancers, including bladder cancer, cholangiocarcinoma, squamous lung cancer, breast cancer, thyroid cancer, oral cancer, head and neck squamous cell carcinoma and prostate cancer  <cit> .
tropomyosin-receptor kinases  are known to play a role in cancer biology.
rearrangements of the ntrk <dig> gene are consistently observed in a small fraction of papillary thyroid carcinomas  <cit> .
we identified two cases of ntrk <dig> fusions in the tcga cohort.
frattini et al.
<cit>  screened  <dig> samples for nfasc-ntrk <dig> fusion but did not find any.
we identified a single case of a cep85l-ros <dig> fusion in the tcga patient samples.
in a recent study by giacomini et al.
<cit> , a cep85l-ros <dig> fusion was detected for angiosarcoma.
there have been two more reported cases, one angiosarcoma and one epithelioid hemangioendothelioma, with ros <dig> rearrangements.
ros <dig> rearrangements also define a unique molecular subclass of lung cancer that may respond to an alk inhibitor  <cit> .
we identified fusions of egfr in nine patient samples from the tcga cohort, out of which six retained the tyrosine kinase domain and resulted in a carboxyl-terminal truncation.
a study by cho et al.
has shown that cetuximab prolonged the survival of intracranially xenografted mice with oncogenic egfr carboxyl-terminal deletion mutants compared with untreated control mice  <cit> .
it is likely that patients with fusions of egfr leading to carboxyl-terminal truncation will show sensitivity to egfr inhibitors.
frattini et al.
<cit>  showed that egfr-sept <dig> fusions which occur in about 4% of gbms was a functional gene fusion in gbm and confers mitogen independence and sensitivity to egfr inhibition.
a total of  <dig> cases from both cohorts have fusions of genes involved in chromatin remodeling and modification.
these genes include arid1a, arid1b, ash1l, chd <dig>  hdac <dig>  hmga <dig>  jmjd1c, kdm4b, rere, setd1b and yeats <dig>  arid1a-mast <dig> fusion has been shown to be a critical driver fusion in an mda-mb- <dig> breast cancer cell line  <cit> .
in  <dig> samples, the 5′ partner gene fuses with non-coding rna.
these fusions are predicted to have a c-terminal truncation.
these cases also have highly expressed non-coding rnas that are not expressed in other samples.
a recent study by zhang et al.
<cit>  discovered a signature comprising of six long non-coding rna that predicts survival in gbm.
there is now growing evidence of an oncogenic and tumor suppressive role for long, non-coding rnas in tumor biology  <cit> .
their identification in gene fusion events has thus far been neglected, as most studies focus on fusions of the coding genes.
even though gene fusion events in gbm are abundant with scarce recurrent events, they are not random events.
majority of the fusion events occur at 7p <dig>  12q14- <dig>  1q <dig> and 4q <dig> which are also recurrently amplified regions in gbm.
these fusion hotspots are consistent in both ivy and tcga cohorts.
also majority of the fusion events are due to unbalanced genomic rearrangements.
analysis of whole genome sequencing data also showed that 88% of genic rearrangements in gbm are associated with copy number alterations  <cit> .
some of the key genes implicated in gbm biology within these hotspots are egfr, mdm <dig>  cdk <dig>  pik3c2b, mdm <dig> and pdgfra.
a recent study  <cit>  identified a dense breakpoint pattern on 12q14- <dig> indicative of local chromosome instability and defined this region as “breakpoint enriched region” .
they showed that patients with ber pattern had poor survival and this pattern was associated with mdm2/cdk <dig> co-amplification.
there are two other cancers, dedifferentiated liposarcomas and lung adenocarcinomas that also show mdm2/cdk <dig> co-amplification in 90% and 4% of cases respectively  <cit> .
all three types of cancer display distinct genomic aberration patterns in 12q14- <dig> region in spite of having mdm2/cdk <dig> co-amplification.
gbm samples show shattering of the region with alternate high level deletions and gains, lung adenocarcinomas mostly contain large amplified segments and dedifferentiated liposarcomas contain multiple amplified segments .
whole genome sequencing, copy number and rna-seq datasets show that gbms contain deletion bridges that connect these amplified segments and generate a large number of fusion transcripts.
such complex genomic rearrangements are more prevalent on chromosome  <dig> but not limited to as shown in the study by malhotra et al.
<cit>  where they analyzed whole genome sequencing data of  <dig> gbms.
about 40% of fusion transcripts are formed due to such complex genomic rearrangements.
with the advent of rna-seq technology the list of fusion sequences in solid tumors is growing exponentially but little is known about the mechanisms that facilitate fusion events.
the formation of the tmprss2-erg gene fusion that occurs in about 50% of prostate cancers has been shown to be facilitated by androgen signaling which induces proximity of the tmprss <dig> and erg genomic loci and then exposure to gamma irradiation which causes dna double-strand breaks  <cit> .
the overview of the fusion landscape in gbm leads to questions about what mechanisms are responsible for generating highly site specific dna double-strand breaks and then joining of these breaks that result in complex genomic rearrangements.
CONCLUSIONS
gene fusions are frequent genomic abnormalities in gbm.
the majority of the fusions are private fusions, with a minority recurring in multiple patients.
complex genomic rearrangements are the major mechanism by which fusions are formed in gbm.
due to the low frequency and rarity of clinically relevant fusions, rna-seq of gbm patient samples is an essential tool for the identification of patient specific fusions that can drive personalized therapy.

 cancers result from the accumulation of genomic mutations and epigenetic alterations that change gene expression and function. in particular, gene fusions have been recognized as an associated and significant feature of cancer since the characterization of the philadelphia chromosome. the occurrence of gene fusions in solid tumors has long been noted, but their importance has been appreciated only recently, largely due to high throughput technologies such as transcriptome sequencing. rna-seq permits genome-wide transcription analysis for novel transcript discovery. rna-seq has spurred important gene fusion discoveries for a number of different cancers, including lung, prostate, breast, brain, thyroid and bladder carcinomas. one obvious benefit from gene fusion discovery is the potential to develop novel treatments that target these genetic abnormalities. the eml4-alk translocation fusion is an example in which the fusion causes constitutive kinase activity. mouse fibroblasts transfected with eml4-alk formed tumors when this fusion was injected into nude mice, thus demonstrating the oncogenic activity of the resultant protein. crizotinib, a competitive inhibitor of alk, has recently been granted fda approval for the treatment of specific late-stage, non-small cell lung cancers, and presently there are two phase trials in progress. glioblastoma multiforme, a grade iv astrocytoma, is the most common form of primary brain cancer, with a median survival of approximately 1 year after multi-modal treatments. recent studies suggest that nearly 80% of all malignant brain tumors are accounted for by the broad category of gliomas, and 54% of all malignant brain tumors are gbm. the first fusion protein discovered in glioblastoma was the fig-ros fusion, in which an intra-chromosomal deletion of 240 kb leads to a constitutively active kinase, suggesting oncogenic activity. two more studies reported fusions of pdgfra-kdr and leo1-slc12a, each in a single patient sample. the fgfr-tacc fusion is one of the recurrent fusions in gbm and it has been reported in three studies. the oral administration of an fgfr inhibitor has been shown to prolong the survival of mice harboring intracranial fgfr-tacc-initiated glioma. egfr-sept fusions present in about 4% of gbms were shown to be functional and sensitive to egfr inhibition in a recent study. in this study, we focus on identification of gene fusion events from gbm transcriptome data. using our in-house pipeline, we identify and validate fusion events in gbm samples by analyzing solid single-end 50 bp data. we also identify high-confidence gene fusion events in gbm samples by analyzing tcga illumina hiseq paired-end 75 bp transcriptome data. we integrate gene fusion data with copy number data to elucidate fusion mechanisms in gbm.  gene fusion discovery pipeline for solid single-end 50 bp data we profiled the transcriptome of gbm samples and non-tumor samples using the solid sequencer. we generated 50 bp single-end rna-seq reads with sequencing depths ranging from to million reads. a variety of software packages are available for gene fusion discovery for illumina paired-end, illumina single-end and solid paired-end data. we developed an in-house gene fusion discovery pipeline, as no software package was available for single-end solid data. first, we aligned all the reads and calculated reads per kilobase per million for each exon using bioscope software package. gene annotations were combined from three databases: ensembl gene annotation version ucsc and refseq genes. cancer outlier profile analysis was performed for each exon to identify exons with substantially higher expression in a small set of samples. we evaluated the expression variation at 5′ and 3′ exons of all genes that had at least one exon with outlier expression levels. we extracted reads that partially mapped to the junction where there was a significant change in expression levels for a given gene. we then constructed a consensus sequence from the partially extracted reads. after converting the consensus sequence from color space to base space format, we used the ucsc blat tool to map the consensus sequence to the human genome. if part of the consensus sequence mapped to the original gene and the rest mapped uniquely to another region in the genome, then the sequence was considered a fusion sequence. we identified such sequences in eight samples. we were able to validate all of the fusion transcripts using fusion qpcr followed by sanger sequencing. figure  illustrates the mon2-mars gene fusion as one example of a fusion transcript. the outlier expression of the mon and mars exons can be observed with a z-score > panel a shows the rna-seq read distribution across all exons for both genes. the mon read distribution shows higher 5′ expression relative to its 3′ expression, and the mars read distribution shows higher 3′ expression relative to its 5′ expression. partially mapped reads at exon of mon and exon of mars map to the mon2-mars fusion sequence. panel b shows the gel image for fusion qpcr. the product can be observed in sample sn but not in non-tumor brain and mon2-mars fusion negative gbm samples. panel c shows the sanger sequencing trace of the fusion pcr product. detailed images for the other fusion sequences are available in additional file figure gene fusion discovery pipeline for the ivy center solid single-end data. reads were aligned to the hg assembly using bioscope- software by life technologies. rpkm values were calculated for each exon, followed by modified cancer outlier profile analysis. if any of the exons of a gene displayed outlier expression in a sample, then the read distribution across that gene was evaluated for that sample. if either the 3′ or 5′ end of the gene had a considerably lower rpkm value compared to the other, the gene was further evaluated for fusion. all partially mapped sequences to a potential fusion breakpoint were extracted. one or more consensus sequence was generated and translated to base space format from the color space format. the consensus sequences were then aligned to the hg human genome using ucsc blat. if part of the consensus sequence mapped to the known exon and the other part uniquely mapped to the genome, the sequence was considered a potential fusion sequence. all potential fusion sequences were validated with fusion qpcr followed by sanger sequencing. ivy center fusions fusions identified in the ivy center solid single-end dataset. example of a gene fusion identified in the ivy center solid single-end data. this figure illustrates the mon2-mars gene fusion. a. outlier expression of the mon and mars exons can be observed with a z-score > mon read distribution shows a drop after exon and mars read distribution shows a rise at exon partially mapped reads at exon of mon and exon of mars map to the mon2-mars fusion sequence. the sequence is represented both in color space and base space format. the purple color indicates a mismatch in the color space format. b. gel images showing the fusion qpcr result. the product can be observed in sample sn but not in the non-tumor brain and mon2-mars fusion negative gbm samples. c. trace from sanger sequencing of the fusion pcr product. gene fusions in the ivy center solid dataset we identified fusion transcripts in eight out of the gbm samples. two samples, sn and sn harbored multiple fusions. both fusion partners in eight of the fusion transcripts are well annotated genes. five out of these eight transcripts are predicted to be in-frame fusions coding for a chimeric protein product. transcript creb1 → pard3b results in a c-terminal truncation of the 5′ fusion gene partner due to a frameshift. in transcript plekha6 → pik3c2b, the entire pik3c2b coding sequence is preserved, but the fusion junction is at a novel 5′ utr exon for plekha in transcript lancl2 → rp11-745c the 5′ partner gene fuses with a non-coding rna resulting in c-terminal truncation. for the other five fusion transcripts, the 5′ partner gene fuses with genomic sequence without gene annotation, denoted as “uar” in table  three of these transcripts result in c-terminal truncation of the 5′ partner genes. one transcript is predicted to result in a shorter isoform with an extended 3′ utr, and one transcript does not have any predicted protein product. we had tissue available from surgery at recurrence for patient sn and we were able to validate the presence of sec61g → uar at recurrence. predicted protein sequences are provided in additional file all fusions are intra-chromosomal in our cohort, and fusion partners are in close proximity, ranging from a distance of million base pairs to kilo base pairs between the two partners. although there are no recurrent fusions in our small cohort, there are multiple genes, slc35e pik3c2b and znf that occurred in more than one fusion transcript. an fgfr3 → tacc fusion was recently reported by three independent studies as a recurrent gene fusion. all our fusion transcripts are highly overexpressed compared to their wildtype gene partners, as is evident in the third column in table  which shows a much higher number of reads spanning the fusion junction compared to the number of reads spanning the known wildtype junctions. one of the fusions, slc35e3 → uar, has two isoforms. gene fusions in the illumina hiseq tcga dataset we downloaded rna-seq data for tcga samples from cghub to explore the gene fusion landscape of gbm beyond our cohort. the tcga transcriptome data are 75 bp paired-end reads generated using illumina hiseq with sequencing depths ranging from to million reads per sample. we used the tophat-fusion and snowshoes-ftd software packages to identify fusions because both packages are expected to have a very low false-positive rate. there were and fusion sequences identified by tophat-fusion and snowshoes ftd suggesting a large number of false positives. the number of fusion sequences could be reduced by increasing the threshold for the minimum number of fusion spanning reads, but this modification can lead to the failure to identify some truly important fusions, such as cep85l → ros in our solid dataset, the fgfr3 → tacc fusion has the second lowest number of junction spanning reads. because our method resulted in a 100% validation rate, we applied filtering steps based on our method to the fusion sequences identified by both packages. we required that at least one of the breakpoints must be a known exon boundary. to reduce the likelihood of identifying passenger fusions, we required that at least one of the fusion spanning reads must have a ratio of greater than two compared with its corresponding wild-type exon-exon spanning reads. we discarded gene fusions involving adjacent genes. exact details are provided in the methods section. after curating fusion sequences from both packages, we obtained a set of high-confidence fusion sequences, which was referred to as the curated set. curated fusions were present in 53% of patients, and 22% of these patients harbored more than one fusion. the curated fusion set is available in additional file gene fusions and copy number changes the circos plot of all curated fusions shows specific genomic hotspots where fusions occur in gbm. two major genomic hotspots are on chromosomes and. in our solid dataset, of validated fusions were located on 7p and 12q14- other regions with higher frequency of fusions are on chromosomes and these genomic hotspots for fusions are the regions that are frequently amplified in gbm, as observed in figure . because affymetrix snp array data were available for all but two tcga samples, we looked for associations between fusion points and copy number data. we downloaded level segmented copy number data from tcga. the start and end points of each segment were considered to be the genomic breakpoints. for the curated set, copy number data were available for fusion sequences, out of which at least one of the partner genes harbored a genomic breakpoint in cases. we also predicted the fusion mechanism for each of the fusion sequences based on the copy number data. figure  shows the distribution of different fusion mechanisms for all curated fusions. we binned fusion mechanisms into six types:figure gene fusions identified in the tcga illumina hiseq paired-end data and the ivy center solid single-end data. this circos plot shows all the curated fusions identified in tcga dataset. fusions are represented by arcs. the thickness of the arc represents the number of fusion-spanning reads. the colors of the arc represent the likely mechanism of the fusion formation. green arcs represent fusions formed by focal amplifications, cyan arcs represent fusions formed by tandem duplications, magenta arcs represent deletion-based fusions, dark orange arcs represent fusions formed by complex genomic rearrangements and blue arcs show the fusions formed by inter-chromosomal rearrangements. the outer ring shows the fusion breakpoint density histogram at a given genomic location. yellow triangles represent the fusions detected in the ivy center samples. the size of the triangle indicates the number of breakpoints in that location. the two rings outside of the ideogram show frequency of the copy number gain and loss in tcga samples. two major genomic hotspots for fusions can be observed on chromosomes and. these regions also show frequent focal gains. association between gene fusions and copy number changes in the tcga dataset. a. distribution of each fusion mechanism over three chromosomes with most fusions. majority of fusions due to focal amplifications are found on chromosome fusions formed due to complex genomic rearrangements are mostly present on chromosomes and b. fusion without associated copy number changes. c. fusion points within a genomic amplicon. d. fusions due to tandem duplications. e. black arc showing fusion due to interstitial genomic deletion. f. fusions due to complex genomic rearrangements. g. inter-chromosomal fusion with at least one fusion point near a genomic breakpoint. h. distribution of fusion mechanisms for fusion sequences. eight percent of the fusion sequences do not have associated copy number changes. forty percent of fusion sequences are formed by complex genomic rearrangements. fusions in the proneural subtype are mostly formed by complex genomic rearrangements. no copy number changes - there are no genomic breakpoints around fusion points. these could be either inter- or intra-chromosomal fusions, see figure 4b. focal amplifications - fusion points are within a genomic amplicon, see figure 4c. tandem duplications - fusion points are around the start and end of an amplified genomic segment, see figure 4d. deletion-based - fusion points are around the start and end of a relatively deleted genomic segment, see figure 4e. complex genomic rearrangements - both fusion points are around genomic breakpoints with multiple segments between the two breakpoints, see figure 4f. inter-chromosomal - fusion partners are located on different chromosomes with at least one fusion point near a genomic breakpoint, see figure 4g. only 8% of the fusions are without accompanying copy number changes suggesting that the majority of the fusions in gbm are associated with unbalanced genomic rearrangements. majority of the fusions in focal amplicons are present on chromosome and restricted to the egfr locus, see figure  and figure 4a. approximately 40% of all the fusions in gbm result from complex genomic rearrangements, see figure 4h. some of the inter-chromosomal rearrangements also display complex fusion mechanisms, see figure 4g. a recent study analyzed whole genome sequencing data and showed a high incidence of cgr in gbm resulting from chromothripsis—39% in gbm compared to 9% in other tumor types. fusions generated through cgrs are largely present on chromosomes and see figure 4a. the distribution of cgr based fusions on chromosome is largely restricted to 12q14- amplicon, see figure  even though partners of fusion sequences formed due to cgrs belong to different copy number segments, they have highly correlated copy number value, see figure 4f. this suggests co-amplification of segments involving fusion. gene fusions and molecular features we checked to see if samples with at least one curated fusion were enriched in any clinically associated molecular features. we did not find any association with presence of egfr viii, mutation/homozygous deletion of pten or tp mutation of idh or g-cimp status. amplifications of egfr were more prevalent in samples with at least one fusion compared to samples with no fusions. we observed that the samples with classical subtype were more likely to have fusions and that samples with mesenchymal subtype were less likely to have fusions, see figure 5a. this result can be explained by the association of genomic fusion hotspots with subtypes. almost all of the samples with a classical subtype have focal amplification of the egfr locus, and samples with a mesenchymal subtype have a much lower incidence of focal amplifications on chromosomes and. figure 5b shows the chromosomal distribution of fusion breakpoints for each subtype. the majority of the fusions in the classical subtype are located on chromosomes and fusions in the mesenchymal subtype are mostly present on chromosomes and whereas chromosomes and harbor the majority of the fusions with the proneural subtype. proneural subtype shows enrichment of fusions formed by complex genomic rearrangements, see figure 4h. gene fusions in samples with the neural subtype have a broader chromosomal distribution, with the majority of breakpoints on chromosomes and figure association between gene fusions and molecular subtype in the tcga dataset. a. seventy-two percent of the samples with a classical subtype had at least one fusion event compared to 39% of samples with a mesenchymal subtype. b. chromosomal distribution of fusion events for different molecular subtypes. predicted structure of the curated fusions set we predicted the amino acid sequence of all curated fusions based on their chimeric nucleotide sequence. a significant portion of the fusions were predicted to be in-frame fusions with amino acid sequences present from both fusion partner genes. another 18% were predicted to have c-terminal truncation due to either the out of frame fusion with another gene or fusion with an unannotated region. approximately 8% of fusions are predicted to result in the same protein product as its 3′ partner gene by borrowing only the promoter from the 5′ partner. in approximately 10% of the fusions, the 5′ gene partner is predicted to contribute only the promoter, but the n-terminal of the 3′ gene is truncated. we also observed another novel class of fusions that involve non-coding rna genes. in approximately 14% of the fusions, the 5′ partner gene is predicted to have a c-terminal truncation due to fusion with a non-coding rna. these fusions also result in the expression of non-coding rnas that are not expressed in other samples. another important set of fusions involve tyrosine kinases. in cases, the fusion sequences retained the tyrosine kinase domain. egfr, fgfr and ntrk were recurrently fused. other kinase genes included ephb flt and ros recurrent gene fusions although more than half of gbms showed evidence of gene fusions, there were very few fusions that were present in more than one sample. one of those fusions is the already reported fgfr3 → tacc fusion, which was found in two patients in the tcga cohort and in one patient in the ivy center cohort. egfr → sept an in-frame fusion with c-terminal deletion of egfr, was found in three patients in the tcga cohort. lancl2 → sept an out of frame fusion that leads to the c-terminal truncation of lancl was found in two tcga patients. two additional patients, one tcga and one ivy center, had fusions of lancl with non-coding rna rp11-745c, which also resulted in the c-terminal truncation of lancl the same non-coding rna rp11-745c fused with egfr in two tcga patients, resulting in the c-terminal truncation of egfr. there are genes that are fusion partners in more than one patient sample. the majority of these genes are on genomic fusion hotspots located on chromosomes and table fusions involving genes that partner in more than one fusion discussion our study highlights the prevalence of gene fusions as one of the major genomic abnormalities in gbm. fusions occur in approximately 30-50% of gbm patient samples. in the ivy center cohort of patients, 33% of samples harbored fusions that were validated by qpcr and sanger sequencing. we were able to identify high-confidence gene fusions from rna-seq data in 53% of samples in a tcga cohort of patients. we identified cases with fusions retaining the tyrosine kinase domain in the tcga cohort and one case in the ivy center cohort. recent advances in the development of tyrosine kinase inhibitors have demonstrated that these drugs can provide significant benefit to patients whose tumors have a specific genetic abnormality. we also identified a novel class of fusions that result in the c-terminal truncation of its 5′ partner due to fusion with non-coding rna genes. one such case was also present in the ivy center cohort. this study reveals the diversity of gene fusions in gbm samples. the majority of the fusions are private fusions occurring in one patient. there are a few fusions that recur at low frequency in gbm. our study is the first to provide a comprehensive view of the gene fusion landscape in gbm by examining sequences from patients from two independent cohorts. we successfully utilized our in-house pipeline for fusion discovery using solid single-end, 50 bp rna-seq data with a 100% validation rate. for the tcga cohort, we used two different gene fusion detection software packages to comprehensively identify fusions from illumina paired-end, 75 bp rna-seq data. ours is the first study to describe recurrent fusions involving non-coding genes. we combined copy number data with gene fusion discovery to elucidate mechanisms of the formation of gene fusions in gbm. all of the fusions detected in this study can be further visualized and analyzed on our website. we were able to validate all of the fusions in our solid single-end rna-seq data by using strict filtering criteria. it is likely that we may have underestimated fusions for ivy center data. due to lack of access to the tissue samples, we could not determine the validation rate for our set of curated fusions in the tcga cohort. the curated fusion set did have a significantly higher percentage of fusions associated with copy number changes relative to the low-confidence set. we applied filters to discard likely passenger fusions, but the functional significance of these fusions still needs to be evaluated. singh et al. was the first study to describe multiple fusions of fgfr-tacc in gbm, reporting this phenomenon in of the tumors examined. they showed that the fusion protein has oncogenic activity when introduced into astrocytes and oral administration of an fgfr inhibitor prolongs the survival of mice harboring intracranial fgfr-tacc-initiated glioma. a second study by parker et al. showed that the fusion gene is overexpressed by escaping mir-99a regulation due to loss of the 3′ utr of fgfr. in their cohort, out of samples harbored the fgfr3 → tacc fusion. in our ivy center cohort, the fgfr3 → tacc fusion was detected in one out of samples. we tested for this fusion in an additional samples in addition to the rna-seq samples, but did not detect any fusion events. in the tcga cohort, of samples harbored the fgfr3 → tacc fusion. fusions of fgfr genes are identified in other cancers, including bladder cancer, cholangiocarcinoma, squamous lung cancer, breast cancer, thyroid cancer, oral cancer, head and neck squamous cell carcinoma and prostate cancer. tropomyosin-receptor kinases are known to play a role in cancer biology. rearrangements of the ntrk gene are consistently observed in a small fraction of papillary thyroid carcinomas. we identified two cases of ntrk fusions in the tcga cohort. frattini et al. screened samples for nfasc-ntrk fusion but did not find any. we identified a single case of a cep85l-ros fusion in the tcga patient samples. in a recent study by giacomini et al., a cep85l-ros fusion was detected for angiosarcoma. there have been two more reported cases, one angiosarcoma and one epithelioid hemangioendothelioma, with ros rearrangements. ros rearrangements also define a unique molecular subclass of lung cancer that may respond to an alk inhibitor. we identified fusions of egfr in nine patient samples from the tcga cohort, out of which six retained the tyrosine kinase domain and resulted in a carboxyl-terminal truncation. a study by cho et al. has shown that cetuximab prolonged the survival of intracranially xenografted mice with oncogenic egfr carboxyl-terminal deletion mutants compared with untreated control mice. it is likely that patients with fusions of egfr leading to carboxyl-terminal truncation will show sensitivity to egfr inhibitors. frattini et al. showed that egfr-sept fusions which occur in about 4% of gbms was a functional gene fusion in gbm and confers mitogen independence and sensitivity to egfr inhibition. a total of cases from both cohorts have fusions of genes involved in chromatin remodeling and modification. these genes include arid1a, arid1b, ash1l, chd hdac hmga jmjd1c, kdm4b, rere, setd1b and yeats arid1a-mast fusion has been shown to be a critical driver fusion in an mda-mb- breast cancer cell line. in samples, the 5′ partner gene fuses with non-coding rna. these fusions are predicted to have a c-terminal truncation. these cases also have highly expressed non-coding rnas that are not expressed in other samples. a recent study by zhang et al. discovered a signature comprising of six long non-coding rna that predicts survival in gbm. there is now growing evidence of an oncogenic and tumor suppressive role for long, non-coding rnas in tumor biology. their identification in gene fusion events has thus far been neglected, as most studies focus on fusions of the coding genes. even though gene fusion events in gbm are abundant with scarce recurrent events, they are not random events. majority of the fusion events occur at 7p 12q14- 1q and 4q which are also recurrently amplified regions in gbm. these fusion hotspots are consistent in both ivy and tcga cohorts. also majority of the fusion events are due to unbalanced genomic rearrangements. analysis of whole genome sequencing data also showed that 88% of genic rearrangements in gbm are associated with copy number alterations. some of the key genes implicated in gbm biology within these hotspots are egfr, mdm cdk pik3c2b, mdm and pdgfra. a recent study identified a dense breakpoint pattern on 12q14- indicative of local chromosome instability and defined this region as “breakpoint enriched region”. they showed that patients with ber pattern had poor survival and this pattern was associated with mdm2/cdk co-amplification. there are two other cancers, dedifferentiated liposarcomas and lung adenocarcinomas that also show mdm2/cdk co-amplification in 90% and 4% of cases respectively. all three types of cancer display distinct genomic aberration patterns in 12q14- region in spite of having mdm2/cdk co-amplification. gbm samples show shattering of the region with alternate high level deletions and gains, lung adenocarcinomas mostly contain large amplified segments and dedifferentiated liposarcomas contain multiple amplified segments. whole genome sequencing, copy number and rna-seq datasets show that gbms contain deletion bridges that connect these amplified segments and generate a large number of fusion transcripts. such complex genomic rearrangements are more prevalent on chromosome but not limited to as shown in the study by malhotra et al. where they analyzed whole genome sequencing data of gbms. about 40% of fusion transcripts are formed due to such complex genomic rearrangements. with the advent of rna-seq technology the list of fusion sequences in solid tumors is growing exponentially but little is known about the mechanisms that facilitate fusion events. the formation of the tmprss2-erg gene fusion that occurs in about 50% of prostate cancers has been shown to be facilitated by androgen signaling which induces proximity of the tmprss and erg genomic loci and then exposure to gamma irradiation which causes dna double-strand breaks. the overview of the fusion landscape in gbm leads to questions about what mechanisms are responsible for generating highly site specific dna double-strand breaks and then joining of these breaks that result in complex genomic rearrangements.  gene fusions are frequent genomic abnormalities in gbm. the majority of the fusions are private fusions, with a minority recurring in multiple patients. complex genomic rearrangements are the major mechanism by which fusions are formed in gbm. due to the low frequency and rarity of clinically relevant fusions, rna-seq of gbm patient samples is an essential tool for the identification of patient specific fusions that can drive personalized therapy. 
fusions on 7p are formed in focally amplified egfr locus whereas 12q14- fusions are formed by complex genomic rearrangements. the majority of the fusions are private fusions, and a minority of these recur with low frequency. due to the low frequency and rarity of clinically relevant fusions, rna-seq of gbm patient samples will be a vital tool for the identification of patient-specific fusions that can drive personalized therapy. we identified cases with fusions retaining a tyrosine kinase domain in the tcga cohort and one case in the ivy center cohort. in this study, we provide comprehensive view of gene fusion landscape in glioblastoma multiforme patients from two independent cohorts. we were able to identify high-confidence gene fusions from rna-seq data in 53% of the samples in a tcga cohort of patients. gene fusion discovery can potentially lead to the development of novel treatments that target the underlying genetic abnormalities. rna-seq has spurred important gene fusion discoveries in a number of different cancers, including lung, prostate, breast, brain, thyroid and bladder carcinomas. ours is the first study to describe recurrent fusions involving non-coding genes. our study highlights the prevalence of gene fusions as one of the major genomic abnormalities in gbm. in the ivy center cohort of patients, 33% of samples harbored fusions that were validated by qpcr and sanger sequencing. fusions occur in approximately 30-50% of gbm patient samples. gene fusionglioblastomarna-seqegfr fusionsntrk1ros1fgfr3-tacc3pik3c2bnon-coding gene fusionsissue-copyright-statement© biomed central ltd 2013
 rna-seq has spurred important gene fusion discoveries in a number of different cancers, including lung, prostate, breast, brain, thyroid and bladder carcinomas. gene fusion discovery can potentially lead to the development of novel treatments that target the underlying genetic abnormalities.  in this study, we provide comprehensive view of gene fusion landscape in glioblastoma multiforme patients from two independent cohorts. fusions occur in approximately 30-50% of gbm patient samples. in the ivy center cohort of patients, 33% of samples harbored fusions that were validated by qpcr and sanger sequencing. we were able to identify high-confidence gene fusions from rna-seq data in 53% of the samples in a tcga cohort of patients. we identified cases with fusions retaining a tyrosine kinase domain in the tcga cohort and one case in the ivy center cohort. ours is the first study to describe recurrent fusions involving non-coding genes. genomic locations 7p and 12q14- harbor majority of the fusions. fusions on 7p are formed in focally amplified egfr locus whereas 12q14- fusions are formed by complex genomic rearrangements. all the fusions detected in this study can be further visualized and analyzed using our website: http://ivygap.swedish.org/fusions.  our study highlights the prevalence of gene fusions as one of the major genomic abnormalities in gbm. the majority of the fusions are private fusions, and a minority of these recur with low frequency. a small subset of patients with fusions of receptor tyrosine kinases can benefit from existing fda approved drugs and drugs available in various clinical trials. due to the low frequency and rarity of clinically relevant fusions, rna-seq of gbm patient samples will be a vital tool for the identification of patient-specific fusions that can drive personalized therapy. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords gene fusionglioblastomarna-seqegfr fusionsntrk1ros1fgfr3-tacc3pik3c2bnon-coding gene fusionsissue-copyright-statement© biomed central ltd 2013 