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Team Finds New Cancer Gene Fusions Using Paired End Sequencing

NEW YORK (GenomeWeb News) – A paper scheduled to appear online this week in the Proceedings of the National Academy of Sciences suggests paired-end transcriptome sequencing can identify previously undetected gene fusions — even in well-characterized human cancer lines — and offer clues about which fusions may drive cancer development.

In their search for new gene fusions, a team of scientists from the Michigan Center for Translational Pathology, the University of Michigan, and Illumina did paired-end sequencing of RNA transcripts from four human cell lines using the Illumina Genome Analyzer. Their search turned up several known fusions as well as a dozen more that were previously missed.

Based on these results, the team concluded that paired-end transcriptome sequencing is an affordable way to systematically track down chimeric RNA transcripts. And, they argued, quantifying the reads supporting each fusion may offer insights into driver fusions involved in various cancer types.

"By using a paired-end approach, we were able to rediscover known gene fusions, comprehensively discover previously undescribed gene fusions, and hone in on causal gene fusions," Arul Chinnaiyan, Howard Hughes Medical Institute investigator affiliated with the University of Michigan and the Michigan Institute for Translational Pathology, and his co-workers wrote.

More than four-fifths of known gene fusions have been discovered in leukemia, lymphoma, or bone and soft tissue sarcomas. Nevertheless, gene fusions have also turned up in other types of cancer as well, including non-small cell lung cancer and prostate cancer.

Chinnaiyan and his colleagues found the first prostate cancer gene fusion — between a protease gene called TMPRSS2 and either ERG or ETV1, transcription factors in the ETS family — several years ago using gene expression studies. And earlier this year, he and his team reported that they had identified recurrent gene fusions in prostate cancer using Illumina and Roche 454 sequencing platforms.

For the latest paper, Chinnaiyan and his co-workers tested the effectiveness of paired end transcriptome sequencing with one sequencing platform — the Illumina Genome Analyzer II — for finding gene fusions in human cell lines and tumor samples.

First, they looked at four human cell lines: a prostate cancer line called VCaP, a chronic myeloid leukemia line called K562, a human brain reference, and a universal human reference, which is a mixture of different cancer cell lines.

Using this approach, the team generated between 16.9 million and 25.5 million paired end reads or "transcriptome mate pairs" for each of the cell lines. They then mapped these against the human transcriptome — mainly comparing them with reads in the RefSeq database and the University of California at Santa Cruz Known Genes web site, lead author Christopher Maher, a research fellow in Chinnaiyan's lab, told GenomeWeb Daily News.

They initially found between 119 and 294 potential chimeras in each of the cell lines. Using bioinformatics, the researchers weeded out false positives and classified the aberrant transcription events detected in order to narrow in on genuine fusions.

Among the top fusions, the team found both known and previously undetected transcripts in each of the lines. For example, they identified the well-known BCR-ABL1 fusion in the chronic myeloid leukemia cell line K562. But they also found a new fusion between the nucleoporin gene NUP214 on chromosome 9 and the Kell blood group precursor gene XKR3 on chromosome 22.

Because the fusion was not detected in several other CML lines, the researchers propose that the NUP214-XKR3 fusion may have arisen as a consequence of nearby BCR and ABL1 mutations.

Such findings are making researchers optimistic about finding still more yet undetected gene fusions, even in cell lines that are thought to be well-studied and characterized.

The approach also seems to be a promising way to distinguish between driver and passenger fusions, Maher explained, since the best known fusions tended to be those with many supporting reads.

"You can start to prioritize which are the important reads," Maher said, noting that the approach seems particularly promising in the case of fusions that turn normally silent genes from "off" to "on." Even so, he cautioned, that is not always the case and the method may provide different information about driver/passenger fusions depending on the genes affected.

The team also used the paired end transcriptome sequencing on a few tumor samples, including two prostate cancer samples with no known gene fusions. Sure enough, the prostate cancer samples yielded two new fusions — HERPUD1-ERG and AXT47630-ETV1 — both involving ETS transcription factor genes. One of these had been previously detected by other researchers using fluorescence in situ hybridization, Maher noted, suggesting it may represent a new prostate cancer subset.

In the future, Maher said, the team plans to continue exploring prostate cancer in an effort to identify additional gene fusions. "We really want to leverage the strategy to hone in on prostate cancers that are lacking known driver mutations."

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