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Transcriptome Sequencing Uncovers Breast Cancer Rearrangements

NEW YORK (GenomeWeb News) – In a paper scheduled to appear online this week in the Proceedings of the National Academy of Sciences, an international research team has published a proof-of-principle study using high-throughput transcriptome sequencing to pick up genomic rearrangements in a breast cancer cell line.

Researchers from the J. Craig Venter Institute, three branches of the Ludwig Institute for Cancer Research, and New York's Memorial Sloan-Kettering Cancer Center used Roche 454 transcriptome sequencing to look for translocations in a highly rearranged breast cancer cell line. In the process, they identified seven new genomic rearrangements — five truncations and two chimeric proteins — believed to affect at least nine genes.

"We're very excited about it," senior author Robert Strausberg, deputy director of genomic medicine and group leader at JCVI, told GenomeWeb Daily News. Strausberg credits high-throughput sequencing for providing researchers with access to new types of information in the transcriptome. "It's a dream come true to be able to look at this many transcripts in a cell," he said.

Several studies have demonstrated the utility of large-scale genomic analysis for identifying mutations, rearrangements, and gene expression changes associated with various cancers.

For the latest study, Strausberg and his colleagues focused on information hidden in the transcriptome, the collection of transcripts within a cell. Since the transcriptome reflects the active genome, it contains a wealth of information on everything from alternative gene splicing to gene expression.

But Strausberg and his team wanted to interrogate the transcriptome for something else: clues about translocation events in the genome. In an effort to detect the active gene products of genomic rearrangements, they used Roche 454-FLX pyrosequencing to assess the transcriptome of a highly rearranged breast cancer cell line called HCC1954.

The team generated 510,703 cDNA sequence reads from the cell line. Of these, more than 384,900 reads aligned to 9,221 RefSeq gene mRNAs. After tossing out sequences that aligned to RefSeq, the researchers attempted to align the remaining sequences with the human reference genome, leaving them with 47,370 sequences that didn't align with RefSeq mRNAs or the reference.

By putting these remaining sequences into a computational analysis pipeline, the researchers pulled out 496 potential chimeric transcripts containing information from at least two different genomic locations. Roughly half of these represented rearrangements within the same chromosome while the other half represented rearrangements involving different chromosomes.

From there, the team selected 33 putative chimeras for follow-up and experimentally validated 13 chimeric cDNA. Most of the changes the team detected were also present in a control line derived from the same individual's blood cells, though. That prompted the researchers to use long-range PCR, Sanger end sequencing, and fluorescence in situ hybridization to distinguish between trans-splicing events and genuine genomic rearrangements.

While some of the rearrangements they detected overlapped with known changes in the cell line, lead author Qi Zhao, a human genomic scientist at JCVI, told GenomeWeb Daily News, others were new. For instance, they identified four inter-chromosomal translocations and one intra-chromosomal rearrangement in their initial experiments.

Next, the team went back to look for chimeric transcripts that mapped to more than one spot in the genome. In the process, they identified and verified two more inter-chromosomal rearrangements.

Together, the seven rearrangements identified in the study are thought to affect at least nine different genes. And five of the rearrangements produced transcripts coding for truncated proteins — including proteins with known oncogenic functions.

For instance, the researchers detected a truncation in MRE11A, a gene coding for a protein involved in double strand break repair that has been detected in several breast cancers, as well as NSD1, a gene coding a potential transcription regulator that's sometimes fused in acute myeloid leukemia.

"Our results suggest that genomic translocations may be additional mechanisms of inactivation of this gene in breast cancer, and that it may be another frequent mechanism in parallel with point mutations," the authors wrote.

Beyond this proof-of principle study, Strausberg said the ultimate goal is to look at and understand cancer at a clinical level. He and his co-authors emphasized the need to compile and integrate different types of data — including genomic rearrangements and alternative splicing events — to improve the chances to translating research into better outcomes.

"Databases that provide the ability to compare and contrast these changes across a broad range of cancer will be essential for identifying features that might be shared across cancers, thereby affording opportunities to apply new intervention approaches effectively to all cancers for which patient outcomes might be improved," the authors concluded.

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