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Study Suggests Sequencing Late-Stage Tumors May be Key to Finding Relevant Cancer Mutations

NEW YORK (GenomeWeb Daily News) — A new sequencing study by Canadian researchers suggests that primary breast cancers acquire significant numbers of new mutations in their coding regions as they progress towards metastasis — a characteristic that underscores the importance of analyzing cancers at different stages, the researchers said.

In a paper published online in Nature today, researchers from the BC Cancer Agency in Vancouver describe how they sequenced the genome and transcriptome of a metastatic breast cancer at high depth, using the Illumina Genome Analyzer platform, and analyzed the data for somatic coding mutations.

When the scientists determined how many of these mutations were already present in the primary tumor of the same patient, they found that only a fraction of them could be found at all, and an even smaller number had a high frequency in the tumor.

"Taken together, our data show that single nucleotide mutational heterogeneity can be a property of low- or intermediate-grade primary breast cancers and that significant evolution can occur with disease progression," the authors wrote.

"Our results show the importance of sequencing samples of tumor cell populations early as well as late in the evolution of tumors, and of estimating allele frequency in tumor genomes," the authors noted.

Sequencing late-stage tumors might be especially important for finding relevant cancer mutations, they suggest. "Our observations suggest that the sequencing of primary breast cancers and pre-invasive malignancy may reveal significantly fewer candidates for tumor initiating mutations," they wrote.

For their study, the researchers chose lobular breast cancer, an estrogen-receptor positive subtype that makes up about 15 percent of all breast cancers.

The researchers sequenced DNA from a metastatic lobular breast cancer sample, and used RNA-seq to sequence the transcriptome of the same sample in parallel.

They then analyzed the data for putative single-nucleotide variants, insertions and deletions, gene fusions, translocations, inversions, and copy number alterations. The team ultimately identified 437 non-synonymous coding variants — 405 present in the normal germline DNA, and 32 only present in the tumor. Of those 32 somatic point mutations in 32 genes, 30 occurred in both the tumor DNA and transcriptome, whereas two were present only in the transcriptome.

The researchers then tested six of the mutations in 192 additional breast cancer samples — a mix of lobular and ductal subtypes — and found that although none had one of the exact same mutations, three contained different mutations in one of the six genes.

In order to determine how many of the 32 mutations were already present in the primary tumor, which was diagnosed nine years before the metastatic tumor, the team sequenced the positions of 30 of the mutations in the primary tumor, using the read counts to estimate the frequency of the mutations in the tumor.

They found that five of the mutations were prevalent in the primary tumor, and six were present at frequencies between 1 percent and 13 percent, meaning they were probably only present in portions of the tumor. Nineteen of the mutations could not be detected in the primary tumor.

"Thus, significant heterogeneity in tumor somatic mutation content existed in the primary tumor at diagnosis," the authors wrote, and "significant evolution of coding mutational content occurred between primary and metastasis," although it is unclear whether the 19 additional mutations in the metastasis resulted from radiation therapy or "innate tumor progression."

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