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Punctuated Copy Number Changes Identified in Triple-Negative Breast Cancer

NEW YORK (GenomeWeb) – A study appearing online today in Nature Genetics suggests triple-negative breast cancers undergo flurries of copy number changes early in the tumor evolution process, followed by periods of more stable sub-clone expansion.

A University of Texas MD Anderson Cancer Center-led team did single-cell sequencing on 1,000 cells taken from tumor sub-clone samples from a dozen women with estrogen receptor-, progesterone receptor-, and HER2 amplification-negative breast cancers. Such triple-negative forms of the disease are difficult to treat and often associated with poor survival. The team's results indicated that punctuated copy number evolution is common in triple-negative tumors, arguing against the notion that the copy number patterns found in bulk tumor samples occurred through slow, sequential processes.

"This model has important implications for our evolutionary understanding of cancer growth dynamics and for the clinical diagnosis and treatment of [triple-negative breast cancer] patients," senior author Nicholas Navin — a genetics, bioinformatics, and computational biology researcher at MD Anderson — said in a statement. Although tumor copy number patterns can influence oncogene or tumor suppressor gene expression, he and his co-authors explained, "most genomic studies have analyzed tumor samples from a single time point (biopsy or surgery) making it difficult to study the natural progression of chromosome evolution during tumorigenesis."

Using its highly multiplexed single nucleus sequencing (HM-SNS) method, the team profiled copy number patterns in 1,000 cancer cells isolated from triple-negative breast cancer tumors in a dozen individuals with the disease.

The researchers started by making nuclear suspensions from frozen tumor samples. The nuclei were then sorted and isolated into 96-well plates prior to a whole-genome amplification step with degenerative-oligonucleotide PCR. From there, barcoded single nucleus libraries were pooled for single-end Illumina sequencing on the HiSeq 2000.

The team saw anywhere from one to three stable clonal sub-populations per tumor after clustering individual cells based on the copy number data, though some individual cells with non-clonal copy number shifts turned up in the tumors as well. Depending on the particular copy number alterations present, these sub-clones appeared to become more or less frequent in the broader tumor over time. Meanwhile, the researchers' phylogenetic analysis of the copy number alterations in the individual tumor cells was consistent with a spate of copy number changes early in tumor development.

"[O]ur single-cell copy number data and mathematical modeling suggest that clonal stasis and [punctuated copy number evolution] are common in patients with [triple-negative breast cancer]," the study's authors wrote. "This process leads to complex aneuploid copy number profiles that are remarkably stable during tumor growth and ubiquitous throughout the tumor mass."

The same punctuated copy number process may also occur in other types of tumors, they noted, based on initial findings in colon, prostate, liver, and lung cancer samples.