Published today in the journal Nature is the whole-genome sequencing of a basal-like breast cancer tumor, metastasis, and xenograft. There’s also a News and Views article by Joe Gray of Lawrence Berkeley National Laboratory, as well as a news feature on large-scale cancer projects.
This study is a bit unlike our previous cancer genomes (AML1 and AML2). By my count it is the sixth cancer genome to be sequenced, and the third to come out of the Genome Center at Washington University. Obviously, it’s our first solid tumor. What’s particularly interesting about this study, however, is that we sequenced four DNA samples from a single patient with “double-negative” breast cancer: the primary tumor, peripheral blood (normal), a brain metastasis, and a mouse xenograft derived from the primary tumor. The xenograft is a success story in itself – we managed to create a human-in-mouse (HIM) transplant of the primary tumor that was >90% pure when harvested 101 days after engraftment.
The genomes of these four samples (tumor, normal, metastasis, and xenograft), examined with the incredible power of Illumina massively parallel sequencing, offer an unprecedented view of the somatic changes that underlie breast cancer development, growth, and metastasis.
Repertoire of Somatic Mutations
We validated a total of 50 somatic sites in at least one of the three cancer genomes, including:
- 28 missense mutations predicted to alter the sequence of an encoded protein
- 11 synonymous (silent) mutations in coding sequences
- 4 small insertions ranging in size from 1 to 6 bp
- 3 small deletions ranging in size from 1 to 13 bp
- 2 splice site mutations at intron-exon junctions
- 1 nonsense mutation predicted to result in a truncated protein
- 1 RNA mutation in a gene encoding a signal recognition particle (SRP) RNA.
We employed deep Illumina sequencing of PCR amplicons to assess the frequencies of each mutation across all four tissues. Intriguingly, more than half of them exhibited differential frequencies between primary tumor, metastasis, and/or xenograft. Two mutations (a nonsense mutation in MYCBP2 and a missense mutation in TGFBI) were significantly enriched in the primary tumor (88-89% vs 14-44%). Some 26 mutations were significantly enriched in the metastasis and/or xenograft. Perhaps most interesting, however, were two sites (a missense mutation in SNED1 and a silent mutation in FLNC) that appear to be de novo mutations unique to the metastasis.
Acquired Structural Variation
Using our internally developed tools for structural variant prediction (BreakDancer) and de novo assembly (TIGRA), we predicted 59 deletions and 18 inversions that were putative somatic events. Validation by PCR and 454/3730 sequencing showed that 73/77 (94.8%) were real structural variants, of which 34 (28 deletions and 6 inversions) were somatic alterations not present in the normal genome. Among them was a 46.5 kbp heterozygous deletion affecting FBXW7 (a known cancer gene) and two overlapping 500-kb deletions affecting CTNNA1 and a handful of other genes. The latter was particularly interesting, because loss of CTNNA1 has been shown to result in global loss of cell adhesion in human breast cancer cell lines.
We also validated seven translocations with a combination of manual review (Pairoscope), assembly, and PCR/3730 sequencing. One translocation that we assembled in all three tumor samples involves a long terminal repeat (LTR) from the ERVL-MaLR family on chromosome 4 and the ABCA2 gene on chromosome 9. Two other validated translocations that assembled in all three tumors are on chromosome 2, and separated only by a 393-bp TcMar-Tigger repeat.
Insights from Comparisons of Tumor, Metastasis, and Xenograft
One of the most intriguing findings from our study was the differential mutation frequencies and structural variation patterns that we observed in the metastasis and xenograft, compared to the primary tumor. More than half of the somatic mutations (26/50) were significantly enriched in the metastasis and xenograft, while observed at relatively low frequencies in the primary tumor. This suggests that a sub-population of tumor cells, not the primary clone, gave rise to the cerebellar metastasis that eventually killed the patient.
Is there a fitness cost to the mutations that enabled metastasis? Can we develop sensitive tests to detect the cells that are likely to spread? Genome sequencing has brought us to a point where we can begin to ask these questions, and answering them brings us one step closer to unraveling the complex, devastating, deadly disease that is cancer.
Li Ding, Matthew J. Ellis, Shunqiang Li, David E. Larson, Ken Chen, John W. Wallis, Christopher C. Harris, Michael D. McLellan, Robert S. Fulton, Lucinda L. Fulton, Rachel M. Abbott, Jeremy Hoog, David J. Dooling, Daniel C. Koboldt, Heather Schmidt, Joell (2010). Genome remodelling in a basal-like breast cancer metastasis and xenograft Nature, 464 (15), 999-1005 : 10.1038/nature08989