Somatic rearrangements contribute to the mutagenized landscape of cancer genomes. Here, we systematically interrogated rearrangements in 560 breast cancers by using a piecewise constant fitting approach. We identified 33 hotspots of large (>100 kb) tandem duplications, a mutational signature associated with homologous-recombination-repair deficiency. Notably, these tandem-duplication hotspots were enriched in breast cancer germline susceptibility loci (odds ratio (OR) = 4.28) and breast-specific 'super-enhancer' regulatory elements (OR = 3.54). These hotspots may be sites of selective susceptibility to double-strand-break damage due to high transcriptional activity or, through incrementally increasing copy number, may be sites of secondary selective pressure. The transcriptomic consequences ranged from strong individual oncogene effects to weak but quantifiable multigene expression effects. We thus present a somatic-rearrangement mutational process affecting coding sequences and noncoding regulatory elements and contributing a continuum of driver consequences, from modest to strong effects, thereby supporting a polygenic model of cancer development.
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Data used in this analysis were funded through the ICGC Breast Cancer Working group by the Breast Cancer Somatic Genetics Study (BASIS), a European research project funded by the European Community's Seventh Framework Programme (FP7/2010-2014) under grant agreement number 242006; the Triple Negative project, funded by the Wellcome Trust (grant reference 077012/Z/05/Z); and the HER2+ project, funded by Institut National du Cancer (INCa) in France (grant nos. 226-2009, 02-2011, 41-2012, 144-2008 and 06-2012). J.W.M.M. received funding for this project through an ERC Advanced grant (no. 322737). G.K. is supported by National Research Foundation of Korea grants (NRF 2015R1A2A1A10052578). The ICGC Asian Breast Cancer Project was funded through a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A111218-SC01). D.G. is supported by the EU-FP7-SUPPRESSTEM project. S.N.-Z. is funded by a Wellcome Trust Intermediate Fellowship (WT100183MA) and is supported as a Wellcome Beit Fellow.
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Integrated supplementary information
Supplementary Figure 1 Relationship between genomic features and distributions of RS1 and RS3 rearrangements in breast cancer genomes.
(a,b) Values of coefficients associated with genomic features, separately for RS1 (a) and RS3 (b). The values of coefficients and 95% confidence intervals were obtained through negative binomial regression, where we divided the genome into 0.5-Mb bins. The panels show the exponentiated values, ewi, for ease of interpretation. The further a coefficient deviates from 1, the more it influences expected number of breakpoints in genomic regions.
This summarizes the experiments conducted to gauge optimal parameters. Experiments were performed on observed data as well as simulations of rearrangements that took into account the background model of rearrangements. The x-axis indicates the setting of PCF parameters (g and i). The y-axis indicates the number of hotspots found in the observed (black dots) and simulated (grey dots) datasets. The blue rectangles highlight the PCF parameters that were finally selected to categorize hotspots of rearrangements in the observed data. The error bars at the grey dots denote standard deviation of the count when analysing 10 different simulated datasets. Red stars show estimated false discovery rate for the range of algorithm settings.
The images display overlap of the rearrangements across the cohort, by showing cumulative number of samples with a tandem duplication involving each of the genomic regions. Dashed vertical lines represent boundaries of the hotspots. Thick red lines represent breast-tissue specific super enhancers. Blue vertical line represents position of germline susceptibility locus of breast cancer. Black lines above show positions of genes.
Supplementary Figure 4 Tandem-duplication hotspots are enriched in breast-tissue-specific super-enhancers and germline breast cancer–susceptibility loci.
(a) The likelihood of observing germline susceptibility loci coinciding with tandem duplication hotspots. Single-sided Poisson test. OR, odds ratio; error bars denote 95% confidence levels. (b) The likelihood of observing super-enhancers falling into tandem duplication hotspots. Density of breast-tissue specific super-enhancer and germline susceptibility loci for tandem duplication hotspots versus other tandemly duplicated regions that do not fall within hotspots. Single-sided Poisson test. OR, odds ratio; error bars denote 95% confidence levels. (c) Simulations were used to obtain an empirical null distribution of number of super-enhancer elements within the hotspots, presented as a histogram. We observed 59 super-enhancers in the hotspots. The likelihood of that observation according to the simulations is <0.0001.
Supplementary Figure 5 Enrichment of hotspots in breast-tissue super-enhancers and germline breast cancer–susceptibility loci is robust with respect to the parameters of the PCF algorithm.
The x-axis shows the parameter i of the PCF algorithm. First top panel shows which hotspot are detected at more stringent values of the i parameter. Second panel shows number of hotspots detected. Third and fourth panels depict the enrichments of breast cancer SNP loci and super-enhancers at more stringent values of the i parameter. Error bars denote 95% confidence intervals for the enrichment from Fisher’s exact test.
Supplementary Figure 6 Relationship between tandem-duplicated segments and breast-tissue super-enhancer loci and germline breast cancer–susceptibility SNP loci.
In this analysis, all tandem duplication that had a breakpoint that fell within 1 Mb of super-enhancers (SENH, top panel) and/or breast cancer susceptibility SNPs (lower panel) were included. The x-axis reports on a 1-Mb genomic window surrounding SENH and SNPs, respectively. The y-axis reports the fraction of tandem duplications that have duplicated any given location within the 2-Mb window, out of all rearrangements in each group. The data are presented for RS1 tandem duplications in hotspots, RS1 tandem duplications that are not within hotspots and simulated RS1 rearrangements. Note the peak demonstrated for hotspot tandem duplication centered on the regulatory element/SNP, which is not exhibited by tandem duplications that are not within hotspots or simulated data.
Supplementary Figure 7 Tandem duplications wholly or partially increase the number of copies of ESR1, which correlates with high expression of the gene.
The top panel compares the expression of ESR1 between samples with and without tandem duplications in the hotspot. Samples that have tandem duplicated ESR1, even by just a single tandem duplication, have ESR1 expression levels that are in a similar high range as ER-positive tumors and are distinctly elevated when compared to the triple-negative tumors. The boxes highlight median expression level of the gene, with lower and upper quartiles. The second panel shows expression of ESR1 in individual samples with tandem duplications in the hotspot. The bottom panel shows the position of the rearrangements with respect to ESR1 gene body on the left, and across entire chromosome 6 on the right. Copy number (y-axis) depicted as black dots (10-kb bins). Green lines present tandem duplication breakpoints.
Supplementary Figure 8 Tandem duplications in some hotspots (wholly or partially) increase the number of copies of specific driver genes associated with breast cancer, even if by only one or two copies.
Left shows focus on the hotspot. Right shows entire chromosome of the hotspot. Rows correspond to individual samples. Copy number (y-axis) depicted as black dots (10-kb bins). Green lines present tandem duplication breakpoints. The ZNF217 locus is an example of a tandem duplication hotspot. Each patient has an apparent increase in copy number through a long tandem duplication, wholly of the gene. This site is enriched for breast tissue-specific super-enhancers.
Supplementary Figure 9 Tandem duplications in the hotspots are a feature of samples with many or few rearrangements in their genomes.
A histogram of the frequency of each of the 33 RS1-enriched tandem duplication hotspots is shown in the topmost panel with the 33 hotspots noted across the horizontal axis. The number of samples with rearrangements within any of the 33 hotspots is noted on the vertical axis on the left. A histogram of the number of hotspots per sample is provided on the right (purple, BRCA1-intact HR-deficient cancers; blue, BRCA1-null HR-deficient cancers; black, all other groups). Central matrix depicts the relationship between samples and number of hotspots (black, hotspot rearrangement present).
Supplementary Figure 10 Expression of MYC in samples with and without tandem duplications in the hotspot, distinguishing among breast cancer subtypes.
The boxes highlight median expression level of the gene, with lower and upper quartiles. These data were used to fit a linear model, suggesting that a tandem duplication in the hotspot was correlated with increased expression of the gene by 0.99 log2 FPKM, with P = 4.4 x 10-4.
Supplementary Figure 11 Hotspots of tandem duplications can be detected only in cohorts with an adequate number of rearrangements.
We sub-sampled the rearrangement dataset from the breast cancer cohort, in order to assess how many hotspots we could have detected in smaller cohorts. The number of RS1 rearrangements in the ovarian cohort was sufficient to detect hotspots, and indeed, in the ovarian cohort we detected seven hotspots. The number of rearrangements in pancreatic cohort was insufficient to detect hotspots, and indeed we detected none there.
The images display overlap of the rearrangements across the cohort, by showing cumulative number of samples with a tandem duplication involving each of the genomic regions. Thick red lines represent ovarian-tissue specific super enhancers. Black lines above show positions of genes. Dashed vertical lines represent boundaries of the hotspots.
Supplementary Figures 1–12 and Supplementary Note (PDF 2351 kb)
Hotspots of rearrangement signatures RS1 and RS3 identified through a PCF-based method. (a) Description of headers. (b) Summary of hotspots. (XLSX 63 kb)
Genomic consequences of RS1 and RS3 duplications (related to Fig. 4). Numbers of duplications and transections of genomic elements, separately for RS1 and RS3, inside and outside of the hotspots. (XLSX 39 kb)
Hotspots of other rearrangement signatures (RS2, RS4, RS5, RS6) identified through PCF-based method. (a) Description of headers. (b) Summary of hotspots. (XLSX 81 kb)
Genomic features of the RS1 hotspots. Comparison with the rest of tandem-duplicated genome with respect to: breast cancer susceptibility SNPs, breast tissue super-enhancers, non-breast super-enhancers, known oncogenes, promoters, enhancers, broad fragile sites, narrow fragile sites. (a) Description of headers. (b) Associations. (XLSX 44 kb)
Modeling the effects of RS1 tandem duplications on gene expression. Rows, coefficients used in the regression models. Columns, experiments with different sets of genes. In the table we show the fitted values of regression coefficients. (XLSX 37 kb)
Hotspots of rearrangement signatures RS1 and RS3 identified through PCF-based method in ovarian tumors. (a) Description of headers. (b) Summary of hotspots. (XLSX 55 kb)
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Glodzik, D., Morganella, S., Davies, H. et al. A somatic-mutational process recurrently duplicates germline susceptibility loci and tissue-specific super-enhancers in breast cancers. Nat Genet 49, 341–348 (2017) doi:10.1038/ng.3771
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