Sequence analysis of mutations and translocations across breast cancer subtypes

Abstract

Breast carcinoma is the leading cause of cancer-related mortality in women worldwide, with an estimated 1.38 million new cases and 458,000 deaths in 2008 alone. This malignancy represents a heterogeneous group of tumours with characteristic molecular features, prognosis and responses to available therapy. Recurrent somatic alterations in breast cancer have been described, including mutations and copy number alterations, notably ERBB2 amplifications, the first successful therapy target defined by a genomic aberration. Previous DNA sequencing studies of breast cancer genomes have revealed additional candidate mutations and gene rearrangements. Here we report the whole-exome sequences of DNA from 103 human breast cancers of diverse subtypes from patients in Mexico and Vietnam compared to matched-normal DNA, together with whole-genome sequences of 22 breast cancer/normal pairs. Beyond confirming recurrent somatic mutations in PIK3CA, TP53, AKT1, GATA3 and MAP3K1, we discovered recurrent mutations in the CBFB transcription factor gene and deletions of its partner RUNX1. Furthermore, we have identified a recurrent MAGI3-AKT3 fusion enriched in triple-negative breast cancer lacking oestrogen and progesterone receptors and ERBB2 expression. The MAGI3-AKT3 fusion leads to constitutive activation of AKT kinase, which is abolished by treatment with an ATP-competitive AKT small-molecule inhibitor.

Figure 1. Most significantly-mutated genes in breast cancer as determined by whole exome sequencing (n=103)

Upper histogram: rates of sample-specific mutations (substitutions and indels), green = synonymous, blue = non-synonymous. Left histogram: number of mutations per gene and percentage of samples affected (colour coding as in upper histogram). Central heatmap: Distribution of significant mutations across sequenced samples (“Other non synonymous” mutations = nonsense, indel, splice-site). Right histogram: -log10 score of MutSig q value. Red line at q = 0.1. Lower chart: top - rates of non-silent mutations within categories indicated by legend; bottom - key molecular features of samples in each column (Expression subtypes: “Lum” = luminal. Histology: “Duct.” = Infiltrating ductal carcinoma, “DCIS” = Ductal carcinoma in situ, “Lob.” = Infiltrating lobular carcinoma).

Figure 2. CBFB mutations and RUNX1 deletions

A. CBFB coding region diagram: RUNX binding domain in green. Mutations identified in this study (red bullets), previously identified mutations^, (black bullets), and known CBFB-MYH11 fusion indicated. B. Allelic copy ratios for the 3 Mb region surrounding RUNX1 in samples BR-M-045 and BR-M-174. Dots indicate copy-ratios for individual SNP alleles: Red = higher copy-ratio allele for informative SNPs that are heterozygous in matched normal DNA; Blue = lower-copy ratio SNPs; Grey = uninformative SNPs (homozygous in matched normal). Lines indicate inferred segmental copy-ratios. Red = higher copy segment; Blue = lower copy segment; Purple = equal copy segment. C. Histogram depicting bins of segmented copy number (y-axis), with inferred integral copies shown by dotted lines; the length of each horizontal block corresponds to the fraction of the haploid genome at the copy number level, or “genomic fraction” (x-axis).

Figure 3. MAGI3-AKT3 fusion gene

A. Diagram of balanced translocation between MAGI3 and AKT3. B. (top) Genomic DNA PCR for AKT3, MAGI3, and both fusion products in tumour (T) and normal (N). (bottom) cDNA PCR of fusion gene in tumour. C. (above) MAGI3 and AKT3 protein domains; (below) putative fusion protein. D. Immunoblots of lysates from ZR-75 cells transfected with vector, MAGI3-AKT3 fusion, or AKT1 E17K mutant, grown in low-serum media, for the indicated antibodies. (Left) infected cells with and without insulin growth factor 1 (IGF-1) stimulation; (right) treatment of vector or MAGI3-AKT3 overexpressing cells with Akt inhibitors MK-2206 and GSK-690693. E. Focus formation assays with Rat-1 cells expressing pLX control or MAGI3-AKT3, and stained with crystal violet.