Primary triple negative breast cancers (TNBC) represent approximately 16% of all breast cancers1 and are a tumour type defined by exclusion, for which comprehensive landscapes of somatic mutation have not been determined. Here we show in 104 early TNBC cases, that at the time of diagnosis these cancers exhibit a wide and continuous spectrum of genomic evolution, with some exhibiting only a handful of somatic aberrations in a few pathways, whereas others contain hundreds of somatic events and multiple pathways implicated. Integration with matched whole transcriptome sequence data revealed that only ~36% of mutations are expressed. By examining single nucleotide variant (SNV) allelic abundance derived from deep re-sequencing (median >20,000 fold) measurements in 2414 somatic mutations, we determine for the first time in an epithelial tumour, the relative abundance of clonal genotypes among cases in the population. We show that TNBC vary widely and continuously in their clonal frequencies at the time of diagnosis, with basal subtype TNBC2,3 exhibiting more variation than non-basal TNBC. Although p53 and PIK3CA/PTEN somatic mutations appear clonally dominant compared with other pathways, in some tumours their clonal frequencies are incompatible with founder status. Mutations in cytoskeletal and cell shape/motility proteins occurred at lower clonal frequencies, suggesting they occurred later during tumour progression. Taken together our results show that future attempts to dissect the biology and therapeutic responses of TNBC will require the determination of individual tumour clonal genotypes.

Follicular lymphoma (FL) and the GCB subtype of diffuse large B-cell lymphoma (DLBCL) derive from germinal center B-cells 1. Targeted re-sequencing studies have revealed mutations in various genes in the NFkB pathway 2 , 3 that contribute to the activated B-cell Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

We introduce a novel statistical method, PyClone, for inference of clonal population structures in cancers. PyClone is a Bayesian clustering method for grouping sets of deeply sequenced somatic mutations into putative clonal clusters while estimating their cellular prevalences and accounting for allelic imbalances introduced by segmental copy number changes and normal cell contamination. Single cell sequencing validation demonstrates that PyClone infers accurate clustering of mutations that co-occur in individual cells.Human cancer progresses under Darwinian evolution where (epi)genetic variation alters molecular phenotypes in individual cells 1 . Consequently, tumours at diagnosis often consist of multiple, genotypically distinct cell populations ( Supplementary Fig. 1) 2 . These populations, referred to as clones, are related through a phylogeny and act as substrates for selection in tumour micro-environments or with therapeutic intervention 2, 3 . The prevalence of a particular clone measured over time or in anatomic space is a reflection of its growth and proliferative fitness. Thus, ascertaining the dynamic prevalence of clones can identify precise genetic determinants of phenotypes such as acquisition of metastatic potential or chemotherapeutic resistance.In this contribution, we provide a statistical model for analysis of deeply sequenced (coverage >1000x) mutations to identify and quantify clonal populations in tumours, which extends to modelling mutations measured in multiple samples from the same patient. Our approach uses the measurement of allelic prevalence to estimate the proportion of tumour ♣

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Next-generation sequencing of follicular lymphoma and diffuse-large B-cell lymphoma has revealed frequent somatic, heterozygous Y641 mutations in the histone methyltransferase EZH2. Heterozygosity and the presence of equal quantities of both mutant and wild-type mRNA and expressed protein suggest a dominant mode of action. Surprisingly, B-cell lymphoma cell lines and lymphoma samples harboring heterozygous EZH2 Y641 mutations have increased levels of histone H3 Lys-27-specific trimethylation (H3K27me3). Expression of EZH2 Y641F/N mutants in cells with EZH2 WT resulted in an increase of H3K27me3 levels in vivo. Structural modeling of EZH2 Y641 mutants suggests a "Tyr/Phe switch" model whereby structurally neutral, nontyrosine residues at position 641 would decrease affinity for unmethylated and monomethylated H3K27 substrates and potentially favor trimethylation. We demonstrate, using in vitro enzyme assays of reconstituted PRC2 complexes, that Y641 mutations result in a decrease in monomethylation and an increase in trimethylation activity of the enzyme relative to the wild-type enzyme. This represents the first example of a diseaseassociated gain-of-function mutation in a histone methyltransferase, whereby somatic EZH2 Y641 mutations in lymphoma act dominantly to increase, rather than decrease, histone methylation. The dominant mode of action suggests that allelespecific EZH2 inhibitors should be a future therapeutic strategy for this disease. IntroductionNon-Hodgkin lymphomas represent a diverse spectrum of distinct entities with the 2 most common types represented by follicular lymphoma and diffuse large B-cell lymphoma (DLBCL). There are 2 molecular subtypes of DLBCL based on cell-of-origin distinctions: the activated B-cell type and the germinal center B-cell (GCB) type. Both follicular lymphoma and the GCB subtype of DLBCL derive from germinal center B cells. We have shown that, in 7% of follicular lymphomas and 22% of GCB-type DLBCL, a single point mutation in EZH2, which results in a single amino-acid change at position 641, is selected for; EZH2 (Tyr641 or WT) was mutated to phenylalanine (Y641F), asparagine (Y641N), histidine (Y641H), or serine (Y641S). 1 EZH2 has been implicated as an oncoprotein often overexpressed in many solid tumors. [2][3][4] Initial analysis of the activity of Y641 variants in cell-free reconstituted Polycomb Repressive Complex 2 (PRC2) complexes using unmethylated peptides suggested that the mutations behaved as a loss of function. 1 EZH2 is the catalytic member of the PRC2; however, EZH2 alone has very weak histone-methylating activity. Other members of the PRC2 complex include EED, SUZ12, AEBP2, and RbAp48 and are required for full activity. The PRC2 complex has been shown to exhibit in vitro enzyme activity on histone peptide substrates and nucleosomes. EZH2 is a member of the Su(var)3,9, enhancer of zest, Trithorax (SET) domain containing family of histone methyltransferases (HMTases); all contain a conserved SET domain. Genetic and biochemical analysis of EZH2 SET domain ...

G-quadruplex DNAs form four-stranded helical structures and are proposed to play key roles in different cellular processes. Targeting G-quadruplex DNAs for cancer treatment is a very promising prospect. Here, we show that CX-5461 is a G-quadruplex stabilizer, with specific toxicity against BRCA deficiencies in cancer cells and polyclonal patient-derived xenograft models, including tumours resistant to PARP inhibition. Exposure to CX-5461, and its related drug CX-3543, blocks replication forks and induces ssDNA gaps or breaks. The BRCA and NHEJ pathways are required for the repair of CX-5461 and CX-3543-induced DNA damage and failure to do so leads to lethality. These data strengthen the concept of G4 targeting as a therapeutic approach, specifically for targeting HR and NHEJ deficient cancers and other tumours deficient for DNA damage repair. CX-5461 is now in advanced phase I clinical trial for patients with BRCA1/2 deficient tumours (Canadian trial, NCT02719977, opened May 2016).

The evolution of cancer genomes within a single tumor creates mixed cell populations with divergent somatic mutational landscapes. Inference of tumor subpopulations has been disproportionately focused on the assessment of somatic point mutations, whereas computational methods targeting evolutionary dynamics of copy number alterations (CNA) and loss of heterozygosity (LOH) in whole-genome sequencing data remain underdeveloped. We present a novel probabilistic model, TITAN, to infer CNA and LOH events while accounting for mixtures of cell populations, thereby estimating the proportion of cells harboring each event. We evaluate TITAN on idealized mixtures, simulating clonal populations from whole-genome sequences taken from genomically heterogeneous ovarian tumor sites collected from the same patient. In addition, we show in 23 whole genomes of breast tumors that the inference of CNA and LOH using TITAN critically informs population structure and the nature of the evolving cancer genome. Finally, we experimentally validated subclonal predictions using fluorescence in situ hybridization (FISH) and single-cell sequencing from an ovarian cancer patient sample, thereby recapitulating the key modeling assumptions of TITAN.

We performed phylogenetic analysis of high-grade serous ovarian cancers (68 samples from seven patients), identifying constituent clones and quantifying their relative abundances at multiple intraperitoneal sites. Through whole-genome and single-nucleus sequencing, we identified evolutionary features including mutation loss, convergence of the structural genome and temporal activation of mutational processes that patterned clonal progression. We then determined the precise clonal mixtures comprising each tumor sample. The majority of sites were clonally pure or composed of clones from a single phylogenetic clade. However, each patient contained at least one site composed of polyphyletic clones. Five patients exhibited monoclonal and unidirectional seeding from the ovary to intraperitoneal sites, and two patients demonstrated polyclonal spread and reseeding. Our findings indicate that at least two distinct modes of intraperitoneal spread operate in clonal dissemination and highlight the distribution of migratory potential over clonal populations comprising high-grade serous ovarian cancers.