Characterizing Mutational Signatures in Human Cancer Cell Lines Reveals Episodic APOBEC Mutagenesis.

Multiple signatures of somatic mutations have been identified in cancer genomes. Exome sequences of 1,001 human cancer cell lines and 577 xenografts revealed most common mutational signatures, indicating past activity of the underlying processes, usually in appropriate cancer types. To investigate ongoing patterns of mutational-signature generation, cell lines were cultured for extended periods and subsequently DNA sequenced. Signatures of discontinued exposures, including tobacco smoke and ultraviolet light, were not generated in vitro. Signatures of normal and defective DNA repair and replication continued to be generated at roughly stable mutation rates. Signatures of APOBEC cytidine deaminase DNA-editing exhibited substantial fluctuations in mutation rate over time with episodic bursts of mutations. The initiating factors for the bursts are unclear, although retrotransposon mobilization may contribute. The examined cell lines constitute a resource of live experimental models of mutational processes, which potentially retain patterns of activity and regulation operative in primary human cancers.

Universal Patterns of Selection in Cancer and Somatic Tissues.

Cancer develops as a result of somatic mutation and clonal selection, but quantitative measures of selection in cancer evolution are lacking. We adapted methods from molecular evolution and applied them to 7,664 tumors across 29 cancer types. Unlike species evolution, positive selection outweighs negative selection during cancer development. On average, <1 coding base substitution/tumor is lost through negative selection, with purifying selection almost absent outside homozygous loss of essential genes. This allows exome-wide enumeration of all driver coding mutations, including outside known cancer genes. On average, tumors carry ∼4 coding substitutions under positive selection, ranging from <1/tumor in thyroid and testicular cancers to >10/tumor in endometrial and colorectal cancers. Half of driver substitutions occur in yet-to-be-discovered cancer genes. With increasing mutation burden, numbers of driver mutations increase, but not linearly. We systematically catalog cancer genes and show that genes vary extensively in what proportion of mutations are drivers versus passengers.

Life histories of myeloproliferative neoplasms inferred from phylogenies.

Mutations in cancer-associated genes drive tumour outgrowth, but our knowledge of the timing of driver mutations and subsequent clonal dynamics is limited1-3. Here, using whole-genome sequencing of 1,013 clonal haematopoietic colonies from 12 patients with myeloproliferative neoplasms, we identified 580,133 somatic mutations to reconstruct haematopoietic phylogenies and determine clonal histories. Driver mutations were estimated to occur early in life, including the in utero period. JAK2V617F was estimated to have been acquired by 33 weeks of gestation to 10.8 years of age in 5 patients in whom JAK2V617F was the first event. DNMT3A mutations were acquired by 8 weeks of gestation to 7.6 years of age in 4 patients, and a PPM1D mutation was acquired by 5.8 years of age. Additional genomic events occurred before or following JAK2V617F acquisition and as independent clonal expansions. Sequential driver mutation acquisition was separated by decades across life, often outcompeting ancestral clones. The mean latency between JAK2V617F acquisition and diagnosis was 30 years (range 11-54 years). Estimated historical rates of clonal expansion varied substantially (3% to 190% per year), increased with additional driver mutations, and predicted latency to diagnosis. Our study suggests that early driver mutation acquisition and life-long growth and evolution underlie adult myeloproliferative neoplasms, raising opportunities for earlier intervention and a new model for cancer development.

Clonal dynamics of haematopoiesis across the human lifespan.

Age-related change in human haematopoiesis causes reduced regenerative capacity1, cytopenias2, immune dysfunction3 and increased risk of blood cancer4-6, but the reason for such abrupt functional decline after 70 years of age remains unclear. Here we sequenced 3,579 genomes from single cell-derived colonies of haematopoietic cells across 10 human subjects from 0 to 81 years of age. Haematopoietic stem cells or multipotent progenitors (HSC/MPPs) accumulated a mean of 17 mutations per year after birth and lost 30 base pairs per year of telomere length. Haematopoiesis in adults less than 65 years of age was massively polyclonal, with high clonal diversity and a stable population of 20,000-200,000 HSC/MPPs contributing evenly to blood production. By contrast, haematopoiesis in individuals aged over 75 showed profoundly decreased clonal diversity. In each of the older subjects, 30-60% of haematopoiesis was accounted for by 12-18 independent clones, each contributing 1-34% of blood production. Most clones had begun their expansion before the subject was 40 years old, but only 22% had known driver mutations. Genome-wide selection analysis estimated that between 1 in 34 and 1 in 12 non-synonymous mutations were drivers, accruing at constant rates throughout life, affecting more genes than identified in blood cancers. Loss of the Y chromosome conferred selective benefits in males. Simulations of haematopoiesis, with constant stem cell population size and constant acquisition of driver mutations conferring moderate fitness benefits, entirely explained the abrupt change in clonal structure in the elderly. Rapidly decreasing clonal diversity is a universal feature of haematopoiesis in aged humans, underpinned by pervasive positive selection acting on many more genes than currently identified.

Lineage tracing of human development through somatic mutations.

The ontogeny of the human haematopoietic system during fetal development has previously been characterized mainly through careful microscopic observations1. Here we reconstruct a phylogenetic tree of blood development using whole-genome sequencing of 511 single-cell-derived haematopoietic colonies from healthy human fetuses at 8 and 18 weeks after conception, coupled with deep targeted sequencing of tissues of known embryonic origin. We found that, in healthy fetuses, individual haematopoietic progenitors acquire tens of somatic mutations by 18 weeks after conception. We used these mutations as barcodes and timed the divergence of embryonic and extra-embryonic tissues during development, and estimated the number of blood antecedents at different stages of embryonic development. Our data support a hypoblast origin of the extra-embryonic mesoderm and primitive blood in humans.

The mutational landscape of human somatic and germline cells.

Over the course of an individual's lifetime, normal human cells accumulate mutations1. Here we compare the mutational landscape in 29 cell types from the soma and germline using multiple samples from the same individuals. Two ubiquitous mutational signatures, SBS1 and SBS5/40, accounted for the majority of acquired mutations in most cell types, but their absolute and relative contributions varied substantially. SBS18, which potentially reflects oxidative damage2, and several additional signatures attributed to exogenous and endogenous exposures contributed mutations to subsets of cell types. The rate of mutation was lowest in spermatogonia, the stem cells from which sperm are generated and from which most genetic variation in the human population is thought to originate. This was due to low rates of ubiquitous mutational processes and may be partially attributable to a low rate of cell division in basal spermatogonia. These results highlight similarities and differences in the maintenance of the germline and soma.

The mutational landscape of normal human endometrial epithelium.

All normal somatic cells are thought to acquire mutations, but understanding of the rates, patterns, causes and consequences of somatic mutations in normal cells is limited. The uterine endometrium adopts multiple physiological states over a lifetime and is lined by a gland-forming epithelium1,2. Here, using whole-genome sequencing, we show that normal human endometrial glands are clonal cell populations with total mutation burdens that increase at about 29 base substitutions per year and that are many-fold lower than those of endometrial cancers. Normal endometrial glands frequently carry 'driver' mutations in cancer genes, the burden of which increases with age and decreases with parity. Cell clones with drivers often originate during the first decades of life and subsequently progressively colonize the epithelial lining of the endometrium. Our results show that mutational landscapes differ markedly between normal tissues-perhaps shaped by differences in their structure and physiology-and indicate that the procession of neoplastic change that leads to endometrial cancer is initiated early in life.

Author Correction: The evolutionary history of lethal metastatic prostate cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The evolutionary history of lethal metastatic prostate cancer.

Cancers emerge from an ongoing Darwinian evolutionary process, often leading to multiple competing subclones within a single primary tumour. This evolutionary process culminates in the formation of metastases, which is the cause of 90% of cancer-related deaths. However, despite its clinical importance, little is known about the principles governing the dissemination of cancer cells to distant organs. Although the hypothesis that each metastasis originates from a single tumour cell is generally supported, recent studies using mouse models of cancer demonstrated the existence of polyclonal seeding from and interclonal cooperation between multiple subclones. Here we sought definitive evidence for the existence of polyclonal seeding in human malignancy and to establish the clonal relationship among different metastases in the context of androgen-deprived metastatic prostate cancer. Using whole-genome sequencing, we characterized multiple metastases arising from prostate tumours in ten patients. Integrated analyses of subclonal architecture revealed the patterns of metastatic spread in unprecedented detail. Metastasis-to-metastasis spread was found to be common, either through de novo monoclonal seeding of daughter metastases or, in five cases, through the transfer of multiple tumour clones between metastatic sites. Lesions affecting tumour suppressor genes usually occur as single events, whereas mutations in genes involved in androgen receptor signalling commonly involve multiple, convergent events in different metastases. Our results elucidate in detail the complex patterns of metastatic spread and further our understanding of the development of resistance to androgen-deprivation therapy in prostate cancer.

Fast randomization of large genomic datasets while preserving alteration counts.

MOTIVATION: Studying combinatorial patterns in cancer genomic datasets has recently emerged as a tool for identifying novel cancer driver networks. Approaches have been devised to quantify, for example, the tendency of a set of genes to be mutated in a 'mutually exclusive' manner. The significance of the proposed metrics is usually evaluated by computing P-values under appropriate null models. To this end, a Monte Carlo method (the switching-algorithm) is used to sample simulated datasets under a null model that preserves patient- and gene-wise mutation rates. In this method, a genomic dataset is represented as a bipartite network, to which Markov chain updates (switching-steps) are applied. These steps modify the network topology, and a minimal number of them must be executed to draw simulated datasets independently under the null model. This number has previously been deducted empirically to be a linear function of the total number of variants, making this process computationally expensive. RESULTS: We present a novel approximate lower bound for the number of switching-steps, derived analytically. Additionally, we have developed the R package BiRewire, including new efficient implementations of the switching-algorithm. We illustrate the performances of BiRewire by applying it to large real cancer genomics datasets. We report vast reductions in time requirement, with respect to existing implementations/bounds and equivalent P-value computations. Thus, we propose BiRewire to study statistical properties in genomic datasets, and other data that can be modeled as bipartite networks. AVAILABILITY AND IMPLEMENTATION: BiRewire is available on BioConductor at http://www.bioconductor.org/packages/2.13/bioc/html/BiRewire.html. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Clonal dynamics and somatic evolution of haematopoiesis in mouse.

Haematopoietic stem cells maintain blood production throughout life. While extensively characterised using the laboratory mouse, little is known about how the population is sustained and evolves with age. We isolated stem cells and progenitors from young and old mice, identifying 221,890 somatic mutations genome-wide in 1845 single cell-derived colonies, and used phylogenetic analysis to infer the ontogeny and population dynamics of the stem cell pool. Mouse stem cells and progenitors accrue ~45 somatic mutations per year, a rate only about 2-fold greater than human progenitors despite the vastly different organismal sizes and lifespans. Phylogenetic patterns reveal that stem and multipotent progenitor cell pools are both established during embryogenesis, after which they independently self-renew in parallel over life. The stem cell pool grows steadily over the mouse lifespan to approximately 70,000 cells, self-renewing about every six weeks. Aged mice did not display the profound loss of stem cell clonal diversity characteristic of human haematopoietic ageing. However, targeted sequencing revealed small, expanded clones in the context of murine ageing, which were larger and more numerous following haematological perturbations and exhibited a selection landscape similar to humans. Our data illustrate both conserved features of population dynamics of blood and distinct patterns of age-associated somatic evolution in the short-lived mouse.

Physical vapor deposition as a route to hidden amorphous states.

Stable glasses of indomethacin (IMC) were prepared by using physical vapor deposition. Wide-angle X-ray scattering measurements were performed to characterize the average local structure. IMC glasses prepared at a substrate temperature of 0.84 T(g) (where T(g) is the glass transition temperature) and a deposition rate of 0.2 nm/s show a broad, high-intensity peak at low q values that is not present in the supercooled liquid or melt-quenched glasses. When annealed slightly above T(g), the new WAXS pattern transforms into the melt-quenched glass pattern, but only after very long annealing times. For a series of samples prepared at the lowest deposition rate, the new local packing arrangement is present only for deposition temperatures below T(g) -20 K, suggesting an underlying first-order liquid-to-liquid phase transition.

Detection of early seeding of Richter transformation in chronic lymphocytic leukemia.

Richter transformation (RT) is a paradigmatic evolution of chronic lymphocytic leukemia (CLL) into a very aggressive large B cell lymphoma conferring a dismal prognosis. The mechanisms driving RT remain largely unknown. We characterized the whole genome, epigenome and transcriptome, combined with single-cell DNA/RNA-sequencing analyses and functional experiments, of 19 cases of CLL developing RT. Studying 54 longitudinal samples covering up to 19 years of disease course, we uncovered minute subclones carrying genomic, immunogenetic and transcriptomic features of RT cells already at CLL diagnosis, which were dormant for up to 19 years before transformation. We also identified new driver alterations, discovered a new mutational signature (SBS-RT), recognized an oxidative phosphorylation (OXPHOS)high-B cell receptor (BCR)low-signaling transcriptional axis in RT and showed that OXPHOS inhibition reduces the proliferation of RT cells. These findings demonstrate the early seeding of subclones driving advanced stages of cancer evolution and uncover potential therapeutic targets for RT.

Timing the initiation of multiple myeloma.

The evolution and progression of multiple myeloma and its precursors over time is poorly understood. Here, we investigate the landscape and timing of mutational processes shaping multiple myeloma evolution in a large cohort of 89 whole genomes and 973 exomes. We identify eight processes, including a mutational signature caused by exposure to melphalan. Reconstructing the chronological activity of each mutational signature, we estimate that the initial transformation of a germinal center B-cell usually occurred during the first 2nd-3rd decades of life. We define four main patterns of activation-induced deaminase (AID) and apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) mutagenesis over time, including a subset of patients with evidence of prolonged AID activity during the pre-malignant phase, indicating antigen-responsiveness and germinal center reentry. Our findings provide a framework to study the etiology of multiple myeloma and explore strategies for prevention and early detection.

Highly stable indomethacin glasses resist uptake of water vapor.

Mass uptake of water vapor was measured as a function of relative humidity for indomethacin glasses prepared using physical vapor deposition at different substrate temperatures. Highly stable glasses were produced on substrates at 265 K (0.84Tg) by depositing at 0.2 nm/s while samples similar to melt-cooled glasses were produced at 315 K and 5 nm/s. Samples deposited at 315 K absorb approximately the same amount of water as glasses prepared by supercooling the melt while stable glasses absorb a factor of 5 less water. Unexpectedly, the diffusion of water in the stable glass samples is 5-10 times faster than in the glass prepared by cooling the liquid.

Genomic landscape and chronological reconstruction of driver events in multiple myeloma.

The multiple myeloma (MM) genome is heterogeneous and evolves through preclinical and post-diagnosis phases. Here we report a catalog and hierarchy of driver lesions using sequences from 67 MM genomes serially collected from 30 patients together with public exome datasets. Bayesian clustering defines at least 7 genomic subgroups with distinct sets of co-operating events. Focusing on whole genome sequencing data, complex structural events emerge as major drivers, including chromothripsis and a novel replication-based mechanism of templated insertions, which typically occur early. Hyperdiploidy also occurs early, with individual trisomies often acquired in different chronological windows during evolution, and with a preferred order of acquisition. Conversely, positively selected point mutations, whole genome duplication and chromoplexy events occur in later disease phases. Thus, initiating driver events, drawn from a limited repertoire of structural and numerical chromosomal changes, shape preferred trajectories of evolution that are biologically relevant but heterogeneous across patients.

Genomic patterns of progression in smoldering multiple myeloma.

We analyzed whole genomes of unique paired samples from smoldering multiple myeloma (SMM) patients progressing to multiple myeloma (MM). We report that the genomic landscape, including mutational profile and structural rearrangements at the smoldering stage is very similar to MM. Paired sample analysis shows two different patterns of progression: a "static progression model", where the subclonal architecture is retained as the disease progressed to MM suggesting that progression solely reflects the time needed to accumulate a sufficient disease burden; and a "spontaneous evolution model", where a change in the subclonal composition is observed. We also observe that activation-induced cytidine deaminase plays a major role in shaping the mutational landscape of early subclinical phases, while progression is driven by APOBEC cytidine deaminases. These results provide a unique insight into myelomagenesis with potential implications for the definition of smoldering disease and timing of treatment initiation.

Detecting and measuring selection from gene frequency data.

The recent advent of high-throughput sequencing and genotyping technologies makes it possible to produce, easily and cost effectively, large amounts of detailed data on the genotype composition of populations. Detecting locus-specific effects may help identify those genes that have been, or are currently, targeted by natural selection. How best to identify these selected regions, loci, or single nucleotides remains a challenging issue. Here, we introduce a new model-based method, called SelEstim, to distinguish putative selected polymorphisms from the background of neutral (or nearly neutral) ones and to estimate the intensity of selection at the former. The underlying population genetic model is a diffusion approximation for the distribution of allele frequency in a population subdivided into a number of demes that exchange migrants. We use a Markov chain Monte Carlo algorithm for sampling from the joint posterior distribution of the model parameters, in a hierarchical Bayesian framework. We present evidence from stochastic simulations, which demonstrates the good power of SelEstim to identify loci targeted by selection and to estimate the strength of selection acting on these loci, within each deme. We also reanalyze a subset of SNP data from the Stanford HGDP-CEPH Human Genome Diversity Cell Line Panel to illustrate the performance of SelEstim on real data. In agreement with previous studies, our analyses point to a very strong signal of positive selection upstream of the LCT gene, which encodes for the enzyme lactase-phlorizin hydrolase and is associated with adult-type hypolactasia. The geographical distribution of the strength of positive selection across the Old World matches the interpolated map of lactase persistence phenotype frequencies, with the strongest selection coefficients in Europe and in the Indus Valley.