Search

Driver mutations in the two histone 3.3 (H3.3) genes, H3F3A and H3F3B, were recently identified by whole genome sequencing in 95% of chondroblastoma (CB) and by targeted gene sequencing in 92% of giant cell tumour of bone (GCT). Given the high prevalence of these driver mutations, it may be possible to utilise these alterations as diagnostic adjuncts in clinical practice. Here, we explored the spectrum of H3.3 mutations in a wide range and large number of bone tumours (n = 412) to determine if these alterations could be used to distinguish GCT from other osteoclast-rich tumours such as aneurysmal bone cyst, nonossifying fibroma, giant cell granuloma, and osteoclast-rich malignant bone tumours and others. In addition, we explored the driver landscape of GCT through whole genome, exome and targeted sequencing (14 gene panel). We found that H3.3 mutations, namely mutations of glycine 34 in H3F3A, occur in 96% of GCT. We did not find additional driver mutations in GCT, including mutations in IDH1, IDH2, USP6, TP53. The genomes of GCT exhibited few somatic mutations, akin to the picture seen in CB. Overall our observations suggest that the presence of H3F3A p.Gly34 mutations does not entirely exclude malignancy in osteoclast-rich tumours. However, H3F3A p.Gly34 mutations appear to be an almost essential feature of GCT that will aid pathological evaluation of bone tumours, especially when confronted with small needle core biopsies. In the absence of H3F3A p.Gly34 mutations, a diagnosis of GCT should be made with caution.

DNA replication-associated mutations are repaired by two components: polymerase proofreading and mismatch repair. The mutation consequences of disruption to both repair components in humans are not well studied. We sequenced cancer genomes from children with inherited biallelic mismatch repair deficiency (bMMRD). High-grade bMMRD brain tumors exhibited massive numbers of substitution mutations (>250/Mb), which was greater than all childhood and most cancers (>7,000 analyzed). All ultra-hypermutated bMMRD cancers acquired early somatic driver mutations in DNA polymerase ɛ or δ. The ensuing mutation signatures and numbers are unique and diagnostic of childhood germ-line bMMRD (P < 10(-13)). Sequential tumor biopsy analysis revealed that bMMRD/polymerase-mutant cancers rapidly amass an excess of simultaneous mutations (∼600 mutations/cell division), reaching but not exceeding ∼20,000 exonic mutations in

We assessed IgA antibodies and polymerase chain reaction (PCR) for diagnosis of pertussis in nasopharyngeal aspiration (NPA) samples from outpatients in Australia. A total of 1700 patients (849 adults, 851 children) from Western Australia and the Northern Territory fulfilled the laboratory case definition for pertussis between 2004 and 2013: 732 specimens were positive by NPA IgA alone, 559 by PCR alone, and 409 by both tests. Overall, 968 cases (56.8%) were positive by PCR and 1141 cases (67.2%) by IgA [p0.00025]. Among pediatric patients, PCR was positive in 524 (61.3%) and IgA in 569 (67%). In 849 adult cases, the respective proportions were 52.3% and 67.4% [p0.00025]. The duration of cough in 507 patients was shorter in 262 pediatric cases (mean, 2.51 weeks; standard deviation [SD], 2.25) than 245 adult patients (3.27 weeks; SD, 2.79) [p = 0.0009]. PCR positivity showed a season-dependent variance (range, 5.6 to 85.9%) and peaked in the second week (71.7%) of illness. IgA antibodies peaked in the fifth week (89.5%) postinfection, and the positivity rate for NPA IgA was less variable (range, 38.3-97.2%). Nasopharyngeal Bordetella pertussis-specific IgA antibodies are valuable in diagnosis of pertussis in Australia. Reliance on PCR alone misses a significant proportion of pertussis cases, especially those with a delayed presentation.

Homozygous deletions are rare in cancers and often target tumour suppressor genes. Here, we build a compendium of 2218 primary tumours across 12 human cancer types and systematically screen for homozygous deletions, aiming to identify rare tumour suppressors. Our analysis defines 96 genomic regions recurrently targeted by homozygous deletions. These recurrent homozygous deletions occur either over tumour suppressors or over fragile sites, regions of increased genomic instability. We construct a statistical model that separates fragile sites from regions showing signatures of positive selection for homozygous deletions and identify candidate tumour suppressors within those regions. We find 16 established tumour suppressors and propose 27 candidate tumour suppressors. Several of these genes (including MGMT, RAD17, and USP44) show prior evidence of a tumour suppressive function. Other candidate tumour suppressors, such as MAFTRR, KIAA1551, and IGF2BP2, are novel. Our study demonstrates how rare tumour suppressors can be identified through copy number meta-analysis.

Background Precision medicine is rapidly evolving within the field of oncology and has brought many new concepts and terminologies that are often poorly defined when first introduced, which may subsequently lead to miscommunication within the oncology community. The European Society for Medical Oncology (ESMO) recognises these challenges and is committed to support the adoption of precision medicine in oncology. To add clarity to the language used by oncologists and basic scientists within the context of precision medicine, the ESMO Translational Research and Personalised Medicine Working Group has developed a standardised glossary of relevant terms. Materials and methods Relevant terms for inclusion in the glossary were identified via an ESMO member survey conducted in Autumn 2016, and by the ESMO Translational Research and Personalised Medicine Working Group members. Each term was defined by experts in the field, discussed and, if necessary, modified by the Working Group before reaching consensus approval. A literature search was carried out to determine which of the terms, ‘precision medicine’ and ‘personalised medicine’, is most appropriate to describe this field. Results A total of 43 terms are included in the glossary, grouped into five main themes—(i) mechanisms of decision, (ii) characteristics of molecular alterations, (iii) tumour characteristics, (iv) clinical trials and statistics and (v) new research tools. The glossary classes ‘precision medicine’ or ‘personalised medicine’ as technically interchangeable but the term ‘precision medicine’ is favoured as it more accurately reflects the highly precise nature of new technologies that permit base pair resolution dissection of cancer genomes and is less likely to be misinterpreted. Conclusions The ESMO Precision Medicine Glossary provides a resource to facilitate consistent communication in this field by clarifying and raising awareness of the language employed in cancer research and oncology practice. The glossary will be a dynamic entity, undergoing expansion and refinement over the coming years.

Chordoma is a malignant, often incurable bone tumour showing notochordal differentiation. Here, we defined the somatic driver landscape of 104 cases of sporadic chordoma. We reveal somatic duplications of the notochordal transcription factor brachyury (T) in up to 27% of cases. These variants recapitulate the rearrangement architecture of the pathogenic germline duplications of T that underlie familial chordoma. In addition, we find potentially clinically actionable PI3K signalling mutations in 16% of cases. Intriguingly, one of the most frequently altered genes, mutated exclusively by inactivating mutation, was LYST (10%), which may represent a novel cancer gene in chordoma., Chordoma is a rare often incurable malignant bone tumour. Here, the authors investigate driver mutations of sporadic chordoma in 104 cases, revealing duplications in notochordal transcription factor brachyury (T), PI3K signalling mutations, and mutations in LYST, a potential novel cancer gene in chordoma.

Introduction The level of minimal residual disease (MRD) in marrow predicts outcome and guides treatment in childhood acute lymphoblastic leukemia (ALL) but accurate prediction depends on accurate measurement. Methods Forty-one children with ALL were studied at the end of induction. Two samples were obtained from each iliac spine and each sample was assayed twice. Assay, sample and side-to-side variation were quantified by analysis of variance and presumptively incorrect decisions related to high-risk disease were determined using the result from each MRD assay, the mean MRD in the patient as the measure of the true value, and each of 3 different MRD cut-off levels which have been used for making decisions on treatment. Results Variation between assays, samples and sides each differed significantly from zero and the overall standard deviation for a single MRD estimation was 0.60 logs. Multifocal residual disease seemed to be at least partly responsible for the variation between samples. Decision errors occurred at a frequency of 13–14% when the mean patient MRD was between 10−2 and 10−5. Decision errors were observed only for an MRD result within 1 log of the cut-off value used for assessing high risk. Depending on the cut-off used, 31–40% of MRD results were within 1 log of the cut-off value and 21–16% of such results would have resulted in a decision error. Conclusion When the result obtained for the level of MRD is within 1 log of the cut-off value used for making decisions, variation in the assay and/or sampling may result in a misleading assessment of the true level of marrow MRD. This may lead to an incorrect decision on treatment.

Cancer cells can acquire profound alterations to the structure of their genomes, including rearrangements that fuse distant DNA breakpoints. We analyze the distribution of somatic rearrangements across the cancer genome, using whole-genome sequencing data from 2,693 tumor-normal pairs. We observe substantial variation in the density of rearrangement breakpoints, with enrichment in open chromatin and sites with high densities of repetitive elements. After accounting for these patterns, we identify significantly recurrent breakpoints (SRBs) at 52 loci, including novel SRBs near BRD4 and AKR1C3. Taking into account both loci fused by a rearrangement, we observe different signatures resembling either single breaks followed by strand invasion or two separate breaks that become joined. Accounting for these signatures, we identify 90 pairs of loci that are significantly recurrently juxtaposed (SRJs). SRJs are primarily tumor-type specific and tend to involve genes with tissue-specific expression. SRJs were frequently associated with disruption of topology-associated domains, juxtaposition of enhancer elements, and increased expression of neighboring genes. Lastly, we find that the power to detect SRJs decreases for short rearrangements, and that reliable detection of all driver SRJs will require whole-genome sequencing data from an order of magnitude more cancer samples than currently available.

A key mutational process in cancer is structural variation, in which rearrangements delete, amplify or reorder genomic segments ranging in size from kilobases to whole chromosomes. We developed methods to group, classify and describe structural variants, applied to >2,500 cancer genomes. Nine signatures of structural variation emerged. Deletions have trimodal size distribution; assort unevenly across tumour types and patients; enrich in late-replicating regions; and correlate with inversions. Tandem duplications also have trimodal size distribution, but enrich in early-replicating regions, as do unbalanced translocations. Replication-based mechanisms of rearrangement generate varied chromosomal structures with low-level copy number gains and frequent inverted rearrangements. One prominent structure consists of 1-7 templates copied from distinct regions of the genome strung together within one locus. Such ‘cycles of templated insertions’ correlate with tandem duplications, frequently activating the telomerase gene,TERT,in liver cancer. Cancers access many rearrangement processes, flexibly sculpting the genome to maximise oncogenic potential.

Genomic material within the nucleus is folded into successive layers in order to package and organize the long string of linear DNA. This hierarchical level of folding is closely associated with transcriptional regulation and DNA replication. Recent chromosome conformation studies have revealed that mammalian chromosomes are structured into tissue-invariant topologically associating domains (TADs) where the DNA within a given domain interacts more frequently together than with regions in other domains. Genes within the same TADs represent similar expression and histone-modification profiles. Therefore, regions separating different TADs (boundaries) have important roles in reinforcing the stability of these domain-wide features. Indeed, TAD boundary disruptions in human genetic disorders or human cancers lead to misregulation of certain genes, due to de novo enhancer exposure to promoters. Here, to understand effects and distributions of somatic structural variations across TADs, we utilized single nucleotide variations and genomic rearrangements from 2658 high-coverage whole genome sequencing data across various cancer types with paired normal samples. Our analysis revealed that deletions between repressed and active TADs result in up-regulation of genes on the repressed end of the deletions, whereas active domain genes remain unaffected. Interestingly, we further identified a strong correlation between the mutational distributions in human cancers and the spatial organization of the genome. Transcriptionally active TADs contain less mutation burden compared to inactive TADs, as a result regional mutation rates are drastically different around the boundaries delineating epigenetically distinct domains. However, mutation rates remain similar around the boundaries for samples with the DNA mismatch repair deficiency. Taken together, our analyses reveal new insights about genome architecture, aberrant gene expression and mutational distributions in human cancers.

With a few exceptions, cancers typically carry more than one driver mutation, sometimes five, ten, or more, and these driver mutations do not necessarily assort randomly. In this issue of Cancer Cell, Mina et al. systematically characterize patterns of co-mutation and mutual exclusivity in 6,456 cancers across 23 tumor types.

Summary Patterns of genomic evolution between primary and metastatic breast cancer have not been studied in large numbers, despite patients with metastatic breast cancer having dismal survival. We sequenced whole genomes or a panel of 365 genes on 299 samples from 170 patients with locally relapsed or metastatic breast cancer. Several lines of analysis indicate that clones seeding metastasis or relapse disseminate late from primary tumors, but continue to acquire mutations, mostly accessing the same mutational processes active in the primary tumor. Most distant metastases acquired driver mutations not seen in the primary tumor, drawing from a wider repertoire of cancer genes than early drivers. These include a number of clinically actionable alterations and mutations inactivating SWI-SNF and JAK2-STAT3 pathways., Graphical Abstract, Highlights • Metastases mostly disseminate late from primary breast tumors, keeping most drivers • Drivers at relapse sample from a wider range of cancer genes than in primary tumors • Mutations in SWI-SNF complex and inactivated JAK-STAT signaling enriched at relapse • Mutational processes similar in primary and relapse; radiotherapy can damage genome, By sequencing primary, locally relapsed, and metastatic breast cancers, Yates et al. show that clones seeding metastasis or relapse disseminate late from primary tumors but continue to acquire mutations, including clinically actionable alterations and mutations inactivating the SWI/SNF and JAK2-STAT3 pathways.

The International Cancer Genome Consortium (ICGC)’s Pan-Cancer Analysis of Whole Genomes (PCAWG) project aimed to categorize somatic and germline variations in both coding and non-coding regions in over 2,800 cancer patients. To provide this dataset to the research working groups for downstream analysis, the PCAWG Technical Working Group marshalled ~800TB of sequencing data from distributed geographical locations; developed portable software for uniform alignment, variant calling, artifact filtering and variant merging; performed the analysis in a geographically and technologically disparate collection of compute environments; and disseminated high-quality validated consensus variants to the working groups. The PCAWG dataset has been mirrored to multiple repositories and can be located using the ICGC Data Portal. The PCAWG workflows are also available as Docker images through Dockstore enabling researchers to replicate our analysis on their own data.

Mitochondria are essential cellular organelles that play critical roles in cancer development. Through International Cancer Genome Consortium, we performed a multidimensional characterization of mitochondrial genomes using the whole-genome sequencing data of ~2,700 patients across 37 cancer types and related RNA-sequencing data. Our analysis presents the most definitive mutational landscape of mitochondrial genomes including a novel hypermutated case. We observe similar mutational signatures across cancer types, suggesting powerful endogenous mutational processes in mitochondria. Truncating mutations are remarkably enriched in kidney, colorectal and thyroid cancers and associated with the activation of critical signaling pathways. We find frequent somatic nuclear transfers of mitochondrial DNA (especially in skin and lung cancers), some of which disrupt therapeutic target genes (e.g., ERBB2). The mitochondrial DNA copy number shows great variations within and across cancers and correlates with clinical variables. Co-expression analysis highlights the function of mitochondrial genes in oxidative phosphorylation, DNA repair, and cell cycle; and reveals their connections with clinically actionable genes. Our study, including an open-access data portal, lays a foundation for understanding the interplays between the cancer mitochondrial and nuclear genomes and translating mitochondrial biology into clinical applications.

Osteosarcoma is a primary malignancy of bone that affects children and adults. Here, we present the largest sequencing study of osteosarcoma to date, comprising 112 childhood and adult tumours encompassing all major histological subtypes. A key finding of our study is the identification of mutations in insulin-like growth factor (IGF) signalling genes in 8/112 (7%) of cases. We validate this observation using fluorescence in situ hybridization (FISH) in an additional 87 osteosarcomas, with IGF1 receptor (IGF1R) amplification observed in 14% of tumours. These findings may inform patient selection in future trials of IGF1R inhibitors in osteosarcoma. Analysing patterns of mutation, we identify distinct rearrangement profiles including a process characterized by chromothripsis and amplification. This process operates recurrently at discrete genomic regions and generates driver mutations. It may represent an age-independent mutational mechanism that contributes to the development of osteosarcoma in children and adults alike.

© 2017 The Author(s) Myelodysplastic syndromes and chronic myelomonocytic leukemia are blood disorders characterized by ineffective hematopoiesis and progressive marrow failure that can transform into acute leukemia. The DNA methyltransferase inhibitor 5-azacytidine (AZA) is the most effective pharmacological option, but only ∼50% of patients respond. A response only manifests after many months of treatment and is transient. The reasons underlying AZA resistance are unknown, and few alternatives exist for non-responders. Here, we show that AZA responders have more hematopoietic progenitor cells (HPCs) in the cell cycle. Non-responder HPC quiescence is mediated by integrin α5 (ITGA5) signaling and their hematopoietic potential improved by combining AZA with an ITGA5 inhibitor. AZA response is associated with the induction of an inflammatory response in HPCs in vivo. By molecular bar coding and tracking individual clones, we found that, although AZA alters the sub-clonal contribution to different lineages, founder clones are not eliminated and continue to drive hematopoiesis even in complete responders.

Throughout their lifetime cells are subject to extrinsic and intrinsic mutational processes leaving behind characteristic signatures in the genome. DNA mismatch repair (MMR) deficiency leads to hypermutation and is found in different cancer types. While it is possible to associate mutational signatures extracted from human cancers with possible mutational processes the exact causation is often unknown. Here we useC. elegansgenome sequencing ofpms-2andmlh-1knockouts to reveal the mutational patterns linked toC. elegansMMR deficiency and their dependency on endogenous replication errors and errors caused by deletion of the polymerase ε subunitpole-4. Signature extraction from 215 human colorectal and 289 gastric adenocarcinomas revealed three MMR-associated signatures, one of which closely resembles theC. elegansMMR spectrum and strongly discriminates microsatellite stable and unstable tumors (AUC=98%). A characteristic difference between human andC. elegansMMR deficiency is the lack of elevated levels of NCG>NTG mutations inC. elegans, likely caused by the absence of cytosine (CpG) methylation in worms. The other two human MMR signatures may reflect the interaction between MMR deficiency and other mutagenic processes, but their exact cause remains unknown. In summary, combining information from genetically defined models and cancer samples allows for better aligning mutational signatures to causal mutagenic processes.

We have developed ascatNgs to aid researchers in carrying out Allele-Specific Copy number Analysis of Tumours (ASCAT). ASCAT is capable of detecting DNA copy number changes affecting a tumor genome when comparing to a matched normal sample. Additionally, the algorithm estimates the amount of tumor DNA in the sample, known as Aberrant Cell Fraction (ACF). ASCAT itself is an R-package which requires the generation of many file types. Here, we present a suite of tools to help handle this for the user. Our code is available on our GitHub site (https://github.com/cancerit). This unit describes both 'one-shot' execution and approaches more suitable for large-scale compute farms. © 2016 by John Wiley & Sons, Inc.

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body, by undertaking a Human Cell Atlas Project as an international collaborative effort. The aim would be to define all human cell types in terms of distinctive molecular profiles (e.g., gene expression) and connect this information with classical cellular descriptions (e.g., location and morphology). A comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, as well as provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas.

Several studies have demonstrated the feasibility of molecular screening of tumour samples for matching patients with cancer to targeted therapies. However, most of them have been carried out at institutional or national level. Herein, we report on the pilot phase of AURORA (NCT02102165), a European multinational collaborative molecular screening initiative for advanced breast cancer patients. Forty-one patients were prospectively enroled at four participating centres across Europe. Metastatic tumours were biopsied and profiled using an Ion Torrent sequencing platform at a central facility. Sequencing results were obtained for 63% of the patients in real-time with variable turnaround time stemming from delays between patient consent and biopsy. At least one clinically actionable mutation was identified in 73% of patients. We used the Illumina sequencing technology for orthogonal validation and achieved an average of 66% concordance of substitution calls per patient. Additionally, copy number aberrations inferred from the Ion Torrent sequencing were compared to single nucleotide polymorphism arrays and found to be 59% concordant on average. Although this study demonstrates that powerful next generation genomic techniques are logistically ready for international molecular screening programs in routine clinical settings, technical challenges remain to be addressed in order to ensure the accuracy and clinical utility of the genomic data., info:eu-repo/semantics/published

The authors describe a family with a high penetrance of plasma cell dyscrasias, suggesting inheritance of an autosomal dominant risk allele.The authors performed whole-exome sequencing and reported on a combined approach aimed at the identification of causative variants and risk loci, using the wealth of data provided by this approach.The authors identified gene mutations and single-nucleotide polymorphisms of potential significance, and pinpointed a known risk locus for myeloma as a potential area of transmissible risk in the family.To the authors' knowledge, the current study is the first to provide a whole-exome sequencing approach to such cases, and a framework analysis that could be applied to further understanding of the inherited risk of developing plasma cell dyscrasias. Cancer 2017;123:3701-3708. © 2017 American Cancer Society.

Structural variants (SVs), including small insertion and deletion variants (indels), are challenging to detect through standard alignment-based variant calling methods. Sequence assembly offers a powerful approach to identifying SVs, but is difficult to apply at-scale genome-wide for SV detection due to its computational complexity and the difficulty of extracting SVs from assembly contigs. We describe SvABA, an efficient and accurate method for detecting SVs from short-read sequencing data using genome-wide local assembly with low memory and computing requirements. We evaluated SvABA’s performance on the NA12878 human genome and in simulated and real cancer genomes. SvABA demonstrates superior sensitivity and specificity across a large spectrum of SVs, and substantially improved detection performance for variants in the 20-300 bp range, compared with existing methods. SvABA also identifies complex somatic rearrangements with chains of short (< 1,000 bp) templated-sequence insertions copied from distant genomic regions. We applied SvABA to 344 cancer genomes from 11 cancer types, and found that templated-sequence insertions occur in ~4% of all somatic rearrangements. Finally, we demonstrate that SvABA can identify sites of viral integration and cancer driver alterations containing medium-sized SVs.

International audience; Approximately 1–5% of breast cancers are attributed to inherited mutations in BRCA1 or BRCA2 and are selectively sensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. In other cancer types, germline and/or somatic mutations in BRCA1 and/or BRCA2 (BRCA1/BRCA2) also confer selective sensitivity to PARP inhibitors. Thus, assays to detect BRCA1/BRCA2-deficient tumors have been sought. Recently, somatic substitution, insertion/deletion and rearrangement patterns, or 'mutational signatures', were associated with BRCA1/BRCA2 dysfunction. Herein we used a lasso logistic regression model to identify six distinguishing mutational signatures predictive of BRCA1/BRCA2 deficiency. A weighted model called HRDetect was developed to accurately detect BRCA1/BRCA2-deficient samples. HRDetect identifies BRCA1/BRCA2-deficient tumors with 98.7% sensitivity (area under the curve (AUC) = 0.98). Application of this model in a cohort of 560 individuals with breast cancer, of whom 22 were known to carry a germline BRCA1 or BRCA2 mutation, allowed us to identify an additional 22 tumors with somatic loss of BRCA1 or BRCA2 and 47 tumors with functional BRCA1/BRCA2 deficiency where no mutation was detected. We validated HRDetect on independent cohorts of breast, ovarian and pancreatic cancers and demonstrated its efficacy in alternative sequencing strategies. Integrating all of the classes of mutational signatures thus reveals a larger proportion of individuals with breast cancer harboring BRCA1/BRCA2 deficiency (up to 22%) than hitherto appreciated (~1–5%) who could have selective therapeutic sensitivity to PARP inhibition.

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.

International audience; Somatic cells acquire mutations throughout the course of an individual's life. Mutations occurring early in embryogenesis are often present in a substantial proportion of, but not all, cells in postnatal humans and thus have particular characteristics and effects. Depending on their location in the genome and the proportion of cells they are present in, these mosaic mutations can cause a wide range of genetic disease syndromes and predispose carriers to cancer. They have a high chance of being transmitted to offspring as de novo germline mutations and, in principle, can provide insights into early human embryonic cell lineages and their contributions to adult tissues. Although it is known that gross chromosomal abnormalities are remarkably common in early human embryos, our understanding of early embryonic somatic mutations is very limited. Here we use whole-genome sequences of normal blood from 241 adults to identify 163 early embryonic mutations. We estimate that approximately three base substitution mutations occur per cell per cell-doubling event in early human embryogenesis and these are mainly attributable to two known mutational signatures. We used the mutations to reconstruct developmental lineages of adult cells and demonstrate that the two daughter cells of many early embryonic cell-doubling events contribute asymmetrically to adult blood at an approximately 2:1 ratio. This study therefore provides insights into the mutation rates, mutational processes and developmental outcomes of cell dynamics that operate during early human embryogenesis.

International audience; 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.

Adrenocortical carcinoma (ACC) is a rare malignancy that carries a poor prognosis. There has yet to be a large Australian series that documents the characteristics of ACC and there are a paucity of data on management and the long-term outcomes. We sought to provide a unique insight into the management of ACC in Australia as well as to identify factors associated with prognosis and survival.A multivariate analysis of a cohort of patients identified with ACC between 1998 and 2013 was undertaken. Recurrence-free survival (RFS) and overall survival (OS) were assessed as the main outcome measures and correlated with multiple clinical variables in order to identify prognostic markers.Of the 104 patients identified, a total of 98 patients with complete clinical and outcome data were included in the study. Median OS was 56 months, with the 5-year survival being 48 % (95 % confidence interval 36-59). On multivariate analysis, age ≥50 years, metastases at presentation, and evidence of extra-adrenal invasion were found to be statistically associated with reduced OS. RFS was analyzed in patients without metastases. On multivariate analysis, extra-adrenal invasion and no preoperative endocrine investigations were found to be statistically significant poor prognostic factors, with a non-significant trend for higher individual surgeon volume to be associated with improved resection margins and RFS.We present clinical outcomes and prognostic factors for patients with ACC in a landmark Australian series. We suggest that management in a specialized tertiary endocrine and/or surgical oncology unit is more likely to lead to improved outcomes.

COSMIC, the Catalogue Of Somatic Mutations In Cancer (http://cancer.sanger.ac.uk) is the world's largest and most comprehensive resource for exploring the impact of somatic mutations in human cancer. Our latest release (v70; Aug 2014) describes 2 002 811 coding point mutations in over one million tumor samples and across most human genes. To emphasize depth of knowledge on known cancer genes, mutation information is curated manually from the scientific literature, allowing very precise definitions of disease types and patient details. Combination of almost 20 000 published studies gives substantial resolution of how mutations and phenotypes relate in human cancer, providing insights into the stratification of mutations and biomarkers across cancer patient populations. Conversely, our curation of cancer genomes (over 12 000) emphasizes knowledge breadth, driving discovery of unrecognized cancer-driving hotspots and molecular targets. Our high-resolution curation approach is globally unique, giving substantial insight into molecular biomarkers in human oncology. In addition, COSMIC also details more than six million noncoding mutations, 10 534 gene fusions, 61 299 genome rearrangements, 695 504 abnormal copy number segments and 60 119 787 abnormal expression variants. All these types of somatic mutation are annotated to both the human genome and each affected coding gene, then correlated across disease and mutation types.

Our knowledge of the genetic basis of myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) has considerably improved. To define genotype/phenotype relationships of clinical relevance, we studied 308 patients with MDS, MDS/MPN, or acute myeloid leukemia evolving from MDS. Unsupervised statistical analysis, including the World Health Organization classification criteria and somatic mutations, showed that MDS associated with SF3B1-mutation (51 of 245 patients, 20.8%) is a distinct nosologic entity irrespective of current morphologic classification criteria. Conversely, MDS with ring sideroblasts with nonmutated SF3B1 segregated in different clusters with other MDS subtypes. Mutations of genes involved in DNA methylation, splicing factors other than SF3B1, and genes of the RAS pathway and cohesin complex were independently associated with multilineage dysplasia and identified a distinct subset (51 of 245 patients, 20.8%). No recurrent mutation pattern correlated with unilineage dysplasia without ring sideroblasts. Irrespective of driver somatic mutations, a threshold of 5% bone marrow blasts retained a significant discriminant value for identifying cases with clonal evolution. Comutation of TET2 and SRSF2 was highly predictive of a myeloid neoplasm characterized by myelodysplasia and monocytosis, including but not limited to, chronic myelomonocytic leukemia. These results serve as a proof of concept that a molecular classification of myeloid neoplasms is feasible.

The somatic mutations present in the genome of a cell have been accumulated over the lifetime of a multicellular organism. These mutations can provide insights into the developmental lineage tree1, the number of divisions each cell has undergone and the mutational processes that have been operative2. Here, we conducted whole genome sequencing of clonal lines3 derived from multiple tissues of healthy mice. Using somatic base substitutions, we reconstructed the early cell divisions of each animal demonstrating the contributions of embryonic cells to adult tissues. Differences were observed between tissues in the numbers and types of mutations accumulated by each cell, which likely reflect differences in the number of cell divisions they have undergone and varying contributions of different mutational processes. If somatic mutation rates are similar to those in mice, the results indicate that precise insights into development and mutagenesis of normal human cells will be possible.

Angiosarcoma is an aggressive malignancy that arises spontaneously or secondarily to ionizing radiation or chronic lymphoedema. Previous work has identified aberrant angiogenesis, including occasional somatic mutations in angiogenesis signaling genes, as a key driver of angiosarcoma. Here we employed whole-genome, whole-exome and targeted sequencing to study the somatic changes underpinning primary and secondary angiosarcoma. We identified recurrent mutations in two genes, PTPRB and PLCG1, which are intimately linked to angiogenesis. The endothelial phosphatase PTPRB, a negative regulator of vascular growth factor tyrosine kinases, harbored predominantly truncating mutations in 10 of 39 tumors (26%). PLCG1, a signal transducer of tyrosine kinases, encoded a recurrent, likely activating p.Arg707Gln missense variant in 3 of 34 cases (9%). Overall, 15 of 39 tumors (38%) harbored at least one driver mutation in angiogenesis signaling genes. Our findings inform and reinforce current therapeutic efforts to target angiogenesis signaling in angiosarcoma.

Changes in gene dosage are a major driver of cancer, known to be caused by a finite, but increasingly well annotated, repertoire of mutational mechanisms1. This can potentially generate correlated copy-number alterations across hundreds of linked genes, as exemplified by the 2% of childhood acute lymphoblastic leukaemia (ALL) with recurrent amplification of megabase regions of chromosome 21 (iAMP21)2, 3. We used genomic, cytogenetic and transcriptional analysis, coupled with novel bioinformatic approaches, to reconstruct the evolution of iAMP21 ALL. Here we show that individuals born with the rare constitutional Robertsonian translocation between chromosomes 15 and 21, rob(15;21)(q10;q10)c, have approximately 2,700-fold increased risk of developing iAMP21 ALL compared to the general population. In such cases, amplification is initiated by a chromothripsis event involving both sister chromatids of the Robertsonian chromosome, a novel mechanism for cancer predisposition. In sporadic iAMP21, breakage-fusion-bridge cycles are typically the initiating event, often followed by chromothripsis. In both sporadic and rob(15;21)c-associated iAMP21, the final stages frequently involve duplications of the entire abnormal chromosome. The end-product is a derivative of chromosome 21 or the rob(15;21)c chromosome with gene dosage optimized for leukaemic potential, showing constrained copy-number levels over multiple linked genes. Thus, dicentric chromosomes may be an important precipitant of chromothripsis, as we show rob(15;21)c to be constitutionally dicentric and breakage-fusion-bridge cycles generate dicentric chromosomes somatically. Furthermore, our data illustrate that several cancer-specific mutational processes, applied sequentially, can coordinate to fashion copy-number profiles over large genomic scales, incrementally refining the fitness benefits of aggregated gene dosage changes.

Motivation: Targeted resequencing of cancer genes in large cohorts of patients is important to understand the biological and clinical consequences of mutations. Cancers are often clonally heterogeneous, and the detection of subclonal mutations is important from a diagnostic point of view, but presents strong statistical challenges. Results: Here we present a novel statistical approach for calling mutations from large cohorts of deeply resequenced cancer genes. These data allow for precisely estimating local error profiles and enable detecting mutations with high sensitivity and specificity. Our probabilistic method incorporates knowledge about the distribution of variants in terms of a prior probability. We show that our algorithm has a high accuracy of calling cancer mutations and demonstrate that the detected clonal and subclonal variants have important prognostic consequences. Availability: Code is available as part of the Bioconductor package deepSNV. Contact: mg14@sanger.ac.uk; pc8@sanger.ac.uk

CaVEMan is an expectation maximization-based somatic substitution-detection algorithm that is written in C. The algorithm analyzes sequence data from a test sample, such as a tumor relative to a reference normal sample from the same patient and the reference genome. It performs a comparative analysis of the tumor and normal sample to derive a probabilistic estimate for putative somatic substitutions. When combined with a set of validated post-hoc filters, CaVEMan generates a set of somatic substitution calls with high recall and positive predictive value. Here we provide instructions for using a wrapper script called cgpCaVEManWrapper, which runs the CaVEMan algorithm and additional downstream post-hoc filters. We describe both a simple one-shot run of cgpCaVEManWrapper and a more in-depth implementation suited to large-scale compute farms. © 2016 by John Wiley & Sons, Inc.

Introduction: Mature blood cells harbor a mixture of mutations inherited from ancestral hematopoietic stem cells (HSCs) and mutations accumulated after maturation. The landscape of these somatic mutations in normal blood is poorly mapped, with questions as simple as "how many mutations does a memory T cell accumulate throughout life?" remaining unanswered. This gap in our knowledge is particularly relevant for hematopoietic malignancy- while we know that lymphomas derive from lymphocytes of particular stages of differentiation, we do not know if the patterns we see are reflected in their normal counterparts. Results: In order to characterize normal somatic mutation in lymphocytes, we performed single-cell expansion and whole genome sequencing of over 600 T and B lymphocytes and 200 HSC and progenitor cells across 5 individuals (ages 0-85). All lymphocyte subsets show increased mutation burden with respect to HSCs across all classes of variants (Figure 1). Some of this increase can explained by lymphocyte-specific mutational processes, such as the activity of RAG, accounting for at least 20% of observed structural variants. We also find a striking variation in mutation burden within and between lymphocyte subsets. Microenvironment specific mutational processes dominate the observed differences. Examples of this include germinal center ("non-canonical AID") mutations in memory B cells and UV-like mutations in memory T cells (putatively skin resident cells). Naive B and T cells show a lack of variation in discrete mutational patterns relative to their memory counterparts, and have mutational profiles and mutation burdens more similar to that of HSCs. We also observe differences in the mutational patterns between B and T cells that are indicative of the increased divergence of T lymphocytes from the HSC pool. In general, the mutation burden we observe in normal lymphocytes approach those seen in lymphoma. Conclusions: Our work highlights the substantial genetic diversity in normal lymphocytes, with some cells accumulating thousands of mutations on top of those inherited from the HSC compartment. These mutations can be used to describe the life history of each individual lymphocyte including their exposure to specific microenvironments. Our findings shed light on the biology of these cells and will help differentiate between normal and disease processes. Figure 1 Disclosures No relevant conflicts of interest to declare.

Introduction: Mature normal and tumor B cells express a unique rearranged immunoglobulin (IG) gene that can be used as a marker of the clonal expansion of the cell. Somatic hypermutation (SHM) in the V(D)J region of IG genes are acquired in the germinal center and are a surrogate imprint of the cell of origin of lymphoid neoplasms. In chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), the identification of SHM distinguishes subtypes of tumors with different clinical and biological behavior. Although still less studied, cases carrying highly similar IG sequences (i.e. stereotyped IG), specific light chain (LC) rearrangements, and the presence of class switch recombination (CSR) of the constant region of the heavy chains further sub-classify patients into potentially distinct clinico-biological subgroups. The analysis of the rearranged IG gene is currently performed by specific Sanger sequencing (SSeq) or next-generation sequencing protocols. Whole-genome sequencing (WGS) of B-cell neoplasms should store the information to reconstruct the entire rearranged IG gene [heavy (IGH) and kappa or lambda (IGK, IGL) LC]. However, the high genomic complexity and homology of these regions have prevented the analysis of the rearranged IG genes in WGS using standard bioinformatics pipelines. Aim: To assess the use of WGS data to fully characterize the rearranged IG gene in B-cell neoplasms. Methods: We developed IgCaller, a fast, easy-to-run program that uses already aligned WGS data to dissect the rearranged IGH V(D)J genes, IGK and IGL VJ genes, and the presence of constant heavy chain CSR. IgCaller also determines the homology of the rearranged sequences compared to the patient's germ line or reference genome. We demonstrated the accuracy of IgCaller using WGS data of 331 B-cell neoplasms [240 CLL (152 cohort 1 (C1)-CLL; 88 independent cohort 2 (C2)-CLL), 61 MCL, and 30 multiple myeloma (MM)] and compared with SSeq of the IGH V(D)J and/or LC and isotype expression. Results: IgCaller identified a complete IGH productive rearrangement [V(D)J] in 133 (88%) C1-CLL, 80 (91%) C2-CLL, 61 (100%) MCL, and 21 (70%) MM. A partial (VJ) rearrangement was detected in 8 (5%) C1-CLL and 1 (3%) MM. Available SSeq of the V(D)J or at least V gene for 131 C1-CLL, 10 C2-CLL, and 60 MCL successfully characterized by WGS highlighted only one discordant V(D)J rearrangement. Small discrepancies (only J or V disagreement) were found when the J (n=4) or V (n=1) gene identified by SSeq based on homology (IMGT/V-QUEST tool) did not correspond to the rearranged gene detected by WGS, which was the second scoring gene in IMGT/V-QUEST suggesting that our non-homology WGS-based approach might be more accurate. Of note, IgCaller identified the presence of two distinct IGH subclones in 1 case. Next, the comparison of the percentage of homology of the rearranged sequence to the germ line in 131 C1-CLL and 60 MCL with complete V gene both by SSeq and WGS showed a high correlation and concordance in both cohorts [R>0.95, p A productive IGK or IGL was found in 147 (97%) C1-CLL, 85 (97%) C2-CLL, 61 (100%) MCL, and 26 (87%) MM. These results fully agreed with the LC expression observed by flow cytometry (FC). Of clinical relevance, a total of 26/232 (11%) CLL carried IGLV3-21. The presence of IGLV3-21 was associated with a shorter time to first treatment (TTFT) independently of the IGHV status (p=0.045). Finally, IgCaller identified the CSR matching the isotype expressed by FC analysis in 26/30 (87%) MM. One of the 4 discordant cases with IgM by WGS expressed only LC by FC. Overall, IgCaller could not identify the isotype switch in 3 (10%) MM expressing IgG or IgA. In CLL, CSR was observed in 47/240 (20%) cases. Noteworthy, the presence of CSR identified 24% of M-CLL patients with a shorter TTFT than non-switched M-CLL (p=0.004), and similar to that of unmutated IGHV cases (p=0.15). Conclusions: IgCaller successfully characterized the entire IG gene of >90% B-cell neoplasms studied. The complete characterization of the rearranged IG gene based on WGS data, when available, could facilitate the analysis of LC rearrangements and CSR, and replace the traditional SSeq of the IG loci both in research and clinical settings. Disclosures No relevant conflicts of interest to declare.

Whole genome sequencing (WGS) studies have started to reveal the critical role of structural variants (SVs) in multiple myeloma (MM) pathogenesis and evolution. We have recently revealed the existence of three main classes of complex events in 30 MM patients: chromothripsis, chromoplexy and templated insertions (Maura F et al, Nat Comm, 2019). Here, drawing on a large cohort of 768 MM patients enrolled in the MMRF CoMMpass study (NCT01454297), we comprehensively characterized the landscape of SVs and their functional implications. Low coverage long-insert WGS (median 4-8X) was available from all patients, of whom 591 also had RNAseq data. Overall, we identified a median of 15 total SVs (range 1-253). Fifty-one percent of SVs (n = 8766) were defined as part of complex events, with a median of one per patient (range 0-14). Chromothripsis, chromoplexy and templated insertions involving >2 chromosomes were observed in 21%, 11% and 21 %, respectively. Chromothripsis was the only SV class with clear prognostic implications after adjustment for molecular and clinical features, resulting in adverse PFS (adjusted HR = 1.57; 95% CI 1.13-2.22; p = 0.008) and OS (adjusted HR = 2.4; 95% CI 1.5-3.83; p < 0.001). Templated insertions emerged as the cause of CCND1-IGH and MYC translocations in 34 % and 73 % of cases, respectively. This is particularly important given the capability of templated insertions to connect and amplify multiple regions of the genome, involving several oncogenes and regulatory regions (e.g. super enhancers). Twenty-four patients (3.1 %) had translocation between an immunoglobulin locus and a non-canonical driver gene (e.g. PAX5, CD40 and MAP3K14), showing outlier expression by RNAseq where available. SV hotspot analysis was carried out using the Piecewise Constant Fitting algorithm, comparing the local SV breakpoint density to an empirical background model (Glodzik et al, Nat Genet, 2017). To identify functionally important hotspots, we integrated: 1) local cumulative copy number data, 2) amplification and deletion peaks identified by GISTIC v2 (q < 0.1), 3) gene fusion data and 4) differential expression analysis with adjustment for main molecular subgroups (limma; Bonferroni-Holm adjusted p-values < 0.01). Ninety-eight hotspots were identified (Figure 1), of which 71 (72%) have not previously been reported. Among these novel hotspots, 23 (33 %) contained a known or suspected driver gene, including TNFRSF17 (encoding CAR-T target BCMA), SYK (BCR signal transduction) and KLF2 (key myeloma transcription factor and germline predisposition locus). Active enhancer regions were present in 29 of the novel hotspots (41 %), including 65 % of those with a concurrent putative driver gene involved. For 34 hotspots (48 %) no clear target gene or regulatory region emerged. SV hotspots and GISTIC peaks covered 13 % of the genome. Overall 38 % of simple and complex SVs had at least one breakpoint falling within a recurrently involved region. The majority of chromoplexy, chromothripsis and templated insertions involved recurrent regions (64, 76 and 86 %, respectively). Simple events were most commonly rare, ranging from 74 % of deletions to 45 % for translocations. Quantifying the global functional impact of the remaining 72 % of non-recurrent or rare SVs, we observed that genes involved by a rare SV were significantly enriched for outlier expression (z-score +/- 2) compared to a permutation background model. Rare deletions and duplications exerted their effects within 10 Kb of the gene body. Translocations and templated insertions were associated with overexpression up to 1 Mb from the gene, but had no effect when involving the gene body, consistent with a major enhancer hijacking mechanism. Finally, we sought to understand the role of recurrent and rare SVs in evolutionary dynamics, analyzing 27 patients that progressed with branching evolution. Seventy-two acquired SVs involved a hotspot region (42 driver and/or enhancer; 48 unknown), while 328 were rare. In conclusion, the SV landscape in multiple myeloma is characterized by multiple recurrently involved genes and regulatory regions. These regions account for the majority of complex SVs, indicating strong positive selection of these events. Nonetheless, the majority of SVs remain unaccounted for. Rare SVs were associated with outlier gene expression and may contribute to the tumor evolutionary trajectory of individual patients. Disclosures Papaemmanuil: Celgene: Research Funding. Landgren:Karyopharm: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Other: IDMC; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees; Theradex: Other: IDMC; Adaptive: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees.

B-other ALL represents a working definition for patients with B cell precursor (BCP) ALL without a known primary chromosomal abnormality. In this study we use whole genome sequencing (WGS) to characterize adult B-other cases (age ≥ 25yrs) from the UKALL14 trial (NCT01085617). Of 652 patients aged 25-65yrs enrolled onto UKALL14, 333 (51%) had B-other ALL. Sufficient material was available to screen 156/333 B-other cases for recurrent Ph-like fusion events (CLRF2, JAK2, ABL1, ABL2PDGFRB) using FISH and MLPA (kit P335). This identified 28 (18%) Ph-like fusion events (21 CRLF2, 5 ABL-class fusions and 2 JAK). Of the remaining 128 B-other cases 57 had available samples for tumor normal paired WGS (read depth 60x and 30x respectively). Bioinformatic analysis was performed to determine small somatic mutations (SSMs); single nucleotide variants (SNVs) and insertion/deletions (INDELs) as well as copy number aberrations (CNA) and structural variants (SV). We also undertook de novo motif analysis to identify RAG mediated deletions. We present data for 30/57 cases (median age of diagnosis 44, range 25-65), the remainder are undergoing sequencing. Within this cohort we identified 784 SVs, 49,244 SNVs and 2,881 INDELs, with each case having a median representation of 23 SVs, 1,674 SNVs and 86 INDELs. The Median SSM burden was 0.55 per megabase (range 0.31-0.81), which is in the upper third of previous ALL estimates (median 0.26 range 0.03-2.9) but low compared to most other cancer types (Alexandrov et al. 2013 Nature). Fusion gene analysis identified 11/30 (37%) cases with recurrent rearrangements (2 ZNF384r, 2 PAX5r, 5 DUX4r and FGFR1r); this identified a novel 5' partner for ZNF384r (AKAP8) and MYO18A-FGFR1 a fusion previously seen in a single case of 8p11 myeloproliferative syndrome. A further 7 clonal coding drivers were identified, 3 PAX5alt, 3 Ph-like candidates and a single ETV6-RUNX1-like candidate (ETV6 R399C, a dominant negative mutation). We classified our cohort into 6 driver subgroups as shown in table 1 using Gu et al. as a framework (Gu et al. 2019 Nat Gen). We found no evidence of driver subgroups clustering with age, although the Ph-like candidates were all identified in patients 39yrs or older. There was evidence that known driver subgroups have differing mutation burdens but these were not significant in this preliminary cohort; the single MEF2Dr case had a high SV burden compared to all other known subgroups (137 vs. cohort median 22); Favorable outcome subgroups (ZNF384r and DUX4r n=7) had a lower SNV coding mutation burden (median 14 vs. 20; U = 60, p = 0.057; Mann-Whitney U test); PAX5alt (n=5) cases had a higher INDEL burden (median 174 vs. 82; U = 16.5, p =0.057). As expected we found recurrent CNA in CDKN2A/B (63%; 19/30), PAX5 (33%; 10/30), IKZF1 (27%; 8/30), ETV6 (11%; 3/30) and BTG1 (7%; 2/30) across the cohort. IKZF1 deletions were enriched in the Ph-like candidate (n=4) subgroup compared to all other known groups (75% vs. 12%; p = 0.028; Fisher's Exact). RAG mediated deletions have been established as an oncogenic driver mechanism in childhood ALL, so we sought to ascertain its role in adults. We identified 54% (128/236) of the breakpoint resolved deletions had a RAG heptamer site. There was a significant difference (U = 167.5, p = 0.021) in RAG deletion burden between patients over 40yrs and under 40yrs at diagnosis (median 1.5 vs 3). Within the unknown driver subgroup; three cases carried ZEB2 H1038R mutations, two with concurrent IGH-CEBPA/B fusions, identifying them as likely belonging to the G12 cluster identified by Li et al. (Li et al. 2018 PNAS); one case had a high translocation burden (37 cohort median 2) and a single case had a high RAG deletion burden (45, cohort median 2); of the remaining 7 cases, four had either a NRAS or FLT3 subclonal hotspot mutation. Here we present the WGS analyses of 30 cases classified as B-other on the basis of no cytogenetic findings by standard clinical assays. The landscape of adult B-other ALL is highly heterogeneous but with WGS we were able to find at least one disease defining event in 60% of our cohort. These events often encompass novel fusion partners of established genomic subtypes or a cytogenetically cryptic lesion such as IGH-DUX4. Taken together our findings demonstrate the clinical utility of WGS as a diagnostic assay to inform and improve the management of adult B-other ALL patients in the future. Disclosures Fielding: Pfizer: Consultancy; Incyte: Consultancy; Amgen: Consultancy; Novartis: Consultancy. Papaemmanuil:Celgene: Research Funding.

Introduction: 5'-Azacitidine (AZA), a DNA demethylating agent, is the primary drug for the treatment of high-risk Myelodysplastic Syndrome (MDS) and Chronic Myelomonocytic Leukaemia (CMML). Response is associated with improved survival. However, only half of patients respond, and these responses are rarely durable. We recently reported that primary AZA resistance is associated with a molecular signature of cell cycle quiescence within bone marrow (BM) hematopoietic progenitor cells (Unnikrishnan et al, Cell Reports, 20:572-585 (2017)). As DNA incorporation of the deoxyribonucleic form of AZA (5-aza-2′-deoxycytidine, DAC) occurs during DNA replication, cell cycle quiescence is predicted to lead to less DAC in DNA and concomitantly less DNA demethylation. We recently developed a quantitative multi-parameter assay, AZA-MS (Unnikrishnan, Vo et al, Leukemia 32:900-910 (2018)), to measure the intracellular dynamics of AZA in patients. Using AZA-MS, we reported data supporting the predicted resistance model. CC486 is an oral formulation of AZA. A 28-day cycle of CC486 involves 21 continuous days (21/28) versus the standard 7/28 subcutaneous (SC) injection AZA scheme. Whether levels of in vivo DAC incorporation into DNA during a cycle of CC486 are comparable with that of SC AZA is unknown. AZA-MS provides us with a unique opportunity to empirically assess the in vivo intracellular dynamics of SC versus oral AZA. Study Design and Methods: To directly assess in vivo DAC incorporation and concomitant DNA demethylation with SC AZA and CC486 in the same patient, we initiated a phase II clinical trial (NCT03493646; Fig A). MDS (IPSS; intermediate-2 or high-risk), CMML (bone marrow [BM] blasts 10-29%) and AML (20-30%) patients were recruited for six cycles of SC AZA (75mg/m^2/day for 7/28 days) followed by six cycles of CC486 (100mg bid for 21/28 days in C7-C8 and 150mg bid for 21/28 in C9-C12). Clinical response was assessed at the end of C6 and C12 using International Working Group criteria. Clinical responders and non-responders to SC AZA at C6 received CC486 from C7 onwards. From each patient, 36 peripheral blood (PB) samples and five BM samples were collected over the study period. DNA, RNA and intracellular fractions were isolated from the PB MNCs, for intracellular DAC/AZA measurements by AZA-MS (primary endpoint; Fig A). BM MNCs were utilised for AZA-MS as well as flow cytometry-based cell cycle measurements (secondary endpoint). Results: 31 of 42 consented patients have commenced treatment since trial opening (Fig B-C). We applied the AZA-MS assay on the longitudinal PB and BM samples collected from the seven patients who had completed six months AZA and commenced CC486 as at 26th June 2019 (Fig D). DAC incorporation into DNA and DNA methylation levels were quantified within the same cells, in addition to measuring other parameters (Fig E). As represented by patient 61213-005 (Fig F) who had a complete response (CR) at cycle 6, after 7 days of injection AZA we observed robust incorporation of DAC within PB MNCs (left panel, Fig F) together with concomitant DNA demethylation (right panel, Fig F). DAC levels diminished upon cessation of AZA within a cycle, with corresponding increases in DNA methylation. There were quantitatively higher levels of DAC incorporated in DNA during SC AZA cycles versus CC486. The trend observed is also appreciated from 2.3x higher area under the curve (AUC) measurements in 61213-005 during the SC AZA cycle. DAC incorporation was higher at C9/10 (CC486 150mg bid 21/28) than at C7/8 (CC486 100mg bid 21/28) without appreciable changes in DNA demethylation. During SC AZA cycles, higher DAC levels (top panel, Fig G) and greater DNA methylation (lower panel, Fig G) were seen in the BM MNCs. In a non-responding patient at cycle 6 (61290-002, SD), we saw less DAC incorporation and DNA demethylation (Fig H). We also observed a positive correlation between baseline proportions of cycling BM cells (LIN-CD34+CD38+) and the amount of DAC incorporated in BM MNCs at C1 day 8 (Fig I). Conclusion: AZA-MS can be used to reliably measure in vivo DAC incorporation and concomitant DNA demethylation in PB MNCs and inform appropriate CC486 dosing. Figure Disclosures Unnikrishnan: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Fong:Astellas: Consultancy; Novartis: Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau. Roncolato:St. George Hospital: Employment. Enjeti:Roche: Honoraria, Speakers Bureau; Bayer and Sanofi: Honoraria, Speakers Bureau; Astellas: Consultancy; Novartis: Consultancy; Abbvie: Consultancy. Hertzberg:BMS: Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche Ltd: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Polizzotto:Janssen: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; ViiV: Research Funding. Pimanda:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Background, Rationale and Experimental Approach Oncogenic fusions are generated via chromosomal rearrangements resulting in an exchange of coding or regulatory DNA sequences. These mutations play an important role in disease onset and subsequent cancer progression, however the exact timing and mechanisms by which they arise are unknown. Through the SickKids clinical sequencing program, KiCS, we explored how and when canonical fusions arise by studying the whole-genomes of childhood cancers with diagnostic or driver fusions. Our investigation began with the pediatric bone cancer, Ewing sarcoma, and later expanded to include other solid, blood, and brain cancers such as papillary thyroid carcinoma, myeloid leukemia, and ependymoma among others. Results The starting point of our investigation was ES, where we sequenced the whole-genomes of 124 cases. Ewing sarcoma (ES) represents the prototypical fusion-driven sarcoma as it is characterized and driven by the EWSR1-ETS fusion. In ~42% of cases, we found that the ES fusion gene arises by chromoplexy, a sudden burst of complex, loop-like rearrangements, rather than by simple reciprocal translocations as previously thought. We show that these rearrangements rapidly and dramatically altered the chromosomal landscape of ES tumors, producing the driver EWSR1-ETS fusion and disrupting numerous other genes in a short time. Remarkably, these complex rearrangements are enriched for genes, including those with a clear role in oncogenesis, and are associated with the earliest replicating portions of the genome. We then sequenced the genomes of 30 other childhood cancers with oncogenic fusions to study their timing and formation mechanisms. In doing so, we have identified several novel fusions in many cancer types, which have been validated by RNA sequencing and cytogenetics. In some cases, the presence of these chromoplectic fusions indicates these patients may benefit from targeted therapy due to the generation of druggable fusions. . Conclusions Our findings provide fundamental insights into the pathogenesis of gene fusions in human cancer. They reveal complex DNA rearrangements to be a mutational process underpinning gene fusions in cancer that influences tumorigenesis. Citation Format: Nathaniel D. Anderson, Richard de Borja, Matthew D. Young, Fabio Fuligni, Andrej Rosic, Nicola D. Roberts, Nischalan Pillay, Jeffrey A. Toretsky, Yoshida Akihiko, Tatsuhiro Shibata, Markus Metzler, Gino Somers, Stephen W. Scherer, Adrienne M. Flanagan, Peter J. Campbell, Joshua D. Schiffman, Mary Shago, Ludmil B. Alexandrov, Jay S. Wunder, Irene L. Andrulis, David Malkin, Sam Behjati, Adam Shlien. Exploring the complex etiology of oncogenic fusions in childhood cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2506.

Human endometrium is a highly dynamic tissue that undergoes hundreds of cycles of breakdown, rapid repair and remodelling in response to the oscillating levels of oestrogen and progesterone during female reproductive years. The marked regenerative capacity of this tissue’s epithelial compartment is maintained by intra-glandular adult stem cells (ASCs) that reside within the stratum basalis which is retained during menstruation. Although the endometrial ASCs were first described over a decade ago, they remain poorly characterised in comparison to their counterparts in other tissues, such as the small and the large intestines. In particular, the size of the stem cell pool within individual glands, the rates of their division, and mutational landscape are largely unknown. In this study, we isolated 215 morphologically normal endometrial glands from women aged 19 to 81 using laser capture microscopy. Analysis of whole-genome sequencing data identified that the overwhelming majority of the glands were clonal cell populations, and thus originating from a single ASC. Somatic mutations were found to accumulate at a linear rate during adult life. Elevated body mass index (BMI), a well-recognised risk factor for endometrial cancer, accelerated the rate of mutation acquisition. Surprisingly, despite the heterogeneity in age, reproductive history and BMI in our cohort, we find relatively homogenous mutational processes within normal endometrium. Comparison with cancer, shows lower somatic mutation burden and fewer operative signatures. Remarkably, we not only identify recurrent acquisitions of certain cancer-associated mutations, particularly those that are advantageous to cell growth, proliferation and migration, but also show that such events occur early in life, potentially even before adolescence. Over time, these mutant ASCs serve as a reservoir for the acquisition of further driver mutations to the extent that in some cases, the entire sampled endometrium becomes ‘neoplastic’ on the genomic level while still retaining the apparently normal phenotype. In older individuals, we observe a shift in the spectrum of acquired cancer-associated mutations, possibly reflecting post-menopausal changes in the levels of sex-steroid hormones and the resultant tissue microenvironment. Citation Format: Luiza Moore, Daniel Leongamornlert, Tim Coorens, Mathijs Sanders, Peter Ellis, Francesco Maura, Kevin Dawson, Simon F. Brunner, Jyoti Nangalia, Henry Lee-Six, Raheleh Rahbari, Patrick Tarpey, Yvette Hooks, Krishnaa Mahbubani, Christine A. Iacobuzio-Donahue, Jan J. Brosens, Inigo Martincorena, Kourosh Saeb-Parsy, Peter J. Campbell, Michael R. Stratton. The mutational landscape of normal human endometrial epithelium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 970.

Simon Stevens proposes that emergency departments focus waiting time targets on a few specific conditions.1 But few people enter emergency departments with a diagnosis of sepsis, myocardial infarction, or stroke. Instead, they present with fever, pain radiating down the arm, or numbness. How do we know for whom the “clock should start”? …

Summary The commonest causes of chronic liver disease are excess alcohol intake, viral hepatitis or non-alcoholic fatty liver disease, with the clinical spectrum ranging in severity from hepatic inflammation through cirrhosis to liver failure or hepatocellular carcinoma. The hepatocellular carcinoma genome exhibits diverse mutational signatures, resulting in recurrent mutations across >20-30 cancer genes1–7. Stem cells from normal livers have low mutation burden and limited diversity of signatures8, suggesting that the complexity of hepatocellular carcinoma arises during progression to chronic liver disease and subsequent malignant transformation. We sequenced whole genomes of 482 microdissections of 100-500 hepatocytes from 5 normal and 9 cirrhotic livers. Compared to normal liver, cirrhotic liver had higher mutation burden. Although rare in normal hepatocytes, structural variants, including chromothripsis, were prominent in cirrhosis. Driver mutations, both point mutations and structural variants, affected 1-5% clones. Clonal expansions millimetres in diameter occurred in cirrhosis, sequestered by bands of fibrosis engirdling regenerative nodules. Some mutational signatures were universal and equally active in both non-malignant hepatocytes and HCC; some were substantially more active in HCC than chronic liver disease; and others, arising from exogenous exposures, were present in a subset of patients. Up to 10-fold within-patient variation in activity of exogenous signatures existed between adjacent cirrhotic nodules, arising from clone-specific and microenvironmental forces. Synchronous hepatocellular carcinomas exhibited the same mutational signatures as background cirrhotic liver, but with higher burden. Somatic mutations chronicle the exposures, toxicity, regeneration and clonal structure of liver tissue as it progresses from health to disease.

Major funding agencies should ensure that large biological data sets are stored in cloud services to enable easy access and fast analysis, say Lincoln D. Stein and colleagues.

It is recognized that some mutated cancer genes contribute to the development of many cancer types, whereas others are cancer type specific. For genes that are mutated in multiple cancer classes, mutations are usually similar in the different affected cancer types. Here, however, we report exquisite tumor type specificity for different histone H3.3 driver alterations. In 73 of 77 cases of chondroblastoma (95%), we found p.Lys36Met alterations predominantly encoded in H3F3B, which is one of two genes for histone H3.3. In contrast, in 92% (49/53) of giant cell tumors of bone, we found histone H3.3 alterations exclusively in H3F3A, leading to p.Gly34Trp or, in one case, p.Gly34Leu alterations. The mutations were restricted to the stromal cell population and were not detected in osteoclasts or their precursors. In the context of previously reported H3F3A mutations encoding p.Lys27Met and p.Gly34Arg or p.Gly34Val alterations in childhood brain tumors, a remarkable picture of tumor type specificity for histone H3.3 driver alterations emerges, indicating that histone H3.3 residues, mutations and genes have distinct functions.

Item does not contain fulltext All cancers are caused by somatic mutations; however, understanding of the biological processes generating these mutations is limited. The catalogue of somatic mutations from a cancer genome bears the signatures of the mutational processes that have been operative. Here we analysed 4,938,362 mutations from 7,042 cancers and extracted more than 20 distinct mutational signatures. Some are present in many cancer types, notably a signature attributed to the APOBEC family of cytidine deaminases, whereas others are confined to a single cancer class. Certain signatures are associated with age of the patient at cancer diagnosis, known mutagenic exposures or defects in DNA maintenance, but many are of cryptic origin. In addition to these genome-wide mutational signatures, hypermutation localized to small genomic regions, 'kataegis', is found in many cancer types. The results reveal the diversity of mutational processes underlying the development of cancer, with potential implications for understanding of cancer aetiology, prevention and therapy.

Adenoid cystic carcinoma (ACC) is a rare malignancy that can occur in multiple organ sites and is primarily found in the salivary gland. While the identification of recurrent fusions of the MYB-NFIB genes have begun to shed light on the molecular underpinnings, little else is known about the molecular genetics of this frequently fatal cancer. We have undertaken exome sequencing in a series of 24 ACC to further delineate the genetics of the disease. We identified multiple mutated genes that, combined, implicate chromatin deregulation in half of cases. Further, mutations were identified in known cancer genes, including PIK3CA, ATM, CDKN2A, SF3B1, SUFU, TSC1, and CYLD. Mutations in NOTCH1/2 were identified in 3 cases, and we identify the negative NOTCH signaling regulator, SPEN, as a new cancer gene in ACC with mutations in 5 cases. Finally, the identification of 3 likely activating mutations in the tyrosine kinase receptor FGFR2, analogous to those reported in ovarian and endometrial carcinoma, point to potential therapeutic avenues for a subset of cases.