Single-cell genomic variation induced by mutational processes in cancer.

How cell-to-cell copy number alterations that underpin genomic instability1 in human cancers drive genomic and phenotypic variation, and consequently the evolution of cancer2, remains understudied. Here, by applying scaled single-cell whole-genome sequencing3 to wild-type, TP53-deficient and TP53-deficient;BRCA1-deficient or TP53-deficient;BRCA2-deficient mammary epithelial cells (13,818 genomes), and to primary triple-negative breast cancer (TNBC) and high-grade serous ovarian cancer (HGSC) cells (22,057 genomes), we identify three distinct 'foreground' mutational patterns that are defined by cell-to-cell structural variation. Cell- and clone-specific high-level amplifications, parallel haplotype-specific copy number alterations and copy number segment length variation (serrate structural variations) had measurable phenotypic and evolutionary consequences. In TNBC and HGSC, clone-specific high-level amplifications in known oncogenes were highly prevalent in tumours bearing fold-back inversions, relative to tumours with homologous recombination deficiency, and were associated with increased clone-to-clone phenotypic variation. Parallel haplotype-specific alterations were also commonly observed, leading to phylogenetic evolutionary diversity and clone-specific mono-allelic expression. Serrate variants were increased in tumours with fold-back inversions and were highly correlated with increased genomic diversity of cellular populations. Together, our findings show that cell-to-cell structural variation contributes to the origins of phenotypic and evolutionary diversity in TNBC and HGSC, and provide insight into the genomic and mutational states of individual cancer cells.

Accurate determination of CRISPR-mediated gene fitness in transplantable tumours.

Assessing tumour gene fitness in physiologically-relevant model systems is challenging due to biological features of in vivo tumour regeneration, including extreme variations in single cell lineage progeny. Here we develop a reproducible, quantitative approach to pooled genetic perturbation in patient-derived xenografts (PDXs), by encoding single cell output from transplanted CRISPR-transduced cells in combination with a Bayesian hierarchical model. We apply this to 181 PDX transplants from 21 breast cancer patients. We show that uncertainty in fitness estimates depends critically on the number of transplant cell clones and the variability in clone sizes. We use a pathway-directed allelic series to characterize Notch signaling, and quantify TP53 / MDM2 drug-gene conditional fitness in outlier patients. We show that fitness outlier identification can be mirrored by pharmacological perturbation. Overall, we demonstrate that the gene fitness landscape in breast PDXs is dominated by inter-patient differences.

Clonal fitness inferred from time-series modelling of single-cell cancer genomes.

Progress in defining genomic fitness landscapes in cancer, especially those defined by copy number alterations (CNAs), has been impeded by lack of time-series single-cell sampling of polyclonal populations and temporal statistical models1-7. Here we generated 42,000 genomes from multi-year time-series single-cell whole-genome sequencing of breast epithelium and primary triple-negative breast cancer (TNBC) patient-derived xenografts (PDXs), revealing the nature of CNA-defined clonal fitness dynamics induced by TP53 mutation and cisplatin chemotherapy. Using a new Wright-Fisher population genetics model8,9 to infer clonal fitness, we found that TP53 mutation alters the fitness landscape, reproducibly distributing fitness over a larger number of clones associated with distinct CNAs. Furthermore, in TNBC PDX models with mutated TP53, inferred fitness coefficients from CNA-based genotypes accurately forecast experimentally enforced clonal competition dynamics. Drug treatment in three long-term serially passaged TNBC PDXs resulted in cisplatin-resistant clones emerging from low-fitness phylogenetic lineages in the untreated setting. Conversely, high-fitness clones from treatment-naive controls were eradicated, signalling an inversion of the fitness landscape. Finally, upon release of drug, selection pressure dynamics were reversed, indicating a fitness cost of treatment resistance. Together, our findings define clonal fitness linked to both CNA and therapeutic resistance in polyclonal tumours.

Clonal Decomposition and DNA Replication States Defined by Scaled Single-Cell Genome Sequencing.

Accurate measurement of clonal genotypes, mutational processes, and replication states from individual tumor-cell genomes will facilitate improved understanding of tumor evolution. We have developed DLP+, a scalable single-cell whole-genome sequencing platform implemented using commodity instruments, image-based object recognition, and open source computational methods. Using DLP+, we have generated a resource of 51,926 single-cell genomes and matched cell images from diverse cell types including cell lines, xenografts, and diagnostic samples with limited material. From this resource we have defined variation in mitotic mis-segregation rates across tissue types and genotypes. Analysis of matched genomic and image measurements revealed correlations between cellular morphology and genome ploidy states. Aggregation of cells sharing copy number profiles allowed for calculation of single-nucleotide resolution clonal genotypes and inference of clonal phylogenies and avoided the limitations of bulk deconvolution. Finally, joint analysis over the above features defined clone-specific chromosomal aneuploidy in polyclonal populations.

Dissociation of solid tumor tissues with cold active protease for single-cell RNA-seq minimizes conserved collagenase-associated stress responses.

BACKGROUND: Single-cell RNA sequencing (scRNA-seq) is a powerful tool for studying complex biological systems, such as tumor heterogeneity and tissue microenvironments. However, the sources of technical and biological variation in primary solid tumor tissues and patient-derived mouse xenografts for scRNA-seq are not well understood. RESULTS: We use low temperature (6 °C) protease and collagenase (37 °C) to identify the transcriptional signatures associated with tissue dissociation across a diverse scRNA-seq dataset comprising 155,165 cells from patient cancer tissues, patient-derived breast cancer xenografts, and cancer cell lines. We observe substantial variation in standard quality control metrics of cell viability across conditions and tissues. From the contrast between tissue protease dissociation at 37 °C or 6 °C, we observe that collagenase digestion results in a stress response. We derive a core gene set of 512 heat shock and stress response genes, including FOS and JUN, induced by collagenase (37 °C), which are minimized by dissociation with a cold active protease (6 °C). While induction of these genes was highly conserved across all cell types, cell type-specific responses to collagenase digestion were observed in patient tissues. CONCLUSIONS: The method and conditions of tumor dissociation influence cell yield and transcriptome state and are both tissue- and cell-type dependent. Interpretation of stress pathway expression differences in cancer single-cell studies, including components of surface immune recognition such as MHC class I, may be especially confounded. We define a core set of 512 genes that can assist with the identification of such effects in dissociated scRNA-seq experiments.

Base excision repair deficiency signatures implicate germline and somatic MUTYH aberrations in pancreatic ductal adenocarcinoma and breast cancer oncogenesis.

We report a case of early-onset pancreatic ductal adenocarcinoma in a patient harboring biallelic MUTYH germline mutations, whose tumor featured somatic mutational signatures consistent with defective MUTYH-mediated base excision repair and the associated driver KRAS transversion mutation p.Gly12Cys. Analysis of an additional 730 advanced cancer cases (N = 731) was undertaken to determine whether the mutational signatures were also present in tumors from germline MUTYH heterozygote carriers or if instead the signatures were only seen in those with biallelic loss of function. We identified two patients with breast cancer each carrying a pathogenic germline MUTYH variant with a somatic MUTYH copy loss leading to the germline variant being homozygous in the tumor and demonstrating the same somatic signatures. Our results suggest that monoallelic inactivation of MUTYH is not sufficient for C:G>A:T transversion signatures previously linked to MUTYH deficiency to arise (N = 9), but that biallelic complete loss of MUTYH function can cause such signatures to arise even in tumors not classically seen in MUTYH-associated polyposis (N = 3). Although defective MUTYH is not the only determinant of these signatures, MUTYH germline variants may be present in a subset of patients with tumors demonstrating elevated somatic signatures possibly suggestive of MUTYH deficiency (e.g., COSMIC Signature 18, SigProfiler SBS18/SBS36, SignatureAnalyzer SBS18/SBS36).

Breast Cancers Activate Stromal Fibroblast-Induced Suppression of Progenitors in Adjacent Normal Tissue.

Human breast cancer cells are known to activate adjacent "normal-like" cells to enhance their own growth, but the cellular and molecular mechanisms involved are poorly understood. We now show by both phenotypic and functional measurements that normal human mammary progenitor cells are significantly under-represented in the mammary epithelium of patients' tumor-adjacent tissue (TAT). Interestingly, fibroblasts isolated from TAT samples showed a reduced ability to support normal EGF-stimulated mammary progenitor cell proliferation in vitro via their increased secretion of transforming growth factor ß. In contrast, TAT fibroblasts promoted the proliferation of human breast cancer cells when these were co-transplanted in immunodeficient mice. The discovery of a common stromal cell-mediated mechanism that has opposing growth-suppressive and promoting effects on normal and malignant human breast cells and also extends well beyond currently examined surgical margins has important implications for disease recurrence and its prevention.

Homologous Recombination Deficiency and Platinum-Based Therapy Outcomes in Advanced Breast Cancer.

Purpose: Recent studies have identified mutation signatures of homologous recombination deficiency (HRD) in over 20% of breast cancers, as well as pancreatic, ovarian, and gastric cancers. There is an urgent need to understand the clinical implications of HRD signatures. Whereas BRCA1/2 mutations confer sensitivity to platinum-based chemotherapies, it is not yet clear whether mutation signatures can independently predict platinum response.Experimental Design: In this observational study, we sequenced tumor whole genomes (100× depth) and matched normals (60×) of 93 advanced-stage breast cancers (33 platinum-treated). We computed a published metric called HRDetect, independently trained to predict BRCA1/2 status, and assessed its capacity to predict outcomes on platinum-based chemotherapies. Clinical endpoints were overall survival (OS), total duration on platinum-based therapy (TDT), and radiographic evidence of clinical improvement (CI).Results: HRDetect predicted BRCA1/2 status with an area under the curve (AUC) of 0.94 and optimal threshold of 0.7. Elevated HRDetect was also significantly associated with CI on platinum-based therapy (AUC = 0.89; P = 0.006) with the same optimal threshold, even after adjusting for BRCA1/2 mutation status and treatment timing. HRDetect scores over 0.7 were associated with a 3-month extended median TDT (P = 0.0003) and 1.3-year extended median OS (P = 0.04).Conclusions: Our findings not only independently validate HRDetect, but also provide the first evidence of its association with platinum response in advanced breast cancer. We demonstrate that HRD mutation signatures may offer clinically relevant information independently of BRCA1/2 mutation status and hope this work will guide the development of clinical trials. Clin Cancer Res; 23(24); 7521-30. ©2017 AACR.

Detection and genomic characterization of a mammary-like adenocarcinoma.

Whole-genome and transcriptome sequencing were performed to identify potential therapeutic strategies in the absence of viable treatment options for a patient initially diagnosed with vulvar adenocarcinoma. Genomic events were prioritized by comparison against variant distributions in the TCGA pan-cancer data set and complemented with detailed transcriptome sequencing and copy-number analysis. These findings were considered against published scientific literature in order to evaluate the functional effects of potentially relevant genomic events. Analysis of the transcriptome against a background of 27 TCGA cancer types led to reclassification of the tumor as a primary HER2+ mammary-like adenocarcinoma of the vulva. This revised diagnosis was subsequently confirmed by follow-up immunohistochemistry for a mammary-like adenocarcinoma. The patient was treated with chemotherapy and targeted therapies for HER2+ breast cancer. The detailed pathology and genomic findings of this case are presented herein.

ReMixT: clone-specific genomic structure estimation in cancer.

Somatic evolution of malignant cells produces tumors composed of multiple clonal populations, distinguished in part by rearrangements and copy number changes affecting chromosomal segments. Whole genome sequencing mixes the signals of sampled populations, diluting the signals of clone-specific aberrations, and complicating estimation of clone-specific genotypes. We introduce ReMixT, a method to unmix tumor and contaminating normal signals and jointly predict mixture proportions, clone-specific segment copy number, and clone specificity of breakpoints. ReMixT is free, open-source software and is available at http://bitbucket.org/dranew/remixt .

Scalable whole-genome single-cell library preparation without preamplification.

Single-cell genomics is critical for understanding cellular heterogeneity in cancer, but existing library preparation methods are expensive, require sample preamplification and introduce coverage bias. Here we describe direct library preparation (DLP), a robust, scalable, and high-fidelity method that uses nanoliter-volume transposition reactions for single-cell whole-genome library preparation without preamplification. We examined 782 cells from cell lines and triple-negative breast xenograft tumors. Low-depth sequencing, compared with existing methods, revealed greater coverage uniformity and more reliable detection of copy-number alterations. Using phylogenetic analysis, we found minor xenograft subpopulations that were undetectable by bulk sequencing, as well as dynamic clonal expansion and diversification between passages. Merging single-cell genomes in silico, we generated 'bulk-equivalent' genomes with high depth and uniform coverage. Thus, low-depth sequencing of DLP libraries may provide an attractive replacement for conventional bulk sequencing methods, permitting analysis of copy number at the cell level and of other genomic variants at the population level.

A Biobank of Breast Cancer Explants with Preserved Intra-tumor Heterogeneity to Screen Anticancer Compounds.

The inter- and intra-tumor heterogeneity of breast cancer needs to be adequately captured in pre-clinical models. We have created a large collection of breast cancer patient-derived tumor xenografts (PDTXs), in which the morphological and molecular characteristics of the originating tumor are preserved through passaging in the mouse. An integrated platform combining in vivo maintenance of these PDTXs along with short-term cultures of PDTX-derived tumor cells (PDTCs) was optimized. Remarkably, the intra-tumor genomic clonal architecture present in the originating breast cancers was mostly preserved upon serial passaging in xenografts and in short-term cultured PDTCs. We assessed drug responses in PDTCs on a high-throughput platform and validated several ex vivo responses in vivo. The biobank represents a powerful resource for pre-clinical breast cancer pharmacogenomic studies (http://caldaslab.cruk.cam.ac.uk/bcape), including identification of biomarkers of response or resistance.

A co-culture genome-wide RNAi screen with mammary epithelial cells reveals transmembrane signals required for growth and differentiation.

INTRODUCTION: The extracellular signals regulating mammary epithelial cell growth are of relevance to understanding the pathophysiology of mammary epithelia, yet they remain poorly characterized. In this study, we applied an unbiased approach to understanding the functional role of signalling molecules in several models of normal physiological growth and translated these results to the biological understanding of breast cancer subtypes. METHODS: We developed and utilized a cytogenetically normal clonal line of hTERT immortalized human mammary epithelial cells in a fibroblast-enhanced co-culture assay to conduct a genome-wide small interfering RNA (siRNA) screen for evaluation of the functional effect of silencing each gene. Our selected endpoint was inhibition of growth. In rigorous postscreen validation processes, including quantitative RT-PCR, to ensure on-target silencing, deconvolution of pooled siRNAs and independent confirmation of effects with lentiviral short-hairpin RNA constructs, we identified a subset of genes required for mammary epithelial cell growth. Using three-dimensional Matrigel growth and differentiation assays and primary human mammary epithelial cell colony assays, we confirmed that these growth effects were not limited to the 184-hTERT cell line. We utilized the METABRIC dataset of 1,998 breast cancer patients to evaluate both the differential expression of these genes across breast cancer subtypes and their prognostic significance. RESULTS: We identified 47 genes that are critically important for fibroblast-enhanced mammary epithelial cell growth. This group was enriched for several axonal guidance molecules and G protein-coupled receptors, as well as for the endothelin receptor PROCR. The majority of genes (43 of 47) identified in two dimensions were also required for three-dimensional growth, with HSD17B2, SNN and PROCR showing greater than tenfold reductions in acinar formation. Several genes, including PROCR and the neuronal pathfinding molecules EFNA4 and NTN1, were also required for proper differentiation and polarization in three-dimensional cultures. The 47 genes identified showed a significant nonrandom enrichment for differential expression among 10 molecular subtypes of breast cancer sampled from 1,998 patients. CD79A, SERPINH1, KCNJ5 and TMEM14C exhibited breast cancer subtype-independent overall survival differences. CONCLUSION: Diverse transmembrane signals are required for mammary epithelial cell growth in two-dimensional and three-dimensional conditions. Strikingly, we define novel roles for axonal pathfinding receptors and ligands and the endothelin receptor in both growth and differentiation.

Dynamics of genomic clones in breast cancer patient xenografts at single-cell resolution.

Human cancers, including breast cancers, comprise clones differing in mutation content. Clones evolve dynamically in space and time following principles of Darwinian evolution, underpinning important emergent features such as drug resistance and metastasis. Human breast cancer xenoengraftment is used as a means of capturing and studying tumour biology, and breast tumour xenografts are generally assumed to be reasonable models of the originating tumours. However, the consequences and reproducibility of engraftment and propagation on the genomic clonal architecture of tumours have not been systematically examined at single-cell resolution. Here we show, using deep-genome and single-cell sequencing methods, the clonal dynamics of initial engraftment and subsequent serial propagation of primary and metastatic human breast cancers in immunodeficient mice. In all 15 cases examined, clonal selection on engraftment was observed in both primary and metastatic breast tumours, varying in degree from extreme selective engraftment of minor (<5% of starting population) clones to moderate, polyclonal engraftment. Furthermore, ongoing clonal dynamics during serial passaging is a feature of tumours experiencing modest initial selection. Through single-cell sequencing, we show that major mutation clusters estimated from tumour population sequencing relate predictably to the most abundant clonal genotypes, even in clonally complex and rapidly evolving cases. Finally, we show that similar clonal expansion patterns can emerge in independent grafts of the same starting tumour population, indicating that genomic aberrations can be reproducible determinants of evolutionary trajectories. Our results show that measurement of genomically defined clonal population dynamics will be highly informative for functional studies using patient-derived breast cancer xenoengraftment.

Lessons learned from the application of whole-genome analysis to the treatment of patients with advanced cancers.

Given the success of targeted agents in specific populations it is expected that some degree of molecular biomarker testing will become standard of care for many, if not all, cancers. To facilitate this, cancer centers worldwide are experimenting with targeted "panel" sequencing of selected mutations. Recent advances in genomic technology enable the generation of genome-scale data sets for individual patients. Recognizing the risk, inherent in panel sequencing, of failing to detect meaningful somatic alterations, we sought to establish processes to integrate data from whole-genome analysis (WGA) into routine cancer care. Between June 2012 and August 2014, 100 adult patients with incurable cancers consented to participate in the Personalized OncoGenomics (POG) study. Fresh tumor and blood samples were obtained and used for whole-genome and RNA sequencing. Computational approaches were used to identify candidate driver mutations, genes, and pathways. Diagnostic and drug information were then sought based on these candidate "drivers." Reports were generated and discussed weekly in a multidisciplinary team setting. Other multidisciplinary working groups were assembled to establish guidelines on the interpretation, communication, and integration of individual genomic findings into patient care. Of 78 patients for whom WGA was possible, results were considered actionable in 55 cases. In 23 of these 55 cases, the patients received treatments motivated by WGA. Our experience indicates that a multidisciplinary team of clinicians and scientists can implement a paradigm in which WGA is integrated into the care of late stage cancer patients to inform systemic therapy decisions.

DNA barcoding reveals diverse growth kinetics of human breast tumour subclones in serially passaged xenografts.

Genomic and phenotypic analyses indicate extensive intra- as well as intertumoral heterogeneity in primary human malignant cell populations despite their clonal origin. Cellular DNA barcoding offers a powerful and unbiased alternative to track the number and size of multiple subclones within a single human tumour xenograft and their response to continued in vivo passaging. Using this approach we find clone-initiating cell frequencies that vary from ~1/10 to ~1/10,000 cells transplanted for two human breast cancer cell lines and breast cancer xenografts derived from three different patients. For the cell lines, these frequencies are negatively affected in transplants of more than 20,000 cells. Serial transplants reveal five clonal growth patterns (unchanging, expanding, diminishing, fluctuating or of delayed onset), whose predominance is highly variable both between and within original samples. This study thus demonstrates the high growth potential and diverse growth properties of xenografted human breast cancer cells.

Glutathione-dependent and -independent oxidative stress-control mechanisms distinguish normal human mammary epithelial cell subsets.

Mechanisms that control the levels and activities of reactive oxygen species (ROS) in normal human mammary cells are poorly understood. We show that purified normal human basal mammary epithelial cells maintain low levels of ROS primarily by a glutathione-dependent but inefficient antioxidant mechanism that uses mitochondrial glutathione peroxidase 2. In contrast, the matching purified luminal progenitor cells contain higher levels of ROS, multiple glutathione-independent antioxidants and oxidative nucleotide damage-controlling proteins and consume O2 at a higher rate. The luminal progenitor cells are more resistant to glutathione depletion than the basal cells, including those with in vivo and in vitro proliferation and differentiation activity. The luminal progenitors also are more resistant to H2O2 or ionizing radiation. Importantly, even freshly isolated "steady-state" normal luminal progenitors show elevated levels of unrepaired oxidative DNA damage. Distinct ROS control mechanisms operating in different subsets of normal human mammary cells could have differentiation state-specific functions and long-term consequences.

Diagnostic value of next-generation sequencing in an unusual sphenoid tumor.

Extraordinary advancements in sequencing technology have made what was once a decade-long multi-institutional endeavor into a methodology with the potential for practical use in a clinical setting. We therefore set out to examine the clinical value of next-generation sequencing by enrolling patients with incurable or ambiguous tumors into the Personalized OncoGenomics initiative at the British Columbia Cancer Agency whereby whole genome and transcriptome analyses of tumor/normal tissue pairs are completed with the ultimate goal of directing therapeutics. First, we established that the sequencing, analysis, and communication with oncologists could be completed in less than 5 weeks. Second, we found that cancer diagnostics is an area that can greatly benefit from the comprehensiveness of a whole genome analysis. Here, we present a scenario in which a metastasized sphenoid mass, which was initially thought of as an undifferentiated squamous cell carcinoma, was rediagnosed as an SMARCB1-negative rhabdoid tumor based on the newly acquired finding of homozygous SMARCB1 deletion. The new diagnosis led to a change in chemotherapy and a complete nodal response in the patient. This study also provides additional insight into the mutational landscape of an adult SMARCB1-negative tumor that has not been explored at a whole genome and transcriptome level.