Luminal breast epithelial cells from wildtype and BRCA mutation carriers harbor copy number alterations commonly associated with breast cancer.

Cancer-associated mutations have been documented in normal tissues, but the prevalence and nature of somatic copy number alterations and their role in tumor initiation and evolution is not well understood. Here, using single cell DNA sequencing, we describe the landscape of CNAs in >42,000 breast epithelial cells from women with normal or high risk of developing breast cancer. Accumulation of individual cells with one or two of a specific subset of CNAs (e.g. 1q gain and 16q, 22q, 7q, and 10q loss) is detectable in almost all breast tissues and, in those from BRCA1 or BRCA2 mutations carriers, occurs prior to loss of heterozygosity (LOH) of the wildtype alleles. These CNAs, which are among the most common associated with ductal carcinoma in situ (DCIS) and malignant breast tumors, are enriched almost exclusively in luminal cells not basal myoepithelial cells. Allele-specific analysis of the enriched CNAs reveals that each allele was independently altered, demonstrating convergent evolution of these CNAs in an individual breast. Tissues from BRCA1 or BRCA2 mutation carriers contain a small percentage of cells with extreme aneuploidy, featuring loss of TP53 , LOH of BRCA1 or BRCA2 , and multiple breast cancer-associated CNAs in addition to one or more of the common CNAs in 1q, 10q or 16q. Notably, cells with intermediate levels of CNAs are not detected, arguing against a stepwise gradual accumulation of CNAs. Overall, our findings demonstrate that chromosomal alterations in normal breast epithelium partially mirror those of established cancer genomes and are chromosome- and cell lineage-specific.

Breast Cancer.

Breast cancer kills hundreds of thousands of people every year. Rapid progress over the past two decades has increased our understanding of the genetic and environmental risk factors for disease. It has also shed light on drivers of tumor progression and the molecular landscape underpinning tumor heterogeneity, as well as the role of the microenvironment and the immune system. These strides forward should lead to more effective and tailored therapies for early- and late-stage patients.

Disentangling oncogenic amplicons in esophageal adenocarcinoma.

Esophageal adenocarcinoma is a prominent example of cancer characterized by frequent amplifications in oncogenes. However, the mechanisms leading to amplicons that involve breakage-fusion-bridge cycles and extrachromosomal DNA are poorly understood. Here, we use 710 esophageal adenocarcinoma cases with matched samples and patient-derived organoids to disentangle complex amplicons and their associated mechanisms. Short-read sequencing identifies ERBB2, MYC, MDM2, and HMGA2 as the most frequent oncogenes amplified in extrachromosomal DNAs. We resolve complex extrachromosomal DNA and breakage-fusion-bridge cycles amplicons by integrating of de-novo assemblies and DNA methylation in nine long-read sequenced cases. Complex amplicons shared between precancerous biopsy and late-stage tumor, an enrichment of putative enhancer elements and mobile element insertions are potential drivers of complex amplicons' origin. We find that patient-derived organoids recapitulate extrachromosomal DNA observed in the primary tumors and single-cell DNA sequencing capture extrachromosomal DNA-driven clonal dynamics across passages. Prospectively, long-read and single-cell DNA sequencing technologies can lead to better prediction of clonal evolution in esophageal adenocarcinoma.

Circulating tumour mutation detection in triple-negative breast cancer as an adjunct to tissue response assessment.

Circulating tumour DNA (ctDNA) detection via liquid biopsy is an emerging alternative to tissue biopsy, but its potential in treatment response monitoring and prognosis in triple negative breast cancer (TNBC) is not yet well understood. Here we determined the prevalence of actionable mutations detectable in ctDNA using a clinically validated cancer gene panel assay in patients with TNBC, without recurrence at the time of study entry. Sequencing of plasma DNA and validation of variants from 130 TNBC patients collected within 7 months of primary treatment completion revealed that 7.7% had detectable residual disease with a hotspot panel. Among neoadjuvant treated patients, we observed a trend where patients with incomplete pathologic response and positive ctDNA within 7 months of treatment completion were at much higher risk of reduced progression free survival. We propose that a high risk subset of early TNBC patients treated in neoadjuvant therapy protocols may be identifiable by combining tissue response and sensitive ctDNA detection.

Expansion microscopy using a single anchor molecule for high-yield multiplexed imaging of proteins and RNAs.

Expansion microscopy (ExM), by physically enlarging specimens in an isotropic fashion, enables nanoimaging on standard light microscopes. Key to existing ExM protocols is the equipping of different kinds of molecules, with different kinds of anchoring moieties, so they can all be pulled apart from each other by polymer swelling. Here we present a multifunctional anchor, an acrylate epoxide, that enables proteins and RNAs to be equipped with anchors in a single experimental step. This reagent simplifies ExM protocols and reduces cost (by 2-10-fold for a typical multiplexed ExM experiment) compared to previous strategies for equipping RNAs with anchors. We show that this united ExM (uniExM) protocol can be used to preserve and visualize RNA transcripts, proteins in biologically relevant ultrastructures, and sets of RNA transcripts in patient-derived xenograft (PDX) cancer tissues and may support the visualization of other kinds of biomolecular species as well. uniExM may find many uses in the simple, multimodal nanoscale analysis of cells and tissues.

A MYCN-independent mechanism mediating secretome reprogramming and metastasis in MYCN-amplified neuroblastoma.

MYCN amplification (MNA) is a defining feature of high-risk neuroblastoma (NB) and predicts poor prognosis. However, whether genes within or in close proximity to the MYCN amplicon also contribute to MNA+ NB remains poorly understood. Here, we identify that GREB1, a transcription factor encoding gene neighboring the MYCN locus, is frequently coexpressed with MYCN and promotes cell survival in MNA+ NB. GREB1 controls gene expression independently of MYCN, among which we uncover myosin 1B (MYO1B) as being highly expressed in MNA+ NB and, using a chick chorioallantoic membrane (CAM) model, as a crucial regulator of invasion and metastasis. Global secretome and proteome profiling further delineates MYO1B in regulating secretome reprogramming in MNA+ NB cells, and the cytokine MIF as an important pro-invasive and pro-metastatic mediator of MYO1B activity. Together, we have identified a putative GREB1-MYO1B-MIF axis as an unconventional mechanism promoting aggressive behavior in MNA+ NB and independently of MYCN.

Obesity-associated changes in molecular biology of primary breast cancer.

Obesity is associated with an increased risk of developing breast cancer (BC) and worse prognosis in BC patients, yet its impact on BC biology remains understudied in humans. This study investigates how the biology of untreated primary BC differs according to patients' body mass index (BMI) using data from >2,000 patients. We identify several genomic alterations that are differentially prevalent in overweight or obese patients compared to lean patients. We report evidence supporting an ageing accelerating effect of obesity at the genetic level. We show that BMI-associated differences in bulk transcriptomic profile are subtle, while single cell profiling allows detection of more pronounced changes in different cell compartments. These analyses further reveal an elevated and unresolved inflammation of the BC tumor microenvironment associated with obesity, with distinct characteristics contingent on the estrogen receptor status. Collectively, our analyses imply that obesity is associated with an inflammaging-like phenotype. We conclude that patient adiposity may play a significant role in the heterogeneity of BC and should be considered for BC treatment tailoring.

Multi-Omic Analysis of CIC's Functional Networks Reveals Novel Interaction Partners and a Potential Role in Mitotic Fidelity.

CIC encodes a transcriptional repressor and MAPK signalling effector that is inactivated by loss-of-function mutations in several cancer types, consistent with a role as a tumour suppressor. Here, we used bioinformatic, genomic, and proteomic approaches to investigate CIC's interaction networks. We observed both previously identified and novel candidate interactions between CIC and SWI/SNF complex members, as well as novel interactions between CIC and cell cycle regulators and RNA processing factors. We found that CIC loss is associated with an increased frequency of mitotic defects in human cell lines and an in vivo mouse model and with dysregulated expression of mitotic regulators. We also observed aberrant splicing in CIC-deficient cell lines, predominantly at 3' and 5' untranslated regions of genes, including genes involved in MAPK signalling, DNA repair, and cell cycle regulation. Our study thus characterises the complexity of CIC's functional network and describes the effect of its loss on cell cycle regulation, mitotic integrity, and transcriptional splicing, thereby expanding our understanding of CIC's potential roles in cancer. In addition, our work exemplifies how multi-omic, network-based analyses can be used to uncover novel insights into the interconnected functions of pleiotropic genes/proteins across cellular contexts.

Single-cell DNA replication dynamics in genomically unstable cancers.

Dysregulated DNA replication is both a cause and a consequence of aneuploidy, yet the dynamics of DNA replication in aneuploid cell populations remains understudied. We developed a new method, PERT, for inferring cell-specific DNA replication states from single-cell whole genome sequencing, and investigated clone-specific DNA replication dynamics in >50,000 cells obtained from a collection of aneuploid and clonally heterogeneous cell lines, xenografts and primary cancer tissues. Clone replication timing (RT) profiles correlated with future copy number changes in serially passaged cell lines. Cell type was the strongest determinant of RT heterogeneity, while whole genome doubling and mutational process were associated with accumulation of late S-phase cells and weaker RT associations. Copy number changes affecting chromosome X had striking impact on RT, with loss of the inactive X allele shifting replication earlier, and loss of inactive Xq resulting in reactivation of Xp. Finally, analysis of time series xenografts illustrate how cell cycle distributions approximate clone proliferation, recapitulating expected relationships between proliferation and fitness in treatment-naive and chemotherapeutic contexts.

Reconstructing clonal tree for phylo-phenotypic characterization of cancer using single-cell transcriptomics.

Functional characterization of the cancer clones can shed light on the evolutionary mechanisms driving cancer's proliferation and relapse mechanisms. Single-cell RNA sequencing data provide grounds for understanding the functional state of cancer as a whole; however, much research remains to identify and reconstruct clonal relationships toward characterizing the changes in functions of individual clones. We present PhylEx that integrates bulk genomics data with co-occurrences of mutations from single-cell RNA sequencing data to reconstruct high-fidelity clonal trees. We evaluate PhylEx on synthetic and well-characterized high-grade serous ovarian cancer cell line datasets. PhylEx outperforms the state-of-the-art methods both when comparing capacity for clonal tree reconstruction and for identifying clones. We analyze high-grade serous ovarian cancer and breast cancer data to show that PhylEx exploits clonal expression profiles beyond what is possible with expression-based clustering methods and clear the way for accurate inference of clonal trees and robust phylo-phenotypic analysis of cancer.

Imaging and Molecular Annotation of Xenographs and Tumours (IMAXT): High throughput data and analysis infrastructure.

With the aim of producing a 3D representation of tumors, imaging and molecular annotation of xenografts and tumors (IMAXT) uses a large variety of modalities in order to acquire tumor samples and produce a map of every cell in the tumor and its host environment. With the large volume and variety of data produced in the project, we developed automatic data workflows and analysis pipelines. We introduce a research methodology where scientists connect to a cloud environment to perform analysis close to where data are located, instead of bringing data to their local computers. Here, we present the data and analysis infrastructure, discuss the unique computational challenges and describe the analysis chains developed and deployed to generate molecularly annotated tumor models. Registration is achieved by use of a novel technique involving spherical fiducial marks that are visible in all imaging modalities used within IMAXT. The automatic pipelines are highly optimized and allow to obtain processed datasets several times quicker than current solutions narrowing the gap between data acquisition and scientific exploitation.

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.

Results of the phase I CCTG IND.231 trial of CX-5461 in patients with advanced solid tumors enriched for DNA-repair deficiencies.

CX-5461 is a G-quadruplex stabilizer that exhibits synthetic lethality in homologous recombination-deficient models. In this multicentre phase I trial in patients with solid tumors, 40 patients are treated across 10 dose levels (50-650 mg/m2) to determine the recommended phase II dose (primary outcome), and evaluate safety, tolerability, pharmacokinetics (secondary outcomes). Defective homologous recombination is explored as a predictive biomarker of response. CX-5461 is generally well tolerated, with a recommended phase II dose of 475 mg/m2 days 1, 8 and 15 every 4 weeks, and dose limiting phototoxicity. Responses are observed in 14% of patients, primarily in patients with defective homologous recombination. Reversion mutations in PALB2 and BRCA2 are detected on progression following initial response in germline carriers, confirming the underlying synthetic lethal mechanism. In vitro characterization of UV sensitization shows this toxicity is related to the CX-5461 chemotype, independent of G-quadruplex synthetic lethality. These results establish clinical proof-of-concept for this G-quadruplex stabilizer. Clinicaltrials.gov NCT02719977.

Breast tumor microenvironment structures are associated with genomic features and clinical outcome.

The functions of the tumor microenvironment (TME) are orchestrated by precise spatial organization of specialized cells, yet little is known about the multicellular structures that form within the TME. Here we systematically mapped TME structures in situ using imaging mass cytometry and multitiered spatial analysis of 693 breast tumors linked to genomic and clinical data. We identified ten recurrent TME structures that varied by vascular content, stromal quiescence versus activation, and leukocyte composition. These TME structures had distinct enrichment patterns among breast cancer subtypes, and some were associated with genomic profiles indicative of immune escape. Regulatory and dysfunctional T cells co-occurred in large 'suppressed expansion' structures. These structures were characterized by high cellular diversity, proliferating cells and enrichment for BRCA1 and CASP8 mutations and predicted poor outcome in estrogen-receptor-positive disease. The multicellular structures revealed here link conserved spatial organization to local TME function and could improve patient stratification.

DNA methylation landscapes of 1538 breast cancers reveal a replication-linked clock, epigenomic instability and cis-regulation.

DNA methylation is aberrant in cancer, but the dynamics, regulatory role and clinical implications of such epigenetic changes are still poorly understood. Here, reduced representation bisulfite sequencing (RRBS) profiles of 1538 breast tumors and 244 normal breast tissues from the METABRIC cohort are reported, facilitating detailed analysis of DNA methylation within a rich context of genomic, transcriptional, and clinical data. Tumor methylation from immune and stromal signatures are deconvoluted leading to the discovery of a tumor replication-linked clock with genome-wide methylation loss in non-CpG island sites. Unexpectedly, methylation in most tumor CpG islands follows two replication-independent processes of gain (MG) or loss (ML) that we term epigenomic instability. Epigenomic instability is correlated with tumor grade and stage, TP53 mutations and poorer prognosis. After controlling for these global trans-acting trends, as well as for X-linked dosage compensation effects, cis-specific methylation and expression correlations are uncovered at hundreds of promoters and over a thousand distal elements. Some of these targeted known tumor suppressors and oncogenes. In conclusion, this study demonstrates that global epigenetic instability can erode cancer methylomes and expose them to localized methylation aberrations in-cis resulting in transcriptional changes seen in tumors.

A Scalable Strand-Specific Protocol Enabling Full-Length Total RNA Sequencing From Single Cells.

RNA sequencing (RNAseq) has been widely used to generate bulk gene expression measurements collected from pools of cells. Only relatively recently have single-cell RNAseq (scRNAseq) methods provided opportunities for gene expression analyses at the single-cell level, allowing researchers to study heterogeneous mixtures of cells at unprecedented resolution. Tumors tend to be composed of heterogeneous cellular mixtures and are frequently the subjects of such analyses. Extensive method developments have led to several protocols for scRNAseq but, owing to the small amounts of RNA in single cells, technical constraints have required compromises. For example, the majority of scRNAseq methods are limited to sequencing only the 3' or 5' termini of transcripts. Other protocols that facilitate full-length transcript profiling tend to capture only polyadenylated mRNAs and are generally limited to processing only 96 cells at a time. Here, we address these limitations and present a novel protocol that allows for the high-throughput sequencing of full-length, total RNA at single-cell resolution. We demonstrate that our method produced strand-specific sequencing data for both polyadenylated and non-polyadenylated transcripts, enabled the profiling of transcript regions beyond only transcript termini, and yielded data rich enough to allow identification of cell types from heterogeneous biological samples.

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.

Ubiquitin-mediated DNA damage response is synthetic lethal with G-quadruplex stabilizer CX-5461.

CX-5461 is a G-quadruplex (G4) ligand currently in trials with initial indications of clinical activity in cancers with defects in homologous recombination repair. To identify more genetic defects that could sensitize tumors to CX-5461, we tested synthetic lethality for 480 DNA repair and genome maintenance genes to CX-5461, pyridostatin (PDS), a structurally unrelated G4-specific stabilizer, and BMH-21, which binds GC-rich DNA but not G4 structures. We identified multiple members of HRD, Fanconi Anemia pathways, and POLQ, a polymerase with a helicase domain important for G4 structure resolution. Significant synthetic lethality was observed with UBE2N and RNF168, key members of the DNA damage response associated ubiquitin signaling pathway. Loss-of-function of RNF168 and UBE2N resulted in significantly lower cell survival in the presence of CX-5461 and PDS but not BMH-21. RNF168 recruitment and histone ubiquitination increased with CX-5461 treatment, and nuclear ubiquitination response frequently co-localized with G4 structures. Pharmacological inhibition of UBE2N acted synergistically with CX-5461. In conclusion, we have uncovered novel genetic vulnerabilities to CX-5461 with potential significance for patient selection in future clinical trials.

Epiclomal: Probabilistic clustering of sparse single-cell DNA methylation data.

We present Epiclomal, a probabilistic clustering method arising from a hierarchical mixture model to simultaneously cluster sparse single-cell DNA methylation data and impute missing values. Using synthetic and published single-cell CpG datasets, we show that Epiclomal outperforms non-probabilistic methods and can handle the inherent missing data characteristic that dominates single-cell CpG genome sequences. Using newly generated single-cell 5mCpG sequencing data, we show that Epiclomal discovers sub-clonal methylation patterns in aneuploid tumour genomes, thus defining epiclones that can match or transcend copy number-determined clonal lineages and opening up an important form of clonal analysis in cancer. Epiclomal is written in R and Python and is available at https://github.com/shahcompbio/Epiclomal.