Failure of origin activation in response to fork stalling leads to chromosomal instability at fragile sites.

Perturbed DNA replication in early stages of cancer development induces chromosomal instability preferentially at fragile sites. However, the molecular basis for this instability is unknown. Here, we show that even under normal growth conditions, replication fork progression along the fragile site, FRA16C, is slow and forks frequently stall at AT-rich sequences, leading to activation of additional origins to enable replication completion. Under mild replication stress, the frequency of stalling at AT-rich sequences is further increased. Strikingly, unlike in the entire genome, in the FRA16C region additional origins are not activated, suggesting that all potential origins are already activated under normal conditions. Thus, the basis for FRA16C fragility is replication fork stalling at AT-rich sequences and inability to activate additional origins under replication stress. Our results provide a mechanism explaining the replication stress sensitivity of fragile sites and thus, the basis for genomic instability during early stages of cancer development.

Circulating tumor DNA and circulating tumor cells in metastatic triple negative breast cancer patients.

Circulating tumor DNA (ctDNA) is a new circulating tumor biomarker which might be used as a prognostic biomarker in a way similar to circulating tumor cells (CTCs). Here, we used the high prevalence of TP53 mutations in triple negative breast cancer (TNBC) to compare ctDNA and CTC detection rates and prognostic value in metastatic TNBC patients. Forty patients were enrolled before starting a new line of treatment. TP53 mutations were characterized in archived tumor tissues and in plasma DNA using two next generation sequencing (NGS) platforms in parallel. Archived tumor tissue was sequenced successfully for 31/40 patients. TP53 mutations were found in 26/31 (84%) of tumor samples. The same mutation was detected in the matched plasma of 21/26 (81%) patients with an additional mutation found only in the plasma for one patient. Mutated allele fractions ranged from 2 to 70% (median 5%). The observed correlation between the two NGS approaches (R(2) = 0.903) suggested that ctDNA levels data were quantitative. Among the 27 patients with TP53 mutations, CTC count was ≥1 in 19 patients (70%) and ≥5 in 14 patients (52%). ctDNA levels had no prognostic impact on time to progression (TTP) or overall survival (OS), whereas CTC numbers were correlated with OS (p = 0.04) and marginally with TTP (p = 0.06). Performance status and elevated LDH also had significant prognostic impact. Here, absence of prognostic impact of baseline ctDNA level suggests that mechanisms of ctDNA release in metastatic TNBC may involve, beyond tumor burden, biological features that do not dramatically affect patient outcome.

New Myc-anisms for DNA replication and tumorigenesis?

The c-Myc proto-oncogene is an essential activator of cell proliferation and one of the genes most commonly deregulated in cancer. Although these activities of c-Myc are thought to result from its function as a transcription factor, the scientific literature contains hints that this is not the whole story. A new paper in Nature by Dominguez-Sola et al. reports the surprising observation that c-Myc promotes DNA replication via a nontranscriptional mechanism, and that c-Myc deregulation causes DNA damage predominately during S phase. These results identify c-Myc as a new DNA replication factor and suggest an alternative model for its role in cell growth and tumorigenesis.

Clinical validity of circulating tumour cells in patients with metastatic breast cancer: a pooled analysis of individual patient data.

BACKGROUND: We aimed to assess the clinical validity of circulating tumour cell (CTC) quantification for prognostication of patients with metastatic breast cancer by undertaking a pooled analysis of individual patient data. METHODS: We contacted 51 European centres and asked them to provide reported and unreported anonymised data for individual patients with metastatic breast cancer who participated in studies between January, 2003, and July, 2012. Eligible studies had participants starting a new line of therapy, data for progression-free survival or overall survival, or both, and CTC quantification by the CellSearch method at baseline (before start of new treatment). We used Cox regression models, stratified by study, to establish the association between CTC count and progression-free survival and overall survival. We used the landmark method to assess the prognostic value of CTC and serum marker changes during treatment. We assessed the added value of CTCs or serum markers to prognostic clinicopathological models in a resampling procedure using likelihood ratio (LR) χ(2) statistics. FINDINGS: 17 centres provided data for 1944 eligible patients from 20 studies. 911 patients (46·9%) had a CTC count of 5 per 7·5 mL or higher at baseline, which was associated with decreased progression-free survival (hazard ratio [HR] 1·92, 95% CI 1·73-2·14, p<0·0001) and overall survival (HR 2·78, 95% CI 2·42-3·19, p<0·0001) compared with patients with a CTC count of less than 5 per 7·5 mL at baseline. Increased CTC counts 3-5 weeks after start of treatment, adjusted for CTC count at baseline, were associated with shortened progression-free survival (HR 1·85, 95% CI 1·48-2·32, p<0·0001) and overall survival (HR 2·26, 95% CI 1·68-3·03) as were increased CTC counts after 6-8 weeks (progression-free survival HR 2·20, 95% CI 1·66-2·90, p<0·0001; overall survival HR 2·91, 95% CI 2·01-4·23, p<0·0001). Survival prediction was significantly improved by addition of baseline CTC count to the clinicopathological models (progression-free survival LR 38·4, 95% CI 21·9-60·3, p<0·0001; overall survival LR 64·9, 95% CI 41·3-93·4, p<0·0001). This model was further improved by addition of CTC change at 3-5 weeks (progression-free survival LR 8·2, 95% CI 0·78-20·4, p=0·004; overall survival LR 11·5, 95% CI 2·6-25·1, p=0·0007) and at 6-8 weeks (progression-free survival LR 15·3, 95% CI 5·2-28·3; overall survival LR 14·6, 95% CI 4·0-30·6; both p<0·0001). Carcinoembryonic antigen and cancer antigen 15-3 concentrations at baseline and during therapy did not add significant information to the best baseline model. INTERPRETATION: These data confirm the independent prognostic effect of CTC count on progression-free survival and overall survival. CTC count also improves the prognostication of metastatic breast cancer when added to full clinicopathological predictive models, whereas serum tumour markers do not. FUNDING: Janssen Diagnostics, the Nuovo-Soldati foundation for cancer research.

Detection rate and prognostic value of circulating tumor cells and circulating tumor DNA in metastatic uveal melanoma.

Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) have been recently investigated in several cancer types, but their respective clinical significance remains to be determined. In our prospective study, we compared the detection rate and the prognostic value of these two circulating biomarkers in patients with metastatic uveal melanoma. GNAQ/GNA11 mutations were characterized in archived tumor tissue. Using a highly sensitive and mutation-specific bidirectional pyrophosphorolysis-activated polymerization (bi-PAP) technique, GNAQ c.626A>T, GNAQ c.626A>C and GNA11 c.626A>T copy numbers were quantified in plasma from 12 mL of blood. CTCs were detected at the same time in 7.5 mL of blood by the CellSearch technique. Patient characteristics and outcome were prospectively collected. CTCs (≥1) were detected in 12 of the 40 included patients (30%, range 1-20). Among the 26 patients with known detectable mutations, ctDNA was detected and quantified in 22 (84%, range 4-11,421 copies/mL). CTC count and ctDNA levels were associated with the presence of miliary hepatic metastasis (p = 0.004 and 0.03, respectively), with metastasis volume (p = 0.005 and 0.004) and with each other (p < 0.0001). CTC count and ctDNA levels were both strongly associated with progression-free survival (p = 0.003 and 0.001) and overall survival (p = 0.0009 and <0.0001). In multivariate analyses, ctDNA appeared to be a better prognostic marker than CTC. In conclusion, ctDNA and CTC are correlated and both have poor prognostic significance. CTC detection can be performed in every patient but, in patients with detectable mutations, ctDNA was more frequently detected than CTC and has possibly more prognostic value.

DNA is a co-factor for its own replication in Xenopus egg extracts.

Soluble Xenopus egg extracts efficiently replicate added plasmids using a physiological mechanism, and thus represent a powerful system to understand vertebrate DNA replication. Surprisingly, DNA replication in this system is highly sensitive to plasmid concentration, being undetectable below ∼10 pM and highly efficient above ∼75 pM. DNA replication at the high plasmid concentration does not require plasmid-plasmid contacts, since replication is not inhibited when plasmids are immobilized in agarose prior to addition of egg extract. The absence of replication at low plasmid concentration is due to a defect in the assembly of pre-replication complexes (pre-RCs). pre-RC assembly requires contact-independent communication between plasmids. Our results show that in Xenopus egg extracts, aggregation of multiple replication forks is not required for efficient replication of plasmid DNA, and they suggest that DNA functions as a co-factor for its own duplication.

Systematic benchmarking of single-cell ATAC-sequencing protocols.

Single-cell assay for transposase-accessible chromatin by sequencing (scATAC-seq) has emerged as a powerful tool for dissecting regulatory landscapes and cellular heterogeneity. However, an exploration of systemic biases among scATAC-seq technologies has remained absent. In this study, we benchmark the performance of eight scATAC-seq methods across 47 experiments using human peripheral blood mononuclear cells (PBMCs) as a reference sample and develop PUMATAC, a universal preprocessing pipeline, to handle the various sequencing data formats. Our analyses reveal significant differences in sequencing library complexity and tagmentation specificity, which impact cell-type annotation, genotype demultiplexing, peak calling, differential region accessibility and transcription factor motif enrichment. Our findings underscore the importance of sample extraction, method selection, data processing and total cost of experiments, offering valuable guidance for future research. Finally, our data and analysis pipeline encompasses 169,000 PBMC scATAC-seq profiles and a best practices code repository for scATAC-seq data analysis, which are freely available to extend this benchmarking effort to future protocols.

Functional inference of gene regulation using single-cell multi-omics.

Cells require coordinated control over gene expression when responding to environmental stimuli. Here we apply scATAC-seq and single-cell RNA sequencing (scRNA-seq) in resting and stimulated human blood cells. Collectively, we generate ~91,000 single-cell profiles, allowing us to probe the cis-regulatory landscape of the immunological response across cell types, stimuli, and time. Advancing tools to integrate multi-omics data, we develop functional inference of gene regulation (FigR), a framework to computationally pair scA-TAC-seq with scRNA-seq cells, connect distal cis-regulatory elements to genes, and infer gene-regulatory networks (GRNs) to identify candidate transcription factor (TF) regulators. Utilizing these paired multi-omics data, we define domains of regulatory chromatin (DORCs) of immune stimulation and find that cells alter chromatin accessibility and gene expression at timescales of minutes. Construction of the stimulation GRN elucidates TF activity at disease-associated DORCs. Overall, FigR enables elucidation of regulatory interactions across single-cell data, providing new opportunities to understand the function of cells within tissues.

DNA replication in nucleus-free Xenopus egg extracts.

Extracts derived from Xenopus laevis eggs represent a powerful cell-free system to study eukaryotic DNA replication. A variation of the system allows for DNA replication not only in a cell-free environment, but also in the absence of a nucleus. In this nucleus-free system, DNA templates are licensed with High-Speed Supernatant (HSS) and then replicated with a concentrated NucleoPlasmic Extract (NPE). This method has the advantage of allowing replication of small plasmids with desired modifications and manipulation of the nuclear environment. This chapter describes the protocols needed to prepare HSS and NPE and how these extracts are used to study DNA replication.

DNA replication origin interference increases the spacing between initiation events in human cells.

Mammalian DNA replication origins localize to sites that range from base pairs to tens of kilobases. A regular distribution of initiations in individual cell cycles suggests that only a limited number of these numerous potential start sites are converted into activated origins. Origin interference can silence redundant origins; however, it is currently unknown whether interference participates in spacing functional human initiation events. By using a novel hybridization strategy, genomic Morse code, on single combed DNA molecules from primary keratinocytes, we report the initiation sites present on 1.5 Mb of human chromosome 14q11.2. We confirm that initiation zones are widespread in human cells, map to intergenic regions, and contain sequence motifs found at other mammalian initiation zones. Origins used per cell cycle are less abundant than the potential sites of initiation, and their limited use increases the spacing between initiation events. Between-zone interference decreases in proportion to the distance from the active origin, whereas within-zone interference is 100% efficient. These results identify a hierarchical organization of origin activity in human cells. Functional origins govern the probability that nearby origins will fire in the context of multiple potential start sites of DNA replication, and this is mediated by origin interference.

Droplet-based combinatorial indexing for massive-scale single-cell chromatin accessibility.

Recent technical advancements have facilitated the mapping of epigenomes at single-cell resolution; however, the throughput and quality of these methods have limited their widespread adoption. Here we describe a high-quality (105 nuclear fragments per cell) droplet-microfluidics-based method for single-cell profiling of chromatin accessibility. We use this approach, named 'droplet single-cell assay for transposase-accessible chromatin using sequencing' (dscATAC-seq), to assay 46,653 cells for the unbiased discovery of cell types and regulatory elements in adult mouse brain. We further increase the throughput of this platform by combining it with combinatorial indexing (dsciATAC-seq), enabling single-cell studies at a massive scale. We demonstrate the utility of this approach by measuring chromatin accessibility across 136,463 resting and stimulated human bone marrow-derived cells to reveal changes in the cis- and trans-regulatory landscape across cell types and under stimulatory conditions at single-cell resolution. Altogether, we describe a total of 510,123 single-cell profiles, demonstrating the scalability and flexibility of this droplet-based platform.

DNA replication origin plasticity and perturbed fork progression in human inverted repeats.

The stability of metazoan genomes during their duplication depends on the spatiotemporal activation of origins and the progression of forks. Human rRNA genes represent a unique challenge to DNA replication since a large proportion of them exist as noncanonical palindromes in addition to canonical tandem repeats. Whether origin usage and/or fork elongation can cope with the variable structure of these genes is unknown. By analyzing single combed DNA molecules from HeLa cells, we studied the rRNA gene replication program according to the organization of canonical versus noncanonical rRNA genes. Origin positioning, spacing, and timing were not affected by the underlying rRNA gene physical structure. Conversely, fork arrest, both temporary and permanent, occurred more frequently when rRNA gene palindromes were encountered. These findings reveal that while initiation mechanisms are flexible enough to adapt to an rRNA gene structure of any arrangement, palindromes represent obstacles to fork progression, which is a likely source of genomic instability.

Analysis of individual cells identifies cell-to-cell variability following induction of cellular senescence.

Senescent cells play important roles in both physiological and pathological processes, including cancer and aging. In all cases, however, senescent cells comprise only a small fraction of tissues. Senescent phenotypes have been studied largely in relatively homogeneous populations of cultured cells. In vivo, senescent cells are generally identified by a small number of markers, but whether and how these markers vary among individual cells is unknown. We therefore utilized a combination of single-cell isolation and a nanofluidic PCR platform to determine the contributions of individual cells to the overall gene expression profile of senescent human fibroblast populations. Individual senescent cells were surprisingly heterogeneous in their gene expression signatures. This cell-to-cell variability resulted in a loss of correlation among the expression of several senescence-associated genes. Many genes encoding senescence-associated secretory phenotype (SASP) factors, a major contributor to the effects of senescent cells in vivo, showed marked variability with a subset of highly induced genes accounting for the increases observed at the population level. Inflammatory genes in clustered genomic loci showed a greater correlation with senescence compared to nonclustered loci, suggesting that these genes are coregulated by genomic location. Together, these data offer new insights into how genes are regulated in senescent cells and suggest that single markers are inadequate to identify senescent cells in vivo.

High purity microfluidic sorting and analysis of circulating tumor cells: towards routine mutation detection.

A new generation of the Ephesia cell capture technology optimized for CTC capture and genetic analysis is presented, characterized in depth and compared with the CellSearch system as a reference. This technology uses magnetic particles bearing tumour-cell specific EpCAM antibodies, self-assembled in a regular array in a microfluidic flow cell. 48,000 high aspect-ratio columns are generated using a magnetic field in a high throughput (>3 ml h(-1)) device and act as sieves to specifically capture the cells of interest through antibody-antigen interactions. Using this device optimized for CTC capture and analysis, we demonstrated the capture of epithelial cells with capture efficiency above 90% for concentrations as low as a few cells per ml. We showed the high specificity of capture with only 0.26% of non-epithelial cells captured for concentrations above 10 million cells per ml. We investigated the capture behavior of cells in the device, and correlated the cell attachment rate with the EpCAM expression on the cell membranes for six different cell lines. We developed and characterized a two-step blood processing method to allow for rapid processing of 10 ml blood tubes in less than 4 hours, and showed a capture rate of 70% for as low as 25 cells spiked in 10 ml blood tubes, with less than 100 contaminating hematopoietic cells. Using this device and procedure, we validated our system on patient samples using an automated cell immunostaining procedure and a semi-automated cell counting method. Our device captured CTCs in 75% of metastatic prostate cancer patients and 80% of metastatic breast cancer patients, and showed similar or better results than the CellSearch device in 10 out of 13 samples. Finally, we demonstrated the possibility of detecting cancer-related PIK3CA gene mutation in 20 cells captured in the chip with a good correlation between the cell count and the quantitation value Cq of the post-capture qPCR.

Circulating tumor DNA as a non-invasive substitute to metastasis biopsy for tumor genotyping and personalized medicine in a prospective trial across all tumor types.

Cell-free tumor DNA (ctDNA) has the potential to enable non-invasive diagnostic tests for personalized medicine in providing similar molecular information as that derived from invasive tumor biopsies. The histology-independent phase II SHIVA trial matches patients with targeted therapeutics based on previous screening of multiple somatic mutations using metastatic biopsies. To evaluate the utility of ctDNA in this trial, as an ancillary study we performed de novo detection of somatic mutations using plasma DNA compared to metastasis biopsies in 34 patients covering 18 different tumor types, scanning 46 genes and more than 6800 COSMIC mutations with a multiplexed next-generation sequencing panel. In 27 patients, 28 of 29 mutations identified in metastasis biopsies (97%) were detected in matched ctDNA. Among these 27 patients, one additional mutation was found in ctDNA only. In the seven other patients, mutation detection from metastasis biopsy failed due to inadequate biopsy material, but was successful in all plasma DNA samples providing three more potential actionable mutations. These results suggest that ctDNA analysis is a potential alternative and/or replacement to analyses using costly, harmful and lengthy tissue biopsies of metastasis, irrespective of cancer type and metastatic site, for multiplexed mutation detection in selecting personalized therapies based on the patient's tumor genetic content.

Low-coverage single-cell mRNA sequencing reveals cellular heterogeneity and activated signaling pathways in developing cerebral cortex.

Large-scale surveys of single-cell gene expression have the potential to reveal rare cell populations and lineage relationships but require efficient methods for cell capture and mRNA sequencing. Although cellular barcoding strategies allow parallel sequencing of single cells at ultra-low depths, the limitations of shallow sequencing have not been investigated directly. By capturing 301 single cells from 11 populations using microfluidics and analyzing single-cell transcriptomes across downsampled sequencing depths, we demonstrate that shallow single-cell mRNA sequencing (~50,000 reads per cell) is sufficient for unbiased cell-type classification and biomarker identification. In the developing cortex, we identify diverse cell types, including multiple progenitor and neuronal subtypes, and we identify EGR1 and FOS as previously unreported candidate targets of Notch signaling in human but not mouse radial glia. Our strategy establishes an efficient method for unbiased analysis and comparison of cell populations from heterogeneous tissue by microfluidic single-cell capture and low-coverage sequencing of many cells.

Time-Dependent Prognostic Impact of Circulating Tumor Cells Detection in Non-Metastatic Breast Cancer: 70-Month Analysis of the REMAGUS02 Study.

Introduction. In non-metastatic breast cancer patients, the REMAGUS02 neoadjuvant study was the first to report a significant impact of circulating tumor cells (CTCs) detection by the CellSearch system on the distant metastasis-free survival (DMFS) and overall survival (OS) endpoints. However, these results were only reported after a short follow-up. Here, we present the updated data, with a longer follow-up. Material and Methods. CTC count was performed before and after neoadjuvant chemotherapy in 118 patients and correlated to survival. Results. CTC count results were available before and/or after neoadjuvant chemotherapy in 115 patients. After a median follow-up of 70 months, detection of ≥1 CTC/7.5 mL before chemotherapy (N = 95) was significantly associated with DMFS (P = 0.04) and OS (P = 0.03), whereas postchemotherapy CTC detection (N = 85) had no significant impact. In multivariable analysis, prechemotherapy CTC and triple negative phenotype were the two independent prognostic factors for survival. We observed that the CTC impact is most significant during the first three years of follow-up. Discussion. We confirm that the detection of CTC is independently associated with a significantly worse outcome, but mainly during the first 3-4 years of follow-up. No prognostic impact is seen in patients who are still relapse-free at this moment.

Human ribosomal RNA gene arrays display a broad range of palindromic structures.

The standard model of eukaryotic ribosomal RNA (rRNA) genes involves tandem arrays with hundreds of units in clusters, the nucleolus organizer regions (NORs). A first genomic overview for human cells is reported here for these regions, which have never been sequenced in their totality, by using molecular combing. The rRNA-coding regions are examined by fluorescence on single molecules of DNA with two specific probes that cover their entire length. The standard organization assumed for rDNA units is a transcribed region followed by a nontranscribed spacer. While we confirmed this arrangement in many cases, unorthodox patterns were also observed in normal individuals, with one-third of the rDNA units rearranged to form apparently palindromic structures (noncanonical units) independent of the age of the donors. In cells from individuals with a deficiency in the WRN RecQ helicase (Werner syndrome), the proportion of palindromes increased to one-half. These findings, supported by Southern blot analyses, show that rRNA genes are a mosaic of canonical and (presumably nonfunctional) palindromic units that may be altered by factors associated with genomic instability and pathology.

Single DNA molecule analysis: applications of molecular combing.

Dynamic changes to the genomic structure and to the DNA replication programme are important determinants of normal and abnormal cell development. To understand these changes and how they vary from cell to cell, single DNA molecules from both normal and abnormal cell populations must be examined and compared. Physical characterisation of single genomes at the kilobase level of resolution over large genomic regions is possible with molecular combing technology. An array of combed single DNA molecules is prepared by stretching molecules attached by their extremities to a silanised glass surface with a receding air-water meniscus. By performing fluorescent hybridisation on combed DNA, genomic probe position can be directly visualised, providing a means to construct physical maps and detect micro-rearrangements. Single-molecule DNA replication can also be monitored through fluorescent detection of incorporated nucleotide analogues on combed DNA molecules. These and other single-molecule applications of molecular combing are discussed in this paper and future developments of the technology are considered.