Clonal Dynamics Research Articles

Applying phylogenetic methods for species delimitation to distinguish B-cell clonal families

The adaptive immune system generates a diverse array of B-cell receptors through the processes of V(D)J recombination and somatic hypermutation. B-cell receptors that bind to an antigen will undergo clonal expansion, creating a Darwinian evolutionary dynamic within individuals. A key step in studying these dynamics is to identify sequences derived from the same ancestral V(D)J recombination event (i.e. a clonal family). There are a number of widely used methods for accomplishing this task but a major limitation of all of them is that they rely, at least in part, on the ability to map sequences to a germline reference set. This requirement is particularly problematic in non-model systems where we often know little about the germline allelic diversity in the study population. Recognizing that delimiting B-cell clonal families is analogous to delimiting species from single locus data, we propose a novel strategy of reconstructing the phylogenetic tree of all B-cell sequences in a sample and using a popular species delimitation method, multi-rate Poisson Tree Processes (mPTP), to delimit clonal families. Using extensive simulations, we show that not only does this phylogenetically explicit approach perform well for the purpose of delimiting clonal families when no reference allele set is available, it performs similarly to state-of-the-art techniques developed specifically for B-cell data even when we have a complete reference allele set. Additionally, our analysis of an empirical dataset shows that mPTP performs similarly to leading methods in the field. These findings demonstrate the utility of using off-the-shelf phylogenetic techniques for analyzing B-cell clonal dynamics in non-model systems, and suggests that phylogenetic inference techniques may be potentially combined with mapping based approaches for even more robust inferences, even in model systems.

Evolution of Rapid Clonal Dynamics and Non-Cross-Resistance in Response to Alternating Targeted Therapy and Chemotherapy in BRAF-V600E-Mutant Colon Cancer.

Combined BRAF, MEK, and EGFR inhibition can induce clinical responses in BRAF-V600E-mutant colon cancer, but rapid resistance often occurs. We use serial monitoring of circulating tumor DNA cell-free plasma DNA (cfDNA) in a patient case study in addition to organoids derived from mouse models of BRAF-V600E-mutant intestinal cancer, which emulated the patient's mutational profile to assess drug treatment efficacy. We demonstrate dynamic evolution of resistance to combined EGFR/BRAF/MEK inhibition in a pediatric patient with metastatic BRAF-V600E-mutant, mismatch repair-stable colon cancer. Initial resistance to targeted therapy was associated with development of MET amplification. Sequential treatment with chemotherapy and targeted therapy resulted in clearing of the resistant MET-amplified clone. Rechallenge with combined BRAF/EGFR inhibition resulted in clinical and radiographic response, demonstrating these treatments may be non-cross-resistant. Tumor organoids were used to model clinical findings and demonstrated effectiveness of combined targeted therapy and chemotherapy. These findings suggest rational strategies for combining sequential chemotherapy and BRAF-/EGFR-directed therapy in BRAF-V600E-mutant colon cancer to prevent resistance and improve outcome. The data demonstrate rapid clonal dynamics in response to effective therapies in BRAF-V600E-mutant colon cancer that can be monitored by serial cfDNA analysis. Moreover, in mismatch repair-proficient BRAF-V600E-mutant colon cancers, combined EGFR and BRAF/MEK therapy is not cross-resistant with standard chemotherapy, suggesting new rational combination treatment strategies.

Integrative mapping of human CD8+ T cells in inflammation and cancer.

CD8+ T cells exhibit remarkable phenotypic diversity in inflammation and cancer. However, a comprehensive understanding of their clonal landscape and dynamics remains elusive. Here we introduce scAtlasVAE, a deep-learning-based model for the integration of large-scale single-cell RNA sequencing data and cross-atlas comparisons. scAtlasVAE has enabled us to construct an extensive human CD8+ T cell atlas, comprising 1,151,678 cells from 961 samples across 68 studies and 42 disease conditions, with paired T cell receptor information. Through incorporating information in T cell receptor clonal expansion and sharing, we have successfully established connections between distinct cell subtypes and shed light on their phenotypic and functional transitions. Notably, our approach characterizes three distinct exhausted T cell subtypes and reveals diverse transcriptome and clonal sharing patterns in autoimmune and immune-related adverse event inflammation. Furthermore, scAtlasVAE facilitates the automatic annotation of CD8+ T cell subtypes in query single-cell RNA sequencing datasets, enabling unbiased and scalable analyses. In conclusion, our work presents a comprehensive single-cell reference and computational framework for CD8+ T cell research.

Developmental hematopoietic stem cell variation explains clonal hematopoiesis later in life.

Clonal hematopoiesis becomes increasingly common with age, but its cause is enigmatic because driver mutations are often absent. Serial observations infer weak selection indicating variants are acquired much earlier in life with unexplained initial growth spurts. Here we use fluctuating CpG methylation as a lineage marker to track stem cell clonal dynamics of hematopoiesis. We show, via the shared prenatal circulation of monozygotic twins, that weak selection conferred by stem cell variation created before birth can reliably yield clonal hematopoiesis later in life. Theory indicates weak selection will lead to dominance given enough time and large enough population sizes. Human hematopoiesis satisfies both these conditions. Stochastic loss of weakly selected variants is naturally prevented by the expansion of stem cell lineages during development. The dominance of stem cell clones created before birth is supported by blood fluctuating CpG methylation patterns that exhibit low correlation between unrelated individuals but are highly correlated between many elderly monozygotic twins. Therefore, clonal hematopoiesis driven by weak selection in later life appears to reflect variation created before birth.

Asymmetric stem cell division maintains genetic heterogeneity of tissue cells.

Within a given tissue, the stem cell niche provides the microenvironment for stem cells suitable for their self-renewal. Conceptually, the niche space constrains the size of a stem-cell pool, as the cells sharing the niche compete for its space. It has been suggested that either neutral- or non-neutral-competition of stem cells changes the clone dynamics of stem cells. Theoretically, if the rate of asymmetric division is high, the stem cell competition is limited, thus suppressing clonal expansion. However, the effects of asymmetric division on clone dynamics have never been experimentally tested. Here, using the Drosophila germline stem cell (GSC) system, as a simple model of the in-vivo niche, we examine the effect of division modes (asymmetric or symmetric) on clonal dynamics by combining experimental approaches with mathematical modeling. Our results suggest that the rate of asymmetric division positively correlates with the time a stem cell clone takes to expand. Taken together, our data suggests that asymmetric division is essential for maintaining the genetic variation of stem cells and thus serves as a critical mechanism for safeguarding fertility over the animal age or preventing multiple disorders caused by the clonal expansion of stem cells.

Methylation sequencing enhances interpretation of clonal hematopoiesis dynamics

AbstractWe have developed a cost-effective DNA methylation sequencing assay to improve monitoring of clonal hematopoiesis. By inferring cell-type proportions, this method enhances interpretation of clonal trajectories compared with interpretation based on variant allele fraction only.

A rapid and simple clonality assay for bovine leukemia virus-infected cells by amplified fragment length polymorphism (AFLP) analysis.

Enzootic bovine leukosis (EBL) is routinely diagnosed based on external manifestations at the farm, such as the presence of tumors and/or general lymph node enlargement. However, due to the nonspecific clinical manifestations of EBL, over half of EBL cases are unrecognized at the farm, with most cases being diagnosed during postmortem inspection at the slaughterhouse. Early detection and monitoring of clonal expansion are necessary for managing EBL and reducing economic losses. In this study, we developed BLV-AFLP that represents a significant advancement in the diagnosis of EBL in cattle. This method can rapidly assess the clonal proliferation of BLV-infected cells, crucial for distinguishing between asymptomatic and EBL cattle. Additionally, tracking clonal dynamics offers insights into the disease's progression, potentially providing strategies for avoiding economic losses. Overall, as BLV-AFLP is a simple and rapid test for detecting EBL, it is feasible and efficient for routine veterinary practice.

Spatially Resolved in vivo CRISPR Screen Sequencing via Perturb-DBiT.

Perturb-seq enabled the profiling of transcriptional effects of genetic perturbations in single cells but lacks the ability to examine the impact on tissue environments. We present Perturb-DBiT for simultaneous co- sequencing of spatial transcriptome and guide RNAs (gRNAs) on the same tissue section for in vivo CRISPR screen with genome-scale gRNA libraries, offering a comprehensive understanding of how genetic modifications affect cellular behavior and tissue architecture. This platform supports a variety of delivery vectors, gRNA library sizes, and tissue preparations, along with two distinct gRNA capture methods, making it adaptable to a wide range of experimental setups. In applying Perturb-DBiT, we conducted un-biased knockouts of tens of genes or at genome-wide scale across three cancer models. We mapped all gRNAs in individual colonies and corresponding transcriptomes in a human cancer metastatic colonization model, revealing clonal dynamics and cooperation. We also examined the effect of genetic perturbation on the tumor immune microenvironment in an immune-competent syngeneic model, uncovering differential and synergistic perturbations in promoting immune infiltration or suppression in tumors. Perturb-DBiT allows for simultaneously evaluating the impact of each knockout on tumor initiation, development, metastasis, histopathology, and immune landscape. Ultimately, it not only broadens the scope of genetic inquiry, but also lays the groundwork for developing targeted therapeutic strategies.

Long-term tracking of haematopoietic clonal dynamics and mutations in non-human primate undergoing transplantation of lentivirally barcoded haematopoietic stem and progenitor cells.

Haematopoietic stem and progenitor cell (HSPC) autologous gene therapies are promising treatment for a variety of blood disorders. Investigation of the long-term HSPC clonal dynamics and other measures of safety and durability following lentiviral-mediated gene therapies in predictive models are crucial for assessing risks and benefits in order to inform decisions regarding wider utilization. We established an autologous lentivirally barcoded HSPC transplantation model in rhesus macaque (RM), a model offering insights into haematopoiesis and gene therapies with direct relevance to human. Healthy young adult RMs underwent total body irradiation, followed by transplantation of autologous HSPCs transduced with a lentiviral vector containing a diverse genetic barcode library, uniquely labelling individual HSPCs and their progeny. With up to 131 months of follow-up, we now report quantitative clonal dynamics, characterizing the number, diversity, stability and lineage bias of hundreds of thousands of HSPC clones tracked in five RMs. We documented long-term stable and multi-lineage output from a highly polyclonal pool of HSPCs. Clonal succession after stable haematopoietic reconstitution was minimal. There was no evidence for accelerated acquisition of acquired somatic mutations following autologous lentivirally transduced HSPC transplantation. Our results provide relevant insights into long-term HSPC behaviours invivo following transplantation and gene therapies.

Clonal dynamics after allogeneic haematopoietic cell transplantation

Allogeneic haematopoietic cell transplantation (HCT) replaces the stem cells responsible for blood production with those from a donor1,2. Here, to quantify dynamics of long-term stem cell engraftment, we sequenced genomes from 2,824 single-cell-derived haematopoietic colonies of ten donor–recipient pairs taken 9–31 years after HLA-matched sibling HCT3. With younger donors (18–47 years at transplant), 5,000–30,000 stem cells had engrafted and were still contributing to haematopoiesis at the time of sampling; estimates were tenfold lower with older donors (50–66 years). Engrafted cells made multilineage contributions to myeloid, B lymphoid and T lymphoid populations, although individual clones often showed biases towards one or other mature cell type. Recipients had lower clonal diversity than matched donors, equivalent to around 10–15 years of additional ageing, arising from up to 25-fold greater expansion of stem cell clones. A transplant-related population bottleneck could not explain these differences; instead, phylogenetic trees evinced two distinct modes of HCT-specific selection. In pruning selection, cell divisions underpinning recipient-enriched clonal expansions had occurred in the donor, preceding transplant—their selective advantage derived from preferential mobilization, collection, survival ex vivo or initial homing. In growth selection, cell divisions underpinning clonal expansion occurred in the recipient’s marrow after engraftment, most pronounced in clones with multiple driver mutations. Uprooting stem cells from their native environment and transplanting them to foreign soil exaggerates selective pressures, distorting and accelerating the loss of clonal diversity compared to the unperturbed haematopoiesis of donors.

Characterization of clonal dynamics using duplex sequencing in donor-recipient pairs decades after hematopoietic cell transplantation.

After allogeneic hematopoietic cell transplantation (HCT), a very small number of donor stem cells reconstitute the recipient hematopoietic system, whereas the donor is left with a near-normal pool of stem cells. We hypothesized that the increased replicative stress on transplanted donor cells in the recipient could lead to the disproportionate proliferation of clonal hematopoiesis (CH) variants. We obtained blood samples from 16 related donor-recipient pairs at a median of 33.8 years (range: 6.6 to 45.7) after HCT, including the longest surviving HCT recipients in the world. For 11 of 16 pairs, a donor sample from the time of HCT was available for comparison. We performed ultrasensitive duplex sequencing of genes recurrently mutated in myeloid malignancies and CH, as well as a set of functionally neutral genomic regions representative of human genomic content at large. CH variants were observed in all donors, even those as young as 12 years old. Where donor pre-HCT sample was available, the average mutation rate in donors compared to recipients post-HCT was similar (2.0% versus 2.6% per year, respectively) within genes recurrently mutated in myeloid malignancies. Twenty-two (5.6%) of the 393 variants shared between paired donors and recipients post-HCT showed ≥10-fold higher variant allele frequency (VAF) in the recipient. A longer time since HCT was positively associated with the expansion of shared variant VAFs in the recipient. In conclusion, even decades after HCT, there does not appear to be widespread accelerated clonal expansion in the transplanted cells, highlighting the immense regenerative capacity of the human hematopoietic system.

Abstract A010: Tumor-reactive T cell clonal dynamics across treatment time points drive resistance and progression to frontline chemoimmunotherapy in advanced gastric cancer

Abstract A010: Tumor-reactive T cell clonal dynamics across treatment time points drive resistance and progression to frontline chemoimmunotherapy in advanced gastric cancer

Droplet Hi-C enables scalable, single-cell profiling of chromatin architecture in heterogeneous tissues.

Current methods for analyzing chromatin architecture are not readily scalable to heterogeneous tissues. Here we introduce Droplet Hi-C, which uses a commercial microfluidic device for high-throughput, single-cell chromatin conformation profiling in droplets. Using Droplet Hi-C, we mapped the chromatin architecture of the mouse cortex and analyzed gene regulatory programs in major cortical cell types. In addition, we used this technique to detect copy number variations, structural variations and extrachromosomal DNA in human glioblastoma, colorectal and blood cancer cells, revealing clonal dynamics and other oncogenic events during treatment. We refined the technique to allow joint profiling of chromatin architecture and transcriptome in single cells, facilitating exploration of the links between chromatin architecture and gene expression in both normal tissues and tumors. Thus, Droplet Hi-C both addresses critical gaps in chromatin analysis of heterogeneous tissues and enhances understanding of gene regulation.

Somatic mutations in autoinflammatory and autoimmune disease.

Somatic mutations (also known as acquired mutations) are emerging as common, age-related processes that occur in all cells throughout the body. Somatic mutations are canonically linked to malignant processes but over the past decade have been increasingly causally connected to benign diseases including rheumatic conditions. Here we outline the contribution of somatic mutations to complex and monogenic immunological diseases with a detailed review of unique aspects associated with such causes. Somatic mutations can cause early- or late-onset rheumatic monogenic diseases but also contribute to the pathogenesis of complex inflammatory and immune-mediated diseases, affect disease progression and define new clinical subtypes. Although even variants with a low variant allele fraction can be pathogenic, clonal dynamics could lead to changes over time in the proportion of mutant cells, with possible phenotypic consequences for the individual. Thus, somatic mutagenesis and clonal expansion have relevant implications in genetic testing and counselling. On the basis of both increased recognition of somatic diseases in clinical practice and improved technical and bioinformatic processes, we hypothesize that there will be an ever-expanding list of somatic mutations in various genes leading to inflammatory conditions, particularly in late-onset disease.

Mitochondrial genetic variations in leukemia: a comprehensive overview.

Leukemias are a group of heterogeneous hematological malignancies driven by diverse genetic variations, and the advent of genomic sequencing technologies facilitates the investigation of genetic abnormalities in leukemia. However, these sequencing-based studies mainly focus on nuclear DNAs. Increasing evidence indicates that mitochondrial dysfunction is an important mechanism of leukemia pathogenesis, which is closely related to the mitochondrial genome variations. Here, we provide an overview of current research progress concerning mitochondrial genetic variations in leukemia, encompassing gene mutations and copy number variations. We also summarize currently accessible mitochondrial DNA (mtDNA) sequencing methods. Notably, somatic mtDNA mutations may serve as natural genetic barcodes for lineage tracing and longitudinal assessment of clonal dynamics. Collectively, these findings enhance our understanding of leukemia pathogenesis and foster the identification of novel therapeutic targets and interventions.

Neoantigen immunogenicity landscapes and evolution of tumor ecosystems during immunotherapy with nivolumab.

Neoantigen immunoediting drives immune checkpoint blockade efficacy, yet the molecular features of neoantigens and how neoantigen immunogenicity shapes treatment response remain poorly understood. To address these questions, 80 patients with non-small cell lung cancer were enrolled in the biomarker cohort of CheckMate 153 (CA209-153), which collected radiographic guided biopsy samples before treatment and during treatment with nivolumab. Early loss of mutations and neoantigens during therapy are both associated with clinical benefit. We examined 1,453 candidate neoantigens, including many of which that had reduced cancer cell fraction after treatment with nivolumab, and identified 196 neopeptides that were recognized by T cells. Mapping these neoantigens to clonal dynamics, evolutionary trajectories and clinical response revealed a strong selection against immunogenic neoantigen-harboring clones. We identified position-specific amino acid and physiochemical features related to immunogenicity and developed an immunogenicity score. Nivolumab-induced microenvironmental evolution in non-small cell lung cancer shared some similarities with melanoma, yet critical differences were apparent. This study provides unprecedented molecular portraits of neoantigen landscapes underlying nivolumab's mechanism of action.

Clonal dynamics and somatic evolution of haematopoiesis in mouse.

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

Genome analysis of tigecycline-resistant Acinetobacter baumannii reveals nosocomial lineage shifts and novel resistance mechanisms.

To investigate the characteristics and clonal dynamics of tigecycline-resistant Acinetobacter baumannii (TRAB) isolates from a Chinese hospital from 2016 to 2021. A total of 64 TRAB isolates were screened and WGS was performed. Phylogenetic analysis and non-polymorphic mutation analysis were used to analyse their clonal dynamics and tigecycline resistance-related mutations. RT-PCR was used to analyse the expression of the resistance-nodulation cell-division (RND) efflux pump genes adeB and adeJ. Gene cloning was used to explore the effect of tet(39) variants on tigecycline resistance. Most TRAB isolates were found to be MDR, with 95% (61/64) of the isolates showing resistance to carbapenems. These TRAB isolates were classified into three primary genetic clusters based on core-genome SNPs. The KL2 cluster persisted throughout the study period, whereas the KL7 cluster emerged in 2019 and became the dominant clone. The KL7 cluster carried more antimicrobial resistance genes than the other two clusters. The predominant tigecycline resistance mechanism of the KL2 cluster and KL7 cluster was IS insertion in adeN (82.1%, 23/28) and genetic alterations in adeS (76.2%, 16/21), respectively. Eleven novel AdeS mutations were identified associated with elevated AdeB expression and tigecycline resistance. Moreover, we characterized a plasmid-borne tet(39) variant with an Ala-36-Thr substitution that synergizes with the RND efflux pump to confer high-level tigecycline resistance. This work provides important insights into the diverse mechanisms associated with tigecycline resistance in A. baumannii, highlighting a pressing need for further monitoring of ST2-KL7 A. baumannii in clinical settings.

Long-term longitudinal analysis of 4,187 participants reveals insights into determinants of clonal hematopoiesis

Clonal hematopoiesis of indeterminate potential (CHIP) is linked to diverse aging-related diseases, including hematologic malignancy and atherosclerotic cardiovascular disease (ASCVD). While CHIP is common among older adults, the underlying factors driving its development are largely unknown. To address this, we performed whole-exome sequencing on 8,374 blood DNA samples collected from 4,187 Atherosclerosis Risk in Communities Study (ARIC) participants over a median follow-up of 21 years. During this period, 735 participants developed incident CHIP. Splicing factor genes (SF3B1, SRSF2, U2AF1, and ZRSR2) and TET2 CHIP grow significantly faster than DNMT3A non-R882 clones. We find that age at baseline and sex significantly influence the incidence of CHIP, while ASCVD and other traditional ASCVD risk factors do not exhibit such associations. Additionally, baseline synonymous passenger mutations are strongly associated with CHIP status and are predictive of new CHIP clone acquisition and clonal growth over extended follow-up, providing valuable insights into clonal dynamics of aging hematopoietic stem and progenitor cells. This study also reveals associations between germline genetic variants and incident CHIP. Our comprehensive longitudinal assessment yields insights into cell-intrinsic and -extrinsic factors contributing to the development and progression of CHIP clones in older adults.

Cancer drug-tolerant persister cells: from biological questions to clinical opportunities.

The emergence of drug resistance is the most substantial challenge to the effectiveness of anticancer therapies. Orthogonal approaches have revealed that a subset of cells, known as drug-tolerant 'persister' (DTP) cells, have a prominent role in drug resistance. Although long recognized in bacterial populations which have acquired resistance to antibiotics, the presence of DTPs in various cancer types has come to light only in the past two decades, yet several aspects of their biology remain enigmatic. Here, we delve into the biological characteristics of DTPs and explore potential strategies for tracking and targeting them. Recent findings suggest that DTPs exhibit remarkable plasticity, being capable of transitioning between different cellular states, resulting in distinct DTP phenotypes within a single tumour. However, defining the biological features of DTPs has been challenging, partly due to the complex interplay between clonal dynamics and tissue-specific factors influencing their phenotype. Moreover, the interactions between DTPs and the tumour microenvironment, including their potential to evade immune surveillance, remain to be discovered. Finally, the mechanisms underlying DTP-derived drug resistance and their correlation with clinical outcomes remain poorly understood. This Roadmap aims to provide a comprehensive overview of the field of DTPs, encompassing past achievements and current endeavours in elucidating their biology. We also discuss the prospect of future advancements in technologies in helping to unveil the features of DTPs and propose novel therapeutic strategies that could lead to their eradication.