Copy Number Evolution Research Articles

The History of Chromosomal Instability in Genome-Doubled Tumors.

Tumors frequently display high chromosomal instability and contain multiple copies of genomic regions. Here, we describe Gain Route Identification and Timing In Cancer (GRITIC), a generic method for timing genomic gains leading to complex copy number states, using single-sample bulk whole-genome sequencing data. By applying GRITIC to 6,091 tumors, we found that non-parsimonious evolution is frequent in the formation of complex copy number states in genome-doubled tumors. We measured chromosomal instability before and after genome duplication in human tumors and found that late genome doubling was followed by an increase in the rate of copy number gain. Copy number gains often accumulate as punctuated bursts, commonly after genome doubling. We infer that genome duplications typically affect the landscape of copy number losses, while only minimally impacting copy number gains. In summary, GRITIC is a novel copy number gain timing framework that permits the analysis of copy number evolution in chromosomally unstable tumors. Significance: Complex genomic gains are associated with whole-genome duplications, which are frequent across tumors, span a large fraction of their genomes, and are linked to poorer outcomes. GRITIC infers when these gains occur during tumor development, which will help to identify the genetic events that drive tumor evolution. See related commentary by Taylor, p. 1766.

Differential Conservation and Loss of Chicken Repeat 1 (CR1) Retrotransposons in Squamates Reveal Lineage-Specific Genome Dynamics Across Reptiles.

Transposable elements (TEs) are repetitive DNA sequences which create mutations and generate genetic diversity across the tree of life. In amniote vertebrates, TEs have been mainly studied in mammals and birds, whose genomes generally display low TE diversity. Squamates (Order Squamata; including ∼11,000 extant species of lizards and snakes) show as much variation in TE abundance and activity as they do in species and phenotypes. Despite this high TE activity, squamate genomes are remarkably uniform in size. We hypothesize that novel, lineage-specific genome dynamics have evolved over the course of squamate evolution. To understand the interplay between TEs and host genomes, we analyzed the evolutionary history of the chicken repeat 1 (CR1) retrotransposon, a TE family found in most tetrapod genomes which is the dominant TE in most reptiles. We compared 113 squamate genomes to the genomes of turtles, crocodilians, and birds and used ancestral state reconstruction to identify shifts in the rate of CR1 copy number evolution across reptiles. We analyzed the repeat landscapes of CR1 in squamate genomes and determined that shifts in the rate of CR1 copy number evolution are associated with lineage-specific variation in CR1 activity. We then used phylogenetic reconstruction of CR1 subfamilies across amniotes to reveal both recent and ancient CR1 subclades across the squamate tree of life. The patterns of CR1 evolution in squamates contrast other amniotes, suggesting key differences in how TEs interact with different host genomes and at different points across evolutionary history.

Abstract 1200: A novel algorithm for deconvolving cancer allele-specific clone copy number and copy number evolution

Abstract Both single nucleotide variants (SNVs) and somatic copy number alterations (SCNAs) accumulate in cancer cells during tumor development, fuelling clonal evolution. However, accurate inference of their coevolution from bulk DNA sequencing is challenging. We present ALPACA (ALlele-specific Phylogenetic Analysis of clone Copy number Alterations), a novel algorithm to allow the practical inference of SNV and SCNA coevolution. ALPACA is framed as an optimisation problem, that takes as input bulk tumor sample copy number mixtures and the tumor phylogenetic tree constructed from SNVs, and deconvolves the optimal, allele-specific integer copy number profiles for every distinct clone present in the tumor. To circumvent the challenge of joint SNV/SCNA inference, we postulate that in high mutation burden tumors all relevant SCNA clones are identifiable by their unique SNVs, hence ALPACA leverages phylogenetic trees constructed from bulk tumor sequencing data using SNVs. Secondly, to restrict the clone-specific copy number search space, ALPACA leverages constraints imposed by multisample sequencing, specifically in cases where clones are present across multiple samples or clones from different samples are phylogenetically related. Finally, ALPACA relies on parsimony and biologically informed constraints to further guide the deconvolution process. We demonstrate that ALPACA infers the copy number evolution of complex simulated tumors with higher accuracy than current state-of-the-art methods. We apply ALPACA to the large, multisample non-small cell lung cancer (NSCLC) cohort from the recent longitudinal, prospective TRACERx421 study, and show that ALPACA uncovers loss of heterozygosity and amplification events in minor subclones that were previously missed using standard approaches. ALPACA's assignment of SCNA driver events to branches of the phylogenetic tree reveals the evolutionary ordering of SNVs and SCNAs during NSCLC tumor evolution, such as late focal amplifications affecting the TERT gene locus occurring in subclones which expand and dominate a tumor sample. ALPACA enables an in-depth analysis of the coevolution of mutations and SCNAs in the tumor’s evolutionary history, revealing distinct patterns of copy number evolution. Notably, ALPACA identifies common patterns of copy number changes across the genome characterizing metastatic seeding clones, revealing that they harbor an increased number of SCNAs compared to clones that do not metastasize. Additionally, ALPACA uncovers subclonal enrichment for CCND1 amplification in primary tumor subclones that seed metastasis, and an overall increase of SCNA events occurring in both tumors with polyclonal seeding and extrathoracic metastases. Clone-level results obtained with ALPACA can offer new clinical insights and enable new types of analysis, e.g. copy number signature analysis including temporal order of SCNA acquisition. Citation Format: Piotr Pawlik, Kristiana K. Grigoriadis, Abigail Bunkum, Simone Zaccaria, Nicholas McGranahan, Charles Swanton. A novel algorithm for deconvolving cancer allele-specific clone copy number and copy number evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1200.

Abstract 4338: A pan-cancer single-cell analysis of intratumoral copy number diversity and evolution

Abstract Aneuploidy is a hallmark of human cancers and studies have shown that cancer patients harbor many differences in their copy number aberrations (CNAs) reflecting interpatient heterogeneity (IPH). While IPH has been studied extensively, the intratumoral heterogeneity (ITH) of CNAs within a tumor remains understudied. Here, we conducted a pan-cancer analysis of 94 human tumor samples at single cell genomic resolution, representing seven major cancer types: bladder, breast, colon, glioblastoma, kidney, lung, and ovary. In total, 62,646 aneuploid cells were analyzed by single cell copy number profiling and bulk exome sequencing was performed. In most cancer types, increased subclonal diversity was associated with higher CNA burden, whole genome doubling (WGD) events, and TP53 mutations. In all tumors, our data show that cancer cells share a clonal set of truncal CNAs or mutations, suggesting these tumors evolved from a single ancestral cell. In 18/94 cases we identified subclonal WGD events, which resulted in a higher number of copy number losses after tetraploidization. Macro-spatial sampling showed that increased clonal diversity also correlated with increased spatial geographical diversity. We found that many cancers evolved CNAs in short punctuated bursts of evolution, and that this was not restricted to a specific cancer type. Collectively, these data show that copy number ITH is universally associated with molecular features across these human cancer types. Citation Format: Hanghui Ye, Thomas McDonald, Emi Sei, Darlan Conterno Minussi, Min Hu, Chenling Tang, Junke Wang, Kaile Wang, Anna Casasent, Hui Chen, Franziska Michor, Nicholas Navin. A pan-cancer single-cell analysis of intratumoral copy number diversity and evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4338.

Digest: Does the insertion preference of a transposable element represent a strategy for the mitigation of genetic conflicts with the host?

Transposable elements can exhibit a predilection for specific insertion regions. In a recent study, Munasinghe et al. (2023) consider how variation in where TE families prefer to insert within the genome influences their copy number evolution. The study emphasizes how a preference for neutral insertion sites is only advantageous in conjunction with host restriction mechanisms, which suggests that insertion preference may be a strategy to mitigate genetic conflicts with the host.

Abstract A001: Genomic signatures of past and present chromosomal instability in Barrett’s esophagus and early esophageal adenocarcinoma

Abstract Abstract: The progression of precancerous lesions to malignancy is often accompanied by increasing complexity of chromosomal alterations but how these alterations arise is poorly understood. Here we perform haplotype-specific analysis of chromosomal copy-number evolution in the progression of Barrett’s esophagus (BE) to esophageal adenocarcinoma (EAC) on multiregional whole-genome sequencing data of BE with dysplasia and microscopic EAC foci. We identify distinct patterns of copy-number evolution indicating multigenerational chromosomal instability that is initiated by cell division errors but propagated only after p53 loss. While abnormal mitosis, including whole-genome duplication, underlies chromosomal copy-number changes, segmental alterations display signatures of successive breakage-fusion-bridge cycles and chromothripsis of unstable dicentric chromosomes. Our analysis elucidates how multigenerational chromosomal instability generates copy-number variation in BE cells, precipitates complex alterations including DNA amplifications, and promotes their independent clonal expansion and transformation. In particular, we suggest sloping copy-number variation as a signature of ongoing chromosomal instability that precedes copy-number complexity. These findings suggest copy-number heterogeneity in advanced cancers originates from chromosomal instability in precancerous cells and such instability may be identified from the presence of sloping copy-number variation in bulk sequencing data. Citation Format: Chunyang Bao. Genomic signatures of past and present chromosomal instability in Barrett’s esophagus and early esophageal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A001.

A zero-agnostic model for copy number evolution in cancer.

New low-coverage single-cell DNA sequencing technologies enable the measurement of copy number profiles from thousands of individual cells within tumors. From this data, one can infer the evolutionary history of the tumor by modeling transformations of the genome via copy number aberrations. Copy number aberrations alter multiple adjacent genomic loci, violating the standard phylogenetic assumption that loci evolve independently. Thus, specialized models to infer copy number phylogenies have been introduced. A widely used model is the copy number transformation (CNT) model in which a genome is represented by an integer vector and a copy number aberration is an event that either increases or decreases the number of copies of a contiguous segment of the genome. The CNT distance between a pair of copy number profiles is the minimum number of events required to transform one profile to another. While this distance can be computed efficiently, no efficient algorithm has been developed to find the most parsimonious phylogeny under the CNT model. We introduce the zero-agnostic copy number transformation (ZCNT) model, a simplification of the CNT model that allows the amplification or deletion of regions with zero copies. We derive a closed form expression for the ZCNT distance between two copy number profiles and show that, unlike the CNT distance, the ZCNT distance forms a metric. We leverage the closed-form expression for the ZCNT distance and an alternative characterization of copy number profiles to derive polynomial time algorithms for two natural relaxations of the small parsimony problem on copy number profiles. While the alteration of zero copy number regions allowed under the ZCNT model is not biologically realistic, we show on both simulated and real datasets that the ZCNT distance is a close approximation to the CNT distance. Extending our polynomial time algorithm for the ZCNT small parsimony problem, we develop an algorithm, Lazac, for solving the large parsimony problem on copy number profiles. We demonstrate that Lazac outperforms existing methods for inferring copy number phylogenies on both simulated and real data.

Genomic signatures of past and present chromosomal instability in Barrett’s esophagus and early esophageal adenocarcinoma

The progression of precancerous lesions to malignancy is often accompanied by increasing complexity of chromosomal alterations but how these alterations arise is poorly understood. Here we perform haplotype-specific analysis of chromosomal copy-number evolution in the progression of Barrett’s esophagus (BE) to esophageal adenocarcinoma (EAC) on multiregional whole-genome sequencing data of BE with dysplasia and microscopic EAC foci. We identify distinct patterns of copy-number evolution indicating multigenerational chromosomal instability that is initiated by cell division errors but propagated only after p53 loss. While abnormal mitosis, including whole-genome duplication, underlies chromosomal copy-number changes, segmental alterations display signatures of successive breakage-fusion-bridge cycles and chromothripsis of unstable dicentric chromosomes. Our analysis elucidates how multigenerational chromosomal instability generates copy-number variation in BE cells, precipitates complex alterations including DNA amplifications, and promotes their independent clonal expansion and transformation. In particular, we suggest sloping copy-number variation as a signature of ongoing chromosomal instability that precedes copy-number complexity. These findings suggest copy-number heterogeneity in advanced cancers originates from chromosomal instability in precancerous cells and such instability may be identified from the presence of sloping copy-number variation in bulk sequencing data.

1O SPICE: Probabilistic reconstruction of copy-number evolution in metastatic cancers

1O SPICE: Probabilistic reconstruction of copy-number evolution in metastatic cancers

Abstract P5-06-02: Circulating tumor cells in metastatic breast cancer highlight potential role of copy number evolution in late-stage cancer mutational profile

Abstract Background: Structural variation is a hallmark of breast cancer. Previous single-cell genomic analysis of 10 untreated early-stage primary TNBC tumors identified only one or two subclones per tumor, found that copy number variation (CNV) occurs early in the evolution of the tumor, and asserted that CNV changes do not contribute to genomic variation at later time points of a tumor’s growth. This PCNE model (punctuated copy number evolution) is analogous to the big-bang theory of colorectal cancer. However, cancer exists in a dynamic environment, and systemic chemotherapy provides evolutionary pressure, making stasis quite unlikely. An investigation of late-stage cancers could shed light on CNV evolution. Methods: We performed a longitudinal, prospective observational study of over 130 patients with metastatic breast cancer, with regular follow-up and detailed treatment histories. CTCs were enumerated by CellSearch, and samples with >20 CTCs in 7.5ml of whole blood were further processed for single CTC isolation via DEPArray, single-cell library construction, and whole-genome sequencing. CNV counts for each cell were obtained using previously published binning methods (Ginkgo). Three mathematical models were used to evaluate the evolution of CNVs: a punctuated model, a gradual model, and a gradual-on-punctuated hybrid model. Results: In total, 150 patient-years of data were collected. Among samples with sufficient CTC counts for study inclusion, a total of nine patients and 376 cells were isolated for sequencing. A mean sequencing depth of 0.8× was achieved from each single cell. The degree of CNV heterogeneity varies from patient to patient. CNV changes occurred over time in a gradual manner on a background of punctuated changes. The adjusted R2 value for the punctuated model was 0.46, the adj R2 value for the gradual model was 0.76, and the hybrid model achieved an adj R2 of 0.99. Patient samples with large CNV heterogeneity across single cells tend to be from patients with longer treatment histories and from the last blood draw (i.e., the blood collection closest to patient’s point of death). We also identified multiple whole-genome duplication (WGD) events from the same patient, in contrast to previous findings that WGD events are usually early clonal events in most instances. Finally, X loss events are among the most frequent chromosomal aberrations identified in CTCs (in up to 80% of CTCs in some patients), which supports previous literature on the loss of the Barr body in hastening metastatic spread. Discussion and Conclusion: In contrast to the previous study, our findings support a gradual-on-punctuated hybrid model, and this model can explain how copy number changes can be a source from which tumors gain resistance to systemic therapies. The model explains how CNV heterogeneities are seen in patients with longer treatment histories, suggesting that CNV continues to be a source of genetic variation at the metastatic stage of the disease course. Citation Format: Weelic Chong, Rui Luo, Zhenchao Zhang, Maysa Abu-Khalaf, Daniel Silver, Frederick Fellin, Rebecca Jaslow, AnaMaria Lopez, Terrence Cescon, Ronald Myers, Mariel Becker, Qiang Wei, Bingshan Li, Chun Wang, Hushan Yang. Circulating tumor cells in metastatic breast cancer highlight potential role of copy number evolution in late-stage cancer mutational profile [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-06-02.

Abstract B036: The role of whole genome duplication in progression of pancreatic ductal adenocarcinoma

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDA) is a lethal disease that tends to present at an advanced stage, but the evolutionary mechanisms responsible for rapid progression in PDA are poorly understood. Whole genome duplication (WGD) is a hallmark of cancer observed more frequently in late-stage tumors that predicts poorer overall patient survival across cancer types, including PDA. The genome-wide instability that occurs during WGD events can lead to gene dosage imbalances that promote tumor progression and may explain the rapid progression of aneuploidy. Although WGD has been studied extensively in advanced cancer, there is limited data on WGD in primary tumors. We hypothesize that WGD occurs frequently in primary PDA and promotes the rapid progression and high rate of relapse in early stage PDA. We leveraged scWGS to infer copy number profiles and WGD events from tumor cells to determine the rate of subclonal WGD in primary PDA tumors and understand how aneuploidy and genome instability evolve in the earliest stages of tumor progression. Methods: Single cell barcoded libraries from primary treatment naive tumors were prepared with the 10X Genomics Single Cell CNV platform and sequenced to a depth of 20-60x per tumor. Reads were processed using CellRanger DNA pipeline, demultiplexed, and analyzed with Gingko CNV. A total of 10,323 cells from 8 primary PDA tumors (range, 192 – 3146; median = 1286) were recovered, resulting in an average coverage of 0.06x per single cell (range, 0.02x – 0.14x). Results: 5/8 tumors analyzed had evidence of at least one WGD event. The WGD event was identified as a minor subclone in 3 of these 5. Copy number profiles revealed 1-6 distinct subclones in each tumor, demonstrating copy number evolution is ongoing. WGD+ tumors trend towards having higher diversity and higher percentage of genome altered. Conclusion: WGD occurs more frequently in primary PDA than previously understood, and may confer high levels of genetic instability. Citation Format: Lauren Hummel, Irene Xie, Karen Ng, Amy Zhang, Stephanie Ramotar, Anna Dodd, Grainne O'Kane, Jennifer Knox, Julie Wilson, Steven Gallinger, Faiyaz Notta. The role of whole genome duplication in progression of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B036.

Mutation and selection processes regulating short tandem repeats give rise to genetic and phenotypic diversity across species.

Short tandem repeats (STRs) are units of 1-6 bp that repeat in a tandem fashion in DNA. Along with single nucleotide polymorphisms and large structural variations, they are among the major genomic variants underlying genetic, and likely phenotypic, divergence. STRs experience mutation rates that are orders of magnitude higher than other well-studied genotypic variants. Frequent copy number changes result in a wide range of alleles, and provide unique opportunities for modulating complex phenotypes through variation in repeat length. While classical studies have identified key roles of individual STR loci, the advent of improved sequencing technology, high-quality genome assemblies for diverse species, and bioinformatics methods for genome-wide STR analysis now enable more systematic study of STR variation across wide evolutionary ranges. In this review, we explore mutation and selection processes that affect STR copy number evolution, and how these processes give rise to varying STR patterns both within and across species. Finally, we review recent examples of functional and adaptive changes linked to STRs.

Long-read Sequencing Data Reveals Dynamic Evolution of Mitochondrial Genome Size and the Phylogenetic Utility of Mitochondrial DNA in Hercules Beetles (Dynastes; Scarabaeidae).

The evolutionary dynamics and phylogenetic utility of mitochondrial genomes (mitogenomes) have been of particular interest to systematists and evolutionary biologists. However, certain mitochondrial features, such as the molecular evolution of the control region in insects, remain poorly explored due to technological constraints. Using a combination of long- and short-read sequencing data, we assembled ten complete mitogenomes from ten Hercules beetles. We found large-sized mitogenomes (from 24 to 28 kb), which are among the largest in insects. The variation in genome size can be attributed to copy-number evolution of tandem repeats in the control region. Furthermore, one type of tandem repeat was found flanking the conserved sequence block in the control region. Importantly, such variation, which made up around 30% of the size of the mitogenome, may only become detectable should long-read sequencing technology be applied. We also found that, although different mitochondrial loci often inferred different phylogenetic histories, none of the mitochondrial loci statistically reject a concatenated mitochondrial phylogeny, supporting the hypothesis that all mitochondrial loci share a single genealogical history. We on the other hand reported statistical support for mito-nuclear phylogenetic discordance in 50% of mitochondrial loci. We argue that long-read DNA sequencing should become a standard application in the rapidly growing field of mitogenome sequencing. Furthermore, mitochondrial gene trees may differ even though they share a common genealogical history, and ND loci could be better candidates for phylogenetics than the commonly used COX1.

A feedback mechanism controls rDNA copy number evolution in yeast independently of natural selection.

Ribosomal DNA (rDNA) is the genetic loci that encodes rRNA in eukaryotes. It is typically arranged as tandem repeats that vary in copy number within the same species. We have recently shown that rDNA repeats copy number in the yeast Saccharomyces cerevisiae is controlled by cell volume via a feedback circuit that senses cell volume by means of the concentration of the free upstream activator factor (UAF). The UAF strongly binds the rDNA gene promoter, but is also able to repress SIR2 deacetylase gene transcription that, in turn, represses rDNA amplification. In this way, the cells with a smaller DNA copy number than what is optimal evolve to increase that copy number until they reach a number that sequestrates free UAF and provokes SIR2 derepression that, in turn, blocks rDNA amplification. Here we propose a mathematical model to show that this evolutionary process can amplify rDNA repeats independently of the selective advantage of yeast cells having bigger or smaller rDNA copy numbers. We test several variants of this process and show that it can explain the observed experimental results independently of natural selection. These results predict that an autoregulated feedback circuit may, in some instances, drive to non Darwinian deterministic evolution for a limited time period.

CONET: copy number event tree model of evolutionary tumor history for single-cell data

Copy number alterations constitute important phenomena in tumor evolution. Whole genome single-cell sequencing gives insight into copy number profiles of individual cells, but is highly noisy. Here, we propose CONET, a probabilistic model for joint inference of the evolutionary tree on copy number events and copy number calling. CONET employs an efficient, regularized MCMC procedure to search the space of possible model structures and parameters. We introduce a range of model priors and penalties for efficient regularization. CONET reveals copy number evolution in two breast cancer samples, and outperforms other methods in tree reconstruction, breakpoint identification and copy number calling.

Abstract IA025: Decoding breast cancer evolution with single cell genomics

Abstract I will discuss our efforts to develop and apply single cell DNA sequencing technologies to study breast cancer progression and evolution. We have developed a method called Acoustic Cell Tagmentation (ACT) which can provide high-throughput single cell copy number profiling of hundreds of cells in parallel at single-molecule resolution. We have applied this approach to study copy number evolution in Triple-negative breast cancer, which has revealed a ‘transient instability’ model of early copy number evolution, after the acquisition of TP53 mutations and genome doubling. We have also developed a computational toolkit called CopyKit to analyze single cell DNA sequencing data, and applied it to study metastasis in breast and colon cancer, as well as the spatial organization of subclones in metastatic tumors. Collectively these studies and tools provide new insights into the evolution of chromosome aberrations in human tumors. Citation Format: Nicholas Navin. Decoding breast cancer evolution with single cell genomics [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr IA025.

Ongoing transposition in cell culture reveals the phylogeny of diverse Drosophila S2 sublines.

Cultured cells are widely used in molecular biology despite poor understanding of how cell line genomes change in vitro over time. Previous work has shown that Drosophila cultured cells have a higher transposable element content than whole flies, but whether this increase in transposable element content resulted from an initial burst of transposition during cell line establishment or ongoing transposition in cell culture remains unclear. Here, we sequenced the genomes of 25 sublines of Drosophila S2 cells and show that transposable element insertions provide abundant markers for the phylogenetic reconstruction of diverse sublines in a model animal cell culture system. DNA copy number evolution across S2 sublines revealed dramatically different patterns of genome organization that support the overall evolutionary history reconstructed using transposable element insertions. Analysis of transposable element insertion site occupancy and ancestral states support a model of ongoing transposition dominated by episodic activity of a small number of retrotransposon families. Our work demonstrates that substantial genome evolution occurs during long-term Drosophila cell culture, which may impact the reproducibility of experiments that do not control for subline identity.

Gain-loss-duplication models for copy number evolution on a phylogeny: Exact algorithms for computing the likelihood and its gradient

Gene gain-loss-duplication models are commonly based on continuous-time birth–death processes. Employed in a phylogenetic context, such models have been increasingly popular in studies of gene content evolution across multiple genomes. While the applications are becoming more varied and demanding, bioinformatics methods for probabilistic inference on copy numbers (or integer-valued evolutionary characters, in general) are scarce.We describe a flexible probabilistic framework for phylogenetic gain-loss-duplication models. The framework is based on a novel elementary representation by dependent random variables with well-characterized conditional distributions: binomial, Pólya (negative binomial), and Poisson.The corresponding graphical model yields exact numerical procedures for computing the likelihood and the posterior distribution of ancestral copy numbers. The resulting algorithms take quadratic time in the total number of copies. In addition, we show how the likelihood gradient can be computed by a linear-time algorithm.

Branching Copy-Number Evolution and Parallel Immune Profiles across the Regional Tumor Space of Resected Pancreatic Cancer.

Evolutionary copy-number patterns may be associated with survival in patients with resected PDAC.

Single-Cell DNA Sequencing Reveals Punctuated and Gradual Clonal Evolution in Hepatocellular Carcinoma

Copy number alterations (CNAs), elicited by genome instability, are a major source of intratumor heterogeneity. How CNAs evolve in hepatocellular carcinoma (HCC) remains unknown. We performed single-cell DNA sequencing (scDNA-seq) on 1275 cells isolated from 10 patients with HCC, ploidy-resolved scDNA-seq on 356 cells from 1 additional patient, and single-cell RNA sequencing on 27,344 cells from 3 additional patients. Three statistical fitting models were compared to investigate the CNA accumulation pattern. Cells in the tumor were categorized into the following 3 subpopulations: euploid, pseudoeuploid, and aneuploid. Our scDNA-seq analysis revealed that CNA accumulation followed a dual-phase copy number evolution model, that is, a punctuated phase followed by a gradual phase. Patients who exhibited prolonged gradual phase showed higher intratumor heterogeneity and worse disease-free survival. Integrating bulk RNA sequencing of 17 patients with HCC, published datasets of 1196 liver tumors, and immunohistochemical staining of 202 HCC tumors, we found that high expression of CAD, a gene involved in pyrimidine synthesis, was correlated with rapid tumorigenesis and reduced survival. The dual-phase copy number evolution model was validated by our single-cell RNA sequencing data and published scDNA-seq datasets of other cancer types. Furthermore, ploidy-resolved scDNA-seq revealed the common clonal origin of diploid- and polyploid-aneuploid cells, suggesting that polyploid tumor cells were generated by whole genome doubling of diploid tumor cells. Our work revealed a novel dual-phase copy number evolution model, showed HCC with longer gradual phase was more severe, identified CAD as a promising biomarker for early recurrence of HCC, and supported the diploid origin of polyploid HCC.