Distinct Evolutionary Trajectories Research Articles

Abstract LB228: Evolutionary trajectories of lung cancer in never smokers

Abstract Purpose of the study Elucidating evolutionary trajectories of cancers allows us to understand the key events, and the order in which they occur, throughout their development. This can help us to find important associations with tumor progression and prognosis. Our aim was to perform de novo identification of the evolutionary trajectories within Sherlock-lung, with a dataset containing the largest collection of lung cancer in never smokers (LCINS) samples ever analyzed. Experimental procedures Our Plackett-Luce ordering model utilized copy number data from Battenberg and mutation cancer cell fraction (CCF) data from DPClust. Frequently-occurring copy number events and driver mutations are ordered within each sample using their copy number states and CCFs. An aggregate ordering is then calculated for a sample set. Mixture model analysis identifies subsets of samples displaying distinct orders of events, uncovering diverse evolutionary trajectories within a tumor set. Dataset The Sherlock-lung whole genome sequencing dataset (n=1217) was filtered to the samples that allowed us to identify subclonal expansions. Samples required at least 10 reads per chromosome copy and a minimum cellularity of 30%. This provided 458 LCINS samples of various histologies. 155 smoker samples were also analyzed for comparison. Results We identified two subsets of LCINS tumors following distinct evolutionary trajectories. The “loss-based” subset commonly saw whole genome duplication (WGD) combined with copy number losses occurring earlier, and at higher prevalence, than gains. Contrastingly, in the “gain-based” subset, WGD was relatively rare but ploidy increased via copy number gains, which were more prevalent than losses. Interestingly, these different trajectories converged on similar overall copy number states. The loss-based subset had a higher mutational burden and a higher proportion of the genome altered, and followed a more smoker-like trajectory than the gain-based subset. Considering these differences alongside the convergence in copy number states, it is intriguing that survival times were similar between the two subsets. Copy number events defined the difference between the two trajectories. However, driver mutations also played important roles in tumor evolution in LCINS. TP53 and EGFR mutations were associated with greater genomic instability. Conversely, KRAS mutations were associated with more stable genomes. Samples with early clonal mutations in TP53, ERBB2, and PIK3CA, as well as those with a copy number gain of ERBB2, exhibited shorter survival times. Conclusions Two distinct evolutionary trajectories of LCINS were identified by de novo Plackett-Luce event ordering analysis. The contrast between the subgroups was defined by different paths of copy number activity, but they ultimately converged on similar overall copy number states and outcomes. Key early driver mutations influenced genomic instability and survival times. Citation Format: Christopher Wirth, Tongwu Zhang, Wei Zhao, Phuc Hoang, Jian Sang, Nathaniel Rothman, Marcos Díaz-Gay, Ruxandra Teslo, Naser Ansari-Pour, Máire Ní Leathlobhair, Iliana Peneva, William Eagles, Lixing Yang, Ludmil Alexandrov, David C. Wedge, Maria Teresa Landi. Evolutionary trajectories of lung cancer in never smokers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB228.

Two classes of DNA gyrase inhibitors elicit distinct evolutionary trajectories toward resistance in gram-negative pathogens

Comprehensive knowledge of mechanisms driving the acquisition of antimicrobial resistance is essential for the development of new drugs with minimized resistibility. To gain this knowledge, we combine experimental evolution in a continuous culturing device, the morbidostat, with whole genome sequencing of evolving cultures followed by characterization of drug-resistant isolates. Here, this approach was used to assess evolutionary dynamics of resistance acquisition against DNA gyrase/topoisomerase TriBE inhibitor GP6 in Escherichia coli and Acinetobacter baumannii. The evolution of GP6 resistance in both species was driven by a combination of two classes of mutational events: (i) amino acid substitutions near the ATP-binding site of the GyrB subunit of the DNA gyrase target; and (ii) various mutations and genomic rearrangements leading to upregulation of efflux pumps, species-specific (AcrAB/TolC in E. coli and AdeIJK in A. baumannii) and shared by both species (MdtK). A comparison with the experimental evolution of resistance to ciprofloxacin (CIP), previously performed using the same workflow and strains, revealed fundamental differences between these two distinct classes of compounds. Most notable were non-overlapping spectra of target mutations and distinct evolutionary trajectories that, in the case of GP6, were dominated by upregulation of efflux machinery prior to (or even in lieu) of target modification. Most of the efflux-driven GP6-resistant isolates of both species displayed a robust cross-resistance to CIP, while CIP-resistant clones showed no appreciable increase in GP6-resistance.

Evolving immune evasion and transmissibility of SARS-CoV-2: The emergence of JN.1 variant and its global impact.

The continuous evolution of SARS-CoV-2 variants constitutes a significant impediment to the public health. The World Health Organization (WHO) has designated the SARS-CoV-2 variant JN.1, which has evolved from its progenitor BA.2.86, as a Variant of Interest (VOI) in light of its enhanced immune evasion and transmissibility. The proliferating dissemination of JN.1 globally accentuates its competitive superiority and the potential to instigate fresh surges of infection, notably among cohorts previously infected by antecedent variants. Notably, prevailing evidence does not corroborate an increase in pathogenicity associated with JN.1, and antiviral agents retain their antiviral activity against both BA.2.86 and JN.1. The sustained effectiveness of antiviral agents offers a beacon of hope. Nonetheless, the variant's adeptness at eluding the immunoprotective effects conferred by extant vaccines highlights the imperative for the development of more effective vaccines and therapeutic approaches. Overall, the distinct evolutionary trajectories of BA.2.86 and JN.1 underscore the necessity for ongoing surveillance and scholarly inquiry to elucidate their implications for the pandemic's evolution, which requires the international communities to foster collaboration through the sharing of data, exchange of insights, and collective scientific endeavors.

Abstract IA014: Detecting epistatic effects among somatic cancer mutations across oncogenesis

Abstract The somatic evolution of cancer depends on the rate at which new mutations are acquired, as well as the selective pressure acting on them. Further, mutual exclusivity or co-occurrence of somatic variants suggests that current somatic variants can exert antagonistic or synergistic epistatic effects on the selection of new mutations, contributing to a complex landscape of evolutionary trajectories. However, quantifying these epistatic effects—which are essential to estimation of the trajectory of likely cancer genotoypes—has been a challenge, especially for higher-order epistatic effects. To estimate these epistatic effects, we have developed a continuous-time Markov chain model that enables the estimation of mutation origination and fixation, dependent on somatic cancer genotype. Coupling the continuous-time Markov chain model with a mutation-rate deconvolution approach, we have estimated the underlying mutation rates and selection intensities across trajectories of oncogenesis. Specifically, we report the mutation rates, selection intensities, and consequent variant fluxes for lung adenocarcinoma, informed by a compilation of samples from multiple data sets including The Cancer Genome Atlas and the AACR Project GENIE. Furthermore, we compare smoker and nonsmoker tumor cohorts describing the distinct evolutionary trajectories jointly, influenced by differential mutation and also by the differential physiological effects that smoking can have on the selective regime of lung tissue. Our approach enables inference of the most likely routes of site-specific variant evolution, as well as estimation of the selection strength operating on each step along the route—a key component of what we need to know to prioritize, develop, and implement personalized cancer therapies. Citation Format: Jorge A. Alfaro-Murillo, Krishna Dasari, Jeffrey P. Townsend. Detecting epistatic effects among somatic cancer mutations across oncogenesis [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 IA014.

Genomic and phenotypic signatures provide insights into the wide adaptation of a global plant invader

Invasive alien species are primary drivers of biodiversity loss and species extinction. Smooth cordgrass (Spartina alterniflora) is one of the most aggressive invasive plants in coastal ecosystems around the world. However, the genomic bases and evolutionary mechanisms underlying its invasion success have remained largely unknown. Here, we assembled a chromosome-level reference genome and performed phenotypic and population genomic analyses between native US and introduced Chinese populations. Our phenotypic comparisons showed that introduced Chinese populations have evolved competitive traits, such as early flowering time and greater plant biomass, during secondary introductions along China’s coast. Population genomic and transcriptomic inferences revealed distinct evolutionary trajectories of low- and high-latitude Chinese populations. In particular, genetic mixture among different source populations, together with independent natural selection acting on distinct target genes, may have resulted in high genome dynamics of the introduced Chinese populations. Our study provides novel phenotypic and genomic evidence showing how smooth cordgrass rapidly adapts to variable environmental conditions in its introduced ranges. Moreover, candidate genes related to flowering time, fast growth, and stress tolerance (i.e., salinity and submergence) provide valuable genetic resources for future improvement of cereal crops.

Freezing Injury in Mouse Masseter Muscle to Establish an Orofacial Muscle Fibrosis Model.

Orofacial muscle constitutes a subset of skeletal muscle tissue, with a distinct evolutionary trajectory and development origin. Unlike the somite-derived limb muscles, the orofacial muscles originate from the branchial arches, with exclusive contributions from the cranial neural crest. A recent study has revealed that regeneration is also different in the orofacial muscle group. However, the underlying regulatory mechanism remains to be uncovered. Current skeletal muscle regeneration models mainly focus on the limb and trunk muscle. In this protocol, dry ice was used to induce freezing injury in the mouse masseter muscle and tibialis anterior muscle to create an orofacial muscle fibrosis model. The temporal dynamics of muscle satellite cells and fibro-adipogenic progenitors were different between the two muscles, leading to impaired myofiber regeneration and excessive extracellular matrix deposition. With the help of this model, a deeper investigation into muscle regeneration in the orofacial area could be carried out to develop therapeutic approaches for patients with orofacial diseases.

Distinctive plastome evolution in carnivorous angiosperms

BackgroundIndependent origins of carnivory in multiple angiosperm families are fabulous examples of convergent evolution using a diverse array of life forms and habitats. Previous studies have indicated that carnivorous plants have distinct evolutionary trajectories of plastid genome (plastome) compared to their non-carnivorous relatives, yet the extent and general characteristics remain elusive.ResultsWe compared plastomes from 9 out of 13 carnivorous families and their non-carnivorous relatives to assess carnivory-associated evolutionary patterns. We identified inversions in all sampled Droseraceae species and four species of Utricularia, Pinguicula, Darlingtonia and Triphyophyllum. A few carnivores showed distinct shifts in inverted repeat boundaries and the overall repeat contents. Many ndh genes, along with some other genes, were independently lost in several carnivorous lineages. We detected significant substitution rate variations in most sampled carnivorous lineages. A significant overall substitution rate acceleration characterizes the two largest carnivorous lineages of Droseraceae and Lentibulariaceae. We also observe moderate substitution rates acceleration in many genes of Cephalotus follicularis, Roridula gorgonias, and Drosophyllum lusitanicum. However, only a few genes exhibit significant relaxed selection.ConclusionOur results indicate that the carnivory of plants have different effects on plastome evolution across carnivorous lineages. The complex mechanism under carnivorous habitats may have resulted in distinctive plastome evolution with conserved plastome in the Brocchinia hechtioides to strongly reconfigured plastomes structures in Droseraceae. Organic carbon obtained from prey and the efficiency of utilizing prey-derived nutrients might constitute possible explanation.

A new species of pit-viper from the Ayeyarwady and Yangon regions in Myanmar (Viperidae, Trimeresurus).

In a genomic study by Chan and colleagues, pit-vipers of the Trimeresuruserythrurus-purpureomaculatus complex from the Ayeyarwady and Yangon regions in Myanmar were demonstrated to be a distinct species based on robust population genetic and species delimitation analyses. Here, we provide morphological characterizations and a formal description of those populations as a new species. The new species, Trimeresurusayeyarwadyensissp. nov., is most closely related to T.erythrurus and T.purpureomaculatus and shares morphological characteristics with both of those species. Some specimens of T.ayeyarwadyensissp. nov. have green dorsal coloration and no distinct dorsal blotches (a trait shared with T.erythrurus but not T.purpureomaculatus), while others have dark dorsal blotches (a trait shared with T.purpureomaculatus but not T.erythrurus). The distinct evolutionary trajectory of the new species, coupled with the lack of obvious morphological differentiation, represents a classic example of the cryptic nature of species commonly found in the Trimeresurus group of Asian pit-vipers and underscores the need for data-rich analyses to verify species' boundaries more broadly within this genus.

Unraveling the Unusual Subgenomic Organization in the Neopolyploid Free-Living Flatworm Macrostomum lignano.

Whole genome duplication (WGD) is an evolutionary event resulting in a redundancy of genetic material. Different mechanisms of WGD, allo- or autopolyploidization, lead to distinct evolutionary trajectories of newly formed polyploids. Genome studies on such species are important for understanding the early stages of genome evolution. However, assembling neopolyploid is a challenging task due to the presence of 2 homologous (or homeologous) chromosome sets and therefore the existence of the extended paralogous regions in its genome. Post-WGD evolution of polyploids includes cytogenetic diploidization leading to the formation of species, whose polyploid origin might be hidden by disomic inheritance. Earlier we uncovered the hidden polyploid origin of the free-living flatworms of the genus Macrostomum (Macrostomum lignano, M. janickei, and M. mirumnovem). Cytogenetic diploidization in these species is accompanied by intensive chromosomal rearrangements including chromosomes fusions. In this study, we unravel the M. lignano genome organization through generation and sequencing of 2 sublines of the commonly used inbred line of M. lignano (called DV1) differing only in a copy number of the largest chromosome (MLI1). Using nontrivial assembly free comparative analysis of their genomes, we deciphered DNA sequences belonging to MLI1 and validated them by sequencing the pool of microdissected MLI1. Here we presented the uncommon mechanism of genome rediplodization of M. lignano, which consists of (i) presence of 3 subgenomes, which emerged via formation of large fused chromosomes and its variants, and (ii) sustaining their heterozygosity through inter- and intrachromosomal rearrangements.

PICKLE RELATED 2 is a Neofunctionalized Gene Duplicate Under Positive Selection With Antagonistic Effects to the Ancestral PICKLE Gene on the Seed Transcriptome.

The evolution and diversification of proteins capable of remodeling domains has been critical for transcriptional reprogramming during cell fate determination in multicellular eukaryotes. Chromatin remodeling proteins of the CHD3 family have been shown to have important and antagonistic impacts on seed development in the model plant, Arabidopsis thaliana, yet the basis of this functional divergence remains unknown. In this study, we demonstrate that genes encoding the CHD3 proteins PICKLE (PKL) and PICKLE-RELATED 2 (PKR2) originated from a duplication event during the diversification of crown Brassicaceae, and that these homologs have undergone distinct evolutionary trajectories since this duplication, with PKR2 fast evolving under positive selection, while PKL is subject to purifying selection. We find that the rapid evolution of PKR2 under positive selection reduces the encoded protein's intrinsic disorder, possibly suggesting a tertiary structure configuration which differs from that of PKL. Our whole genome transcriptome analysis in seeds of pkr2 and pkl mutants reveals that they act antagonistically on the expression of specific sets of genes, providing a basis for their differing roles in seed development. Our results provide insights into how gene duplication and neofunctionalization can lead to differing and antagonistic selective pressures on transcriptomes during plant reproduction, as well as on the evolutionary diversification of the CHD3 family within seed plants.

Experimental evolution of Pseudomonas aeruginosa to colistin in spatially confined microdroplets identifies evolutionary trajectories consistent with adaptation in microaerobic lung environments.

Antibiotic resistance is a continuing global health crisis. Identifying the evolutionary trajectories leading to increased antimicrobial resistance can be critical to the discovery of biomarkers for clinical diagnostics and new targets for drug discovery. While the combination of patient data and in vitro experimental evolution has been remarkably successful in extending our understanding of antimicrobial resistance, it can be difficult for in vitro methods to recapitulate the spatial structure and consequent microenvironments that characterize in vivo infection. Notably, in cystic fibrosis (CF) patients, changes to either the PmrA/PmrB or PhoP/PhoQ two-component systems have been identified as critical drivers for high levels of colistin and polymyxin resistance. When using microfluidic emulsions to provide spatially structured, low-competition environments, we found that adaptive mutations to phoQ were more successful than pmrB in increasing colistin resistance. Conversely, mutations to pmrB were readily identified using well-mixed unstructured cultures. We found that oxygen concentration gradients within the microdroplet emulsions favored adaptive changes to the PhoP/PhoQ pathway consistent with microaerobic conditions that can be found in the lungs of CF patients. We also observed mutations linked to hallmark adaptations to the CF lung environment, such as loss of motility and loss of O antigen biosynthesis (wbpL). Mutation to wbpL, in addition to causing loss of O antigen, was additionally shown to confer moderately increased colistin resistance. Taken together, our data suggest that distinct evolutionary trajectories to colistin resistance may be shaped by the microaerobic partitioning and spatial separation imposed within the CF lung.IMPORTANCEAntibiotic resistance remains one of the great challenges confronting public health in the world today. Individuals with compromised immune systems or underlying health conditions are often at an increased for bacterial infections. Patients with cystic fibrosis (CF) produce thick mucus that clogs airways and provides a very favorable environment for infection by bacteria that further decrease lung function and, ultimately, mortality. CF patients are often infected by bacteria such as Pseudomonas aeruginosa early in life and experience a series of chronic infections that, over time, become increasingly difficult to treat due to increased antibiotic resistance. Colistin is a major antibiotic used to treat CF patients. Clinical and laboratory studies have identified PmrA/PmrB and PhoP/PhoQ as responsible for increased resistance to colistin. Both have been identified in CF patient lungs, but why, in some cases, is it one and not the other? In this study, we show that distinct evolutionary trajectories to colistin resistance may be favored by the microaerobic partitioning found within the damaged CF lung.

The Geochemical Habitats that Favored the Origin of Thermophilic Lineages

Evidence in the fossil and isotopic records suggests that life inhabited hot springs by ~3.5 Ga. Further, phylogenetic evidence places Bacteria and Archaea from high temperature environments as among the earliest evolving lineages. Moreover, contemporary hot spring communities host an extensive level of biodiversity coinciding with extensive geochemical variation due to spatial and temporal heterogeneity in available oxidants and reductants generated by variable mixing of reduced volcanic and oxidized meteoric fluids. Thus, thermophilic archaeal and bacterial lineages have been co-evolving with their hydrothermal environments since early in the history of life and through dramatic changes in Earth’s geologic history. Yet, little is known of the environmental characteristics that enabled the extensive diversification of microbial life and their metabolic functionalities in these environments. To begin developing a framework to understand the environmental characteristics that enabled expansive microbial taxonomic and functional innovation in thermophilic lineages - coordinated geochemical, metagenomic, and phylogenetic analyses were conducted on 37 high-temperature Yellowstone National Park (YNP) hot spring ecosystems that spanned the range of geochemistry (pH ~1.5-10) in YNP springs. Considerable variation in dissolved solutes and gases were identified, consistent with spatial and temporal variation in the geological, geochemical, and hydrologic processes that influence the YNP hydrothermal system. Shotgun sequencing and bioinformatics analyses yielded 1,154 archaeal and bacterial metagenome-assembled-genomes (MAGs) from the 37 springs. Genomic diversity and metabolic functions encoded by the MAGs were not uniformly distributed among spring types based on geochemistry, with moderately acidic springs (pH 5-7) harboring the greatest overall diversity, despite these spring types being relatively rare among continental hydrothermal systems. Phylogenomic analyses of MAGs indicated that their divergence times (estimated by distance to phylogenetic roots) were variable, but highly associated with spring geochemistry. Specifically, spring types hosting the highest genomic, taxonomic, and functional diversity also predominantly harbored microbial lineages with the oldest inferred divergence times. In addition, MAG-encoded metabolic functions related to carbon fixation, methane, sulfur, iron, arsenic, hydrogen, and nitrogen metabolism were discretely distributed across spring geochemical types. The distribution of metabolic functions coincided with variation in spring geochemical parameters related to those metabolisms. For example, iron metabolism was most prevalent in acidic springs exhibiting the highest iron concentrations, gas metabolism was most prominent in moderately acidic springs that exhibited the highest dissolved gas concentrations, and arsenic metabolism was prevalent among alkaline pH springs where arsenic concentrations were highest. Lastly, preliminary comparisons of microbial communities from YNP and other global continental hydrothermal systems suggest that geologic setting (e.g., bedrock type) significantly influences hot spring geochemistry and ultimately, microbial compositional and functional profiles that coincide with distinct evolutionary trajectories of hot spring taxa and functional genes. Results will be discussed in the context of the early evolution of life and the co-evolution of microbial lineages and their geologic settings in hydrothermal systems.

Abundance and Diversification of Repetitive Elements in Decapoda Genomes.

Repetitive elements are a major component of DNA sequences due to their ability to propagate through the genome. Characterization of Metazoan repetitive profiles is improving; however, current pipelines fail to identify a significant proportion of divergent repeats in non-model organisms. The Decapoda order, for which repeat content analyses are largely lacking, is characterized by extremely variable genome sizes that suggest an important presence of repetitive elements. Here, we developed a new standardized pipeline to annotate repetitive elements in non-model organisms, which we applied to twenty Decapoda and six other Crustacea genomes. Using this new tool, we identified 10% more repetitive elements than standard pipelines. Repetitive elements were more abundant in Decapoda species than in other Crustacea, with a very large number of highly repeated satellite DNA families. Moreover, we demonstrated a high correlation between assembly size and transposable elements and different repeat dynamics between Dendrobranchiata and Reptantia. The patterns of repetitive elements largely reflect the phylogenetic relationships of Decapoda and the distinct evolutionary trajectories within Crustacea. In summary, our results highlight the impact of repetitive elements on genome evolution in Decapoda and the value of our novel annotation pipeline, which will provide a baseline for future comparative analyses.

Evolutionary patterns of archaea predominant in acidic environment

BackgroundArchaea of the order Thermoplasmatales are widely distributed in natural acidic areas and are amongst the most acidophilic prokaryotic organisms known so far. These organisms are difficult to culture, with currently only six genera validly published since the discovery of Thermoplasma acidophilum in 1970. Moreover, known great diversity of uncultured Thermoplasmatales represents microbial dark matter and underlines the necessity of efforts in cultivation and study of these archaea. Organisms from the order Thermoplasmatales affiliated with the so-called “alphabet-plasmas”, and collectively dubbed “E-plasma”, were the focus of this study. These archaea were found predominantly in the hyperacidic site PM4 of Parys Mountain, Wales, UK, making up to 58% of total metagenomic reads. However, these archaea escaped all cultivation attempts.ResultsTheir genome-based metabolism revealed its peptidolytic potential, in line with the physiology of the previously studied Thermoplasmatales isolates. Analyses of the genome and evolutionary history reconstruction have shown both the gain and loss of genes, that may have contributed to the success of the “E-plasma” in hyperacidic environment compared to their community neighbours. Notable genes among them are involved in the following molecular processes: signal transduction, stress response and glyoxylate shunt, as well as multiple copies of genes associated with various cellular functions; from energy production and conversion, replication, recombination, and repair, to cell wall/membrane/envelope biogenesis and archaella production. History events reconstruction shows that these genes, acquired by putative common ancestors, may determine the evolutionary and functional divergences of “E-plasma”, which is much more developed than other representatives of the order Thermoplasmatales. In addition, the ancestral hereditary reconstruction strongly indicates the placement of Thermogymnomonas acidicola close to the root of the Thermoplasmatales.ConclusionsThis study has analysed the metagenome-assembled genome of “E-plasma”, which denotes the basis of their predominance in Parys Mountain environmental microbiome, their global ubiquity, and points into the right direction of further cultivation attempts. The results suggest distinct evolutionary trajectories of organisms comprising the order Thermoplasmatales, which is important for the understanding of their evolution and lifestyle.

Central American Trachemys revisited: New sampling questions current understanding of taxonomy and distribution (Testudines: Emydidae)

Using 3226-bp-long mtDNA sequences and five nuclear loci (Cmos, ODC, R35, Rag1, Rag2, together 3409 bp), we examine genetic differentiation and relationships of Central American slider turtles (Trachemys grayi, T. venusta). Our investigation also included samples from taxa endemic to North America (T. gaigeae, T. scripta), the Antilles (T. decorata, T. decussata, T. stejnegeri, T. terrapen), and South America (T. dorbigni, T. medemi plus the two T. venusta subspecies endemic to northern South America). Our mitochondrial phylogeny retrieves all studied species as distinct, with three well-supported clades in a polytomy: (1) the Central and South American species (T. grayi + T. venusta) + (T. dorbigni + T. medemi), (2) the Antillean species, and (3) T. gaigeae + T. scripta. Our nuclear DNA analyses also suggest three distinct but conflicting clusters: (1) T. scripta plus the Antillean species, (2) T. gaigeae, and (3) the Central and South American species T. dorbigni, T. grayi, T. medemi, and T. venusta. However, in the mitochondrial phylogeny, T. gaigeae is the little divergent sister taxon of T. scripta. This conflicting placement of T. gaigeae suggests a distinct evolutionary trajectory and old hybridization with T. scripta and mitochondrial capture. Despite prominent color pattern differences, genetic divergences within T. grayi and T. venusta are shallow and the taxonomic diversity of each species with several currently recognized subspecies could be overestimated. Finally, we provide for the first time evidence for the occurrence of T. grayi along the Caribbean versant of Costa Rica.

Abstract 862: Pairwise and higher-order epistatic effects among somatic cancer mutations across oncogenesis of lung adenocarcinoma

Abstract The somatic evolution of cancer depends on the rate at which new mutations are acquired, as well as the selective pressure acting on them. Further, mutual exclusivity or co-occurrence of mutations suggests that variants can exert antagonistic or synergistic epistatic effects on the selection of new mutations, contributing to a complex landscape of evolutionary trajectories. Estimation of pairwise and higher-order epistatic effects—essential to estimation of the trajectory of likely cancer genotoypes—has remained a challenge. We have developed a continuous-time Markov chain model that enables the estimation of mutation origination and fixation (flux), dependent on somatic cancer genotype. Coupling the continuous-time Markov chain model with a deconvolution approach provides estimates of underlying mutation rates and selection across the trajectory of oncogenesis. We demonstrate computation of fluxes and selection coefficients in a somatic evolutionary model of lung adenocarcinoma informed by a compilation of samples from multiple data sets including The Cancer Genome Atlas and the AACR Project GENIE. The approach enables inference of the most likely routes of site-specific variant evolution and estimation of the selection strength operating on each step along the route, a key component of what we need to know to develop and implement personalized cancer therapies. Finally, by comparing smoker and nonsmoker tumor cohorts, we reveal and describe distinct evolutionary trajectories, influenced not only by differential mutation, but also by the differential physiological effects that smoking can have on the selective regime of lung tissue. These distinctions disambiguate the physiological and mutational influences of smoking and can inform personalized treatment of the two groups. Citation Format: Jorge A. Alfaro-Murillo, Krishna Dasari, Jeffrey P. Townsend. Pairwise and higher-order epistatic effects among somatic cancer mutations across oncogenesis of lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 862.

Evolutionary characterization of lung adenocarcinoma morphology in TRACERx.

Lung adenocarcinomas (LUADs) display a broad histological spectrum from low-grade lepidic tumors through to mid-grade acinar and papillary and high-grade solid, cribriform and micropapillary tumors. How morphology reflects tumor evolution and disease progression is poorly understood. Whole-exome sequencing data generated from 805 primary tumor regions and 121 paired metastatic samples across 248 LUADs from the TRACERx 421 cohort, together with RNA-sequencing data from 463 primary tumor regions, were integrated with detailed whole-tumor and regional histopathological analysis. Tumors with predominantly high-grade patterns showed increased chromosomal complexity, with higher burden of loss of heterozygosity and subclonal somatic copy number alterations. Individual regions in predominantly high-grade pattern tumors exhibited higher proliferation and lower clonal diversity, potentially reflecting large recent subclonal expansions. Co-occurrence of truncal loss of chromosomes 3p and 3q was enriched in predominantly low-/mid-grade tumors, while purely undifferentiated solid-pattern tumors had a higher frequency of truncal arm or focal 3q gains and SMARCA4 gene alterations compared with mixed-pattern tumors with a solid component, suggesting distinct evolutionary trajectories. Clonal evolution analysis revealed that tumors tend to evolve toward higher-grade patterns. The presence of micropapillary pattern and 'tumor spread through air spaces' were associated with intrathoracic recurrence, in contrast to the presence of solid/cribriform patterns, necrosis and preoperative circulating tumor DNA detection, which were associated with extra-thoracic recurrence. These data provide insights into the relationship between LUAD morphology, the underlying evolutionary genomic landscape, and clinical and anatomical relapse risk.

Characterization of immature ovarian teratomas through single-cell transcriptome.

Immature ovarian teratomas are a type of malignant germ cell tumor composed of complicated cell types and are characterized by pathological features of immature neuroectodermal tubules/rosettes. However, there is a lack of understanding of patient-derived immature ovarian teratomas (PDT) at the single cell level. Moreover, whether stem cell lines derived from immature teratomas (CDT) can be used as models for research on PDT remains to be elucidated. Single-cell RNA sequencing (scRNA-seq) and subsequent bioinformatic analysis was performed on three patient-derived immature ovarian teratomas (PDT) samples to reveal the heterogeneity, evolution trajectory, and cell communication within the tumor microenvironment of PDT. Validations were conducted in additional seven samples through multiplex immunofluorescence. A total of qualified 22,153 cells were obtained and divided into 28 clusters, which can match to the scRNA-seq annotation of CDT as well as human fetal Cell Atlas, but with higher heterogeneity and more prolific cell-cell crosstalk. Radial glia cells (tagged by SOX2) and immature neuron (tagged by DCX) exhibited mutually exclusive expression and differentiated along distinct evolutionary trajectory from cycling neural progenitors. Proportions of these neuroectodermal cell subtypes may play important roles in PDT through contributing to the internal heterogeneity of PDTs. Moreover, the immune cells in PDTs were infiltrated rather than teratoma-derived, with more abundant macrophage in immature neuron than those in radial glia cells, and the infiltrated macrophage subtypes (i.e., M1 and M2) were significantly correlated to clinical grade. Overall, suppressed evolution process and transcriptome regulation in neuroectodermal cells, reduced cell-cell crosstalk, higher M1/M2 proportion ratio, and enhanced T cell effects in tumor microenvironment are enriched in patients with favorable prognosis. This study provides a comprehensive profile of PDT at the single cell level, shedding light on the heterogeneity and evolution of neuroectodermal cells within PDTs and the role of immune cells within the tumor microenvironment. Also, our findings highlight the potential usage of CDTs as a model for research on PDT.

Polyp-Canal Reconstruction Reveals Evolution Toward Complexity in Corals.

Modern scleractinian corals are classified into robust, complex, and basal clades through comparative molecular studies. However, only few morphological or biological criteria can systematically determine the evolutionary trajectories of these major scleractinian coral clades. Here, we obtained the structural information of 21 scleractinian coral species representing robust and complex clades: High-resolution micro-computed tomography was used to reconstruct the polyp-canal systems in their colonies and to visualize the dynamic polyp growth processes. We found that the emergence of mesh-like canals may distinguish representatives of complex and robust clades. The differences in polyp-canal connections suggest distinct evolutionary trajectories among coral species: The formation of the canal network promoted the development of more complex coral structures, and coral polyps within this network formed calices of very similar volume, following precise axial growth directions. The influence of individual polyps on the coral colony becomes less significant as coral structures become more complex, and coral species with more complicated polyp-canal systems occupied niches more efficiently. This work supplements current evolutionary studies on reef-building corals, providing insight for further studies on coral growth patterns.

OGC P04 Spatial sampling of Barrett's oesophagus reveals that tumour heterogeneity is not a useful predictor of progression to cancer

Abstract Background Intra-tumour heterogeneity has been well-described in oesophageal adenocarcinoma and Barrett's oesophagus (BE) segments also consist of multiple clones (groups of cells which share mutations). In BE there is some suggestion that the number of individual genetic clones within the segment and the diversity of the segment can be predictors of progression to cancer. However, this was with pre- whole genome sequencing (WGS) methods. In this study we consider the detailed spatial maps of ten patients with different disease trajectories, using WGS, to determine the heterogeneity and correlate it with their progression. Methods We performed WGS (50x) on 35 high-quality, frozen, endoscopic biopsies taken spatially from ten cases (8 males, 2 females, median age 75 (range 31–83). This included 4 long-term non-dysplastic (ND) patients with long BE segments and 6 patients with dysplasia. We used the copy number and purity information (from Battenberg) and the somatic mutation clustering information (from DPClust) to infer the clonal and subclonal architecture of individual biopsies and looked at the spatial relationship between biopsies within the segment. Results In both ND and dysplastic BE we saw cells with unrelated patterns of mutations, or clones, implying completely separate origins for different parts of the segments. This suggests independent or parallel evolution. There was a higher burden of shared (truncal) mutations in the dysplastic biopsies (p=0.049), indicating a more recent clonal sweep with some clones being outcompeted. Dysplastic samples had more subclonal copy number aberrations and more clonal driver mutations. In addition, most of the driver gene alterations in the dysplastic trees occurred early, whereas CDKN2A, the p16 locus, was the only truncal mutation found on the ND clone trees. We did not find the number of clones or the diversity of the segment to differ between the two groups. Conclusions Each individual patient showed complex, distinct genomic features and evolutionary trajectories. Some cases arose from a single ancestral clone, while others had few shared mutational variants suggesting multiple clones. Importantly, in this sample of cases we did not see any trend that the clonal diversity or number of clones correlated with progression to cancer. This is different to previous studies and highlights the importance of multiple sampling to determine the propensity for cancer progression even if the samples look similar histologically.