Whole-genome Profiling Research Articles

Viral genomic methylation and the interspecies evolutionary relationships of ranavirus.

Ranaviruses are capable of infecting both wild and farmed fish, amphibians, and reptiles, leading to significant economic losses and ecological risks. Currently, ranaviruses have been found in at least 175 species spanning six continents. Except for Singapore grouper iridovirus (SGIV), ranavirus genomes are generally regarded as highly methylated. Nevertheless, our comprehension of the methylation characteristics within ranaviruses remains limited. Despite the numerous genomes currently included in the GenBank database, a complete phylogenetic tree for ranaviruses has not yet been determined, and interspecific evolutionary relationships among ranaviruses have not been thoroughly investigated. In this study, the whole-genome methylation profile of mandarin fish ranavirus (MRV; a ranavirus) was investigated, revealing a methylation level of 16.04%, and hypomethylation of the MRV genome was detrimental to viral replication, speculating the genome methylation may play an important role in MRV replication. Furthermore, by combining with whole-genome DNA sequence phylogenetic analyses, we propose the possibility of an interspecies evolutionary relationship among ranaviruses, with the presence of four distinct evolutionary lineages within ranavirus evolution: "SGIV, SCRAV(MRV/LMBV), EHNV/ENARV/ATV, and CMTV/FV3", which might be also supported by the genomic collinearity, natural host range and host habitats. Furthermore, ranavirus genomic methylation levels may provide additional evidence for this hypothesis, but further proof is needed. Our work enhances the understanding of the role of genome methylation in ranaviruses and is beneficial for the prevention and control of ranavirus diseases; simultaneously, the proposed evolutionary hypothesis of ranavirus provides novel insights and ideas for exploring the evolutionary trajectory of viruses.

Abstract A062: Insightful analysis: Harnessing aqueous humor ctDNA for retinoblastoma stratification

Abstract Retinoblastoma (RB) the most common intraocular malignancy in children poses a significant challenge as traditional tissue biopsies are not feasible. Due to its critical location within the eye and the high risk of tumor seeding, clinicians are limited in performing molecular analysis-based risk stratification prior to enucleation. In this study, we explored the potential of aqueous humor (AH) as a source for minimally invasive liquid biopsies, aiming to establish a molecular profiling methodology for circulating tumor DNA (ctDNA) for risk stratification. We analyzed AH samples and corresponding tumor tissues from 19 enucleated eyes of patients diagnosed with RB. The extracted ctDNA from AH underwent low-coverage whole-genome sequencing (lcWGS) and methylation profiling. These data were then compared with the genomic and methylation landscapes of the matched tumor DNA. Additionally, we assessed the ability of AH- derived ctDNA to predict retinoblastoma methylation cluster. Our findings demonstrate a high concordance between ctDNA from AH and the primary tumor DNA in both methylation patterns and genomic alterations detected by lcWGS allowing the correct prediction of the retinoblastoma molecular group. The shared aberrations underscore the potential of AH as a reliable surrogate for tumor tissue in retinoblastoma. Additionally, the methylation profiles revealed consistent epigenetic modifications, further supporting the accuracy of AH-derived ctDNA in reflecting the tumor’s molecular characteristics. This study highlights the feasibility and effectiveness of using AH for molecular ctDNA genetic analysis in retinoblastoma. The overall high concordance between ctDNA profiles with primary tumor DNA and the ability to accurately predict RB methylation classes, highlights the superiority of the liquid biopsy approach overcoming the limitations of traditional biopsies and transforming the potential for early molecular analysis and risk stratification. This revolutionary technique could redefine pre-enucleation diagnostics, offering unprecedented insights and paving the way for tailored, precision medicine in retinoblastoma care. Citation Format: Sophia H Montigel, Tatsiana Ryl, Stefanie Volz, Elena Afanasyeva, Tatjana Wedig, Nathalie Schwarz, Stefan M Pfister, Kristian W Pajtler, Petra Ketteler, Kendra K Maaß. Insightful analysis: Harnessing aqueous humor ctDNA for retinoblastoma stratification [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A062.

Antibiotic resistance and draft genome profiles of 10 Streptococcus pneumoniae and 3 Streptococcus pseudopneumoniae strains isolated from the nasopharynx of people living with human immunodeficiency virus in Ghana.

Genomic data on clinically important bacteria species such as Streptococcus pneumoniae and Streptococcus pseudopneumoniae from low- and middle-income countries, including Ghana, are scarce. In this study, we provide data on antimicrobial resistance (AMR) and whole-genome profiles of a collection of streptococci species to support AMR surveillance efforts in the country.

Gut Microbiome-Host Genetics Co-Evolution Shapes Adiposity by Modulating Energy and Lipid Metabolism in Selectively Bred Broiler Chickens.

Optimizing fat deposition is crucial for improving chicken production and meat quality. This study investigated the interactive roles of host genetics and gut microbiome in regulating abdominal fat deposition in selectively bred broiler chicken lines. We compared the gut microbiome composition and host whole-genome profiles between fat-line and lean-line broiler chickens that had been selectively bred for divergent abdominal fat levels over 15 generations. Despite identical dietary and environmental conditions, the two chicken lines exhibited significant differences in their gut microbiota. Lean-line broiler chickens exhibited an increased abundance of intestinal Lactobacillus and a decreased presence of potentially pathogenic species, such as Campylobacter coli, Corynebacterium casei, and Enterococcus faecalis. These microbial alterations were accompanied by shifts in the functional metagenome, with enrichment in pathways involved in energy metabolism and nutrient utilization in the lean-line chickens. Notably, the selective breeding process also led to genomic variations in the lean broilers, with single nucleotide polymorphisms predominantly observed in genes related to energy and lipid metabolism. Our findings suggest that the host-microbiome interactions play a key role in the divergent abdominal fat deposition phenotypes observed in these selectively bred chicken lines. The co-evolution of the gut microbiome and host genetics highlights the importance of considering both factors to optimize poultry production efficiency and meat quality. This study offers new insights into the intricate gut-genome interactions in chicken fat metabolism, paving the way for more effective breeding and microbiome-based strategies to manage adiposity in poultry.

Genomes and epigenomes of matched normal and tumor breast tissue reveal diverse evolutionary trajectories and tumor-host interactions

Normal tissues adjacent to the tumor (NATs) may harbor early breast carcinogenesis events driven by field cancerization. Although previous studies have characterized copy-number (CN) and transcriptomic alterations, the evolutionary history of NATs in breast cancer (BC) remains poorly characterized. Utilizing whole-genome sequencing (WGS), methylation profiling, and RNA sequencing (RNA-seq), we analyzed paired germline, NATs, and tumor samples from 43 individuals with BC in Hong Kong (HK). We found that single-nucleotide variants (SNVs) were common in NATs, with one-third of NAT samples exhibiting SNVs in driver genes, many of which were present in paired tumor samples. The most frequently mutated genes in both tumor and NAT samples were PIK3CA, TP53, GATA3, and AKT1. In contrast, large-scale aberrations such as somatic CN alterations (SCNAs) and structural variants (SVs) were rarely detected in NAT samples. We generated phylogenetic trees to investigate the evolutionary history of paired NAT and tumor samples. They could be categorized into tumor only, shared, and multiple-tree groups, the last of which is concordant with non-genetic field cancerization. These groups exhibited distinct genomic and epigenomic characteristics in both NAT and tumor samples.Specifically, NAT samples in the shared-tree group showed higher number of mutations, while NAT samples belonging to the multiple-tree group showed a less inflammatory tumor microenvironment (TME), characterized by a higher proportion ofregulatory Tcells (Tregs) and lower presence of CD14 cell populations. In summary, our findings highlight the diverse evolutionary history in BC NAT/tumor pairs and the impact of field cancerization and TME in shaping the genomic evolutionary historyof tumors.

Temporal Genomic Dynamics Shape Clinical Trajectory in Multiple Myeloma.

To comprehensively unravel the temporal relationship between initiating and driver events and its impact on clinical outcomes, we analyzed 421 whole-genome sequencing profiles from 382 patients. Using clock-like mutational signatures, we estimated a time lag of 2-4 decades between initiating events and diagnosis. In patients with hyperdiploidy, we demonstrate that trisomies of odd-numbered chromosomes can be acquired simultaneously with other chromosomal gains, such as 1q gain. We provide evidence that hyperdiploidy is acquired after canonical IGH translocation when both events are present. Finally, patients with early 1q gain had adverse outcomes similar to those with 1q amplification (>1 extra-copies), but faring worse than those with late 1q gain. This underscores that the prognostic impact of 1q gain/amp depends more on the timing of acquisition than on the number of extra copies gained. Overall, this study contributes to a better understanding of the life history of MM and may have prognostic implications.

1978P Accurate detection of urothelial carcinoma by whole-genome methylation profiling of urinary cell-free DNA

1978P Accurate detection of urothelial carcinoma by whole-genome methylation profiling of urinary cell-free DNA

High-fidelity, large-scale targeted profiling of microsatellites.

Microsatellites are highly mutable sequences that can serve as markers for relationships among individuals or cells within a population. The accuracy and resolution of reconstructing these relationships depends on the fidelity of microsatellite profiling and the number of microsatellites profiled. However, current methods for targeted profiling of microsatellites incur significant "stutter" artifacts that interfere with accurate genotyping, and sequencing costs preclude whole-genome microsatellite profiling of a large number of samples. We developed a novel method for accurate and cost-effective targeted profiling of a panel of more than 150,000 microsatellites per sample, along with a computational tool for designing large-scale microsatellite panels. Our method addresses the greatest challenge for microsatellite profiling-"stutter" artifacts-with a low-temperature hybridization capture that significantly reduces these artifacts. We also developed a computational tool for accurate genotyping of the resulting microsatellite sequencing data that uses an ensemble approach integrating three microsatellite genotyping tools, which we optimize by analysis of de novo microsatellite mutations in human trios. Altogether, our suite of experimental and computational tools enables high-fidelity, large-scale profiling of microsatellites, which may find utility in diverse applications such as lineage tracing, population genetics, ecology, and forensics.

Reprogramming of DNA methylation and changes of gene expression in grafted Hevea brasiliensis.

Rubber tree (Hevea brasiliensis) is reproduced by bud grafting for commercial planting, but significant intraclonal variations exist in bud-grafted clones. DNA methylation changes related to grafting may be partly responsible for intraclonal variations. In the current study, whole-genome DNA methylation profiles of grafted rubber tree plants (GPs) and their donor plants (DPs) were evaluated by whole-genome bisulfite sequencing. Data showed that DNA methylation was downregulated and DNA methylations in CG, CHG, and CHH sequences were reprogrammed in GPs, suggesting that grafting induced the reprogramming of DNA methylation. A total of 5,939 differentially methylated genes (DMGs) were identified by comparing fractional methylation levels between GPs and DPs. Transcriptional analysis revealed that there were 9,798 differentially expressed genes (DEGs) in the DP and GP comparison. A total of 1,698 overlapping genes between DEGs and DMGs were identified. These overlapping genes were markedly enriched in the metabolic pathway and biosynthesis of secondary metabolites by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Global DNA methylation and transcriptional analyses revealed that reprogramming of DNA methylation is correlated with gene expression in grafted rubber trees. The study provides a whole-genome methylome of rubber trees and an insight into the molecular mechanisms underlying the intraclonal variations existing in the commercial planting of grafted rubber trees.

Whole-genome DNA methylation profiling revealed epigenetic regulation of NF-κB signaling pathway involved in response to Vibrio alginolyticus infection in the Pacific oyster, Crassostrea gigas

DNA methylation, an essential epigenetic alteration, is tightly linked to a variety of biological processes, such as immune response. To identify the epigenetic regulatory mechanism in Pacific oyster (Crassostrea gigas), whole-genome bisulfite sequencing (WGBS) was conducted on C. gigas at 0 h, 6 h, and 48 h after infection with Vibrio alginolyticus. At 6 h and 48 h, a total of 11,502 and 14,196 differentially methylated regions (DMRs) were identified (p<0.05, FDR<0.001) compared to 0 h, respectively. Gene ontology (GO) analysis showed that differentially methylated genes (DMGs) were significantly enriched in various biological pathways including immunity, cytoskeleton, epigenetic modification, and metabolic processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that transcription machinery (ko03021) is one of the most important pathways. Integrated transcriptome and methylome analyses allowed the identification of 167 and 379 DMG-related DEGs at 6 h and 48 h, respectively. These genes were significantly enriched in immune-related pathways, including nuclear factor kappa B (NF-κB) signaling pathway (ko04064) and tumor necrosis factor (TNF) signaling pathway (ko04668). Interestingly, it's observed that the NF-κB pathway could be activated jointly by TNF Receptor Associated Factor 2 (TRAF2) and Baculoviral IAP Repeat Containing 3 (BIRC3, the homolog of human BIRC2) which were regulated by DNA methylation in response to the challenge posed by V. alginolyticus infection. Through this study, we provided insightful information about the epigenetic regulation of immunity-related genes in the C. gigas, which will be valuable for the understanding of the innate immune system modulation and defense mechanism against bacterial infection in invertebrates.

Multi-omics profiling the genomic origin and adeno-squamous transdifferentiation mechanism in adenosquamous carcinoma.

e20032 Background: Adenosquamous carcinoma (ASC) represents a rare subtype (0.4-4%) of non-small-cell lung cancer (NSCLC), with its multi-omic landscape yet to be fully elucidated. This study aims to explore the genomic origins and molecular mechanisms of adeno-squamous transdifferentiation in lung ASC through comprehensive multi-omics. Methods: We analyzed 69 samples from 31 lung ASC patients, including 16 FFPE samples with microdissected paired adenocarcinoma (AC) and squamous cell carcinoma (SCC) components, and additionally, incorporated pure Lung Adenocarcinoma (LUAD, n = 51) and pure Squamous Cell Carcinoma (LUSC, n = 28) for comparative analysis. Techniques included whole-exome sequencing, whole-genome methylation profiling, transcriptomics, single-cell RNA sequencing, and spatial transcriptomics. Results: AC and SCC exhibited similar genomic mutation landscapes, indicating a common genomic origin for AD and SCC. The SCC component in ASC displayed higher homologous recombination repair deficiency (HRD) and a notable 16p13.3 deletion in 31% of ASC transformations. Differential methylation analysis highlighted TP63's lower methylation in SCC, correlating negatively with RNA expression (P &lt; 0.001), indicative of methylation-driven regulation. DMR binding motif analysis revealed hypo-methylation in p63, p53, and AP-1 motifs, and hyper-methylation in MKX2-1, FOXA1/2 motifs (P &lt; 0.05). The transition from AC to SCC in ASC was marked by upregulated MYC signaling, proliferation, and DNA repair, with EMT changes not methylation-driven. Metabolic pathways were upregulated, and EMT/KRAS pathways downregulated in SCC compared to LUSC. Immune infiltration in LUAD-AC-ASC transformation showed an initial increase and subsequent decrease, transitioning from immune-infiltrated and immune-excluded to immune-desert phenotypes. IL36G of the IL1 family was highly expressed in the SCC component and strongly correlated with the squamous marker P63 (Pearson r = 0.72, P &lt; 0.001). Single-cell RNA-seq data demonstrated a significant increase in IL36G in squamous cells of lung ASC. In vivo models showed adeno-squamous differentiation in adenocarcinoma cells expressing IL36G. Conclusions: This study provides a comprehensive multi-omic insight into the complex genomic and epigenomic landscape of ASC, highlighting a shared genomic origin and distinct molecular mechanisms in the adeno-squamous transdifferentiation process. The findings, particularly the role of TP63 methylation and IL36G expression in the SCC component, offer novel insights into the molecular heterogeneity of ASC. These results not only deepen our understanding of ASC but also pave the way for future investigations into targeted therapies that could potentially exploit these molecular distinctions for more effective treatment strategies in ASC.

Multi-omic integration reveals alterations in nasal mucosal biology that mediate air pollutant effects on allergic rhinitis.

Allergic rhinitis is a common inflammatory condition of the nasal mucosa that imposes a considerable health burden. Air pollution has been observed to increase the risk of developing allergic rhinitis. We addressed the hypotheses that early life exposure to air toxics is associated with developing allergic rhinitis, and that these effects are mediated by DNA methylation and gene expression in the nasal mucosa. In a case-control cohort of 505 participants, we geocoded participants' early life exposure to air toxics using data from the US Environmental Protection Agency, assessed physician diagnosis of allergic rhinitis by questionnaire, and collected nasal brushings for whole-genome DNA methylation and transcriptome profiling. We then performed a series of analyses including differential expression, Mendelian randomization, and causal mediation analyses to characterize relationships between early life air toxics, nasal DNA methylation, nasal gene expression, and allergic rhinitis. Among the 505 participants, 275 had allergic rhinitis. The mean age of the participants was 16.4 years (standard deviation = 9.5 years). Early life exposure to air toxics such as acrylic acid, phosphine, antimony compounds, and benzyl chloride was associated with developing allergic rhinitis. These air toxics exerted their effects by altering the nasal DNA methylation and nasal gene expression levels of genes involved in respiratory ciliary function, mast cell activation, pro-inflammatory TGF-β1 signaling, and the regulation of myeloid immune cell function. Our results expand the range of air pollutants implicated in allergic rhinitis and shed light on their underlying biological mechanisms in nasal mucosa.

Liquid Biopsy Profiling with Multiple Tests in Patients with Metastatic Breast Cancer

The chief goal of the Blood Profiling Atlas in Cancer (BloodPAC) consortium is to promote collaborative efforts that support the development and implementation of liquid biopsy tests. Here, we report the results of a pilot study conducted by three BloodPAC members that aimed to demonstrate a multisite liquid biopsy testing framework using longitudinal blood specimens from 38 patients with metastatic breast cancer. Three laboratories receiving identical samples from two clinical sites each applied a different targeted sequencing platform to analyze mutations in cell-free DNA (cfDNA). The resulting mutational profiles reflected common breast cancer alterations, including clinically actionable mutations for 40% of hormone- receptor-positive patients. In 12 genes with shared target regions across sequencing panels, perfect inter-assay concordance was also observed for mutations detected above the lowest common assay limit of detection. Whole-genome copy number profiling of cfDNA and circulating tumor cells (CTCs) further revealed marked heterogeneity in copy number alterations and cfDNA tumor fractions across patients. Additionally, comparison of tumor fraction and CTC abundance demonstrated the complementary nature of cfDNA and CTC analyses. Overall, the framework described in this study may serve as a resource for future trials aiming to identify multimodal liquid biopsy biomarkers to guide clinical care.

Gene Co-Expression Analysis of Multiple Brain Tissues Reveals Correlation of FAM222A Expression with Multiple Alzheimer's Disease-Related Genes.

Alzheimer's disease (AD) is the most common form of dementia in the elderly marked by central nervous system (CNS) neuronal loss and amyloid plaques. FAM222A, encoding an amyloid plaque core protein, is an AD brain atrophy susceptibility gene that mediates amyloid-β aggregation. However, the expression interplay between FAM222A and other AD-related pathway genes is unclear. Our goal was to study FAM222A's whole-genome co-expression profile in multiple tissues and investigate its interplay with other AD-related genes. We analyzed gene expression correlations in Genotype-Tissue Expression (GTEx) tissues to identify FAM222A co-expressed genes and performed functional enrichment analysis on identified genes in CNS system. Genome-wide gene expression profiling identified 673 genes significantly correlated with FAM222A (p < 2.5×10-6) in 48 human tissues, including 298 from 13 CNS tissues. Functional enrichment analysis revealed that FAM222A co-expressed CNS genes were enriched in multiple AD-related pathways. Gene co-expression network analysis for identified genes in each brain region predicted other disease associated genes with similar biological function. Furthermore, co-expression of 25 out of 31 AD-related pathways genes with FAM222A was replicated in brain samples from 107 aged subjects from the Aging, Dementia and TBI Study. This gene co-expression study identified multiple AD-related genes that are associated with FAM222A, indicating that FAM222A and AD-associated genes can be active simultaneously in similar biological processes, providing evidence that supports the association of FAM222A with AD.

Phylogenomic profiles of whole-genome duplications in Poaceae and landscape of differential duplicate retention and losses among major Poaceae lineages

Poaceae members shared a whole-genome duplication called rho. However, little is known about the evolutionary pattern of the rho-derived duplicates among Poaceae lineages and implications in adaptive evolution. Here we present phylogenomic/phylotranscriptomic analyses of 363 grasses covering all 12 subfamilies and report nine previously unknown whole-genome duplications. Furthermore, duplications from a single whole-genome duplication were mapped to multiple nodes on the species phylogeny; a whole-genome duplication was likely shared by woody bamboos with possible gene flow from herbaceous bamboos; and recent paralogues of a tetraploid Oryza are implicated in tolerance of seawater submergence. Moreover, rho duplicates showing differential retention among subfamilies include those with functions in environmental adaptations or morphogenesis, including ACOT for aquatic environments (Oryzoideae), CK2β for cold responses (Pooideae), SPIRAL1 for rapid cell elongation (Bambusoideae), and PAI1 for drought/cold responses (Panicoideae). This study presents a Poaceae whole-genome duplication profile with evidence for multiple evolutionary mechanisms that contribute to gene retention and losses.

Biocontrol performance of a novel Bacillus velezensis L33a on tomato gray mold and its complete genome sequence analysis

As one of the most destructive diseases, gray mold caused by Botrytis cinerea is harmful to many important economically crops including tomatoes. The use of microorganisms for biological control is considered safe and effective. Here, a newly isolated strain L33a, which showed a 75.22% inhibition ratio against B. cinerea and broad-spectrum resistance to the tested eight phytopathogenic fungi, was characterized by a series of experiments. Strain L33a was identified as Bacillus velezensis based on its biochemical, morphological, and whole-genome profiles. Microstructural analysis revealed that L33a significantly inhibited the mycelial growth of B. cinerea by compromising the integrity of the cell membrane. The acupuncture inoculation assay demonstrated that L33a significantly alleviated disease symptoms caused by gray mold in tomato fruit through downregulation of pathogenicity-related genes of B. cinerea and activation of the transcription of genes involved in the salicylic acid (SA), jasmonic acid (JA), and antioxidant pathways of the tomato fruit. Whole-genome sequencing indicated that the total size of the L33a genome was 4032063 bp, which contained 14 biosynthetic gene clusters reported to be responsible for the biosynthesis of antimicrobial substances. Forty-one compounds were identified in the volatile organic compounds of strain L33a by using Gas Chromatography-Mass Spectrometer (GC-MS) analysis. Among these, 2,3-butanediol, acetoin, 2-tridecanol, and 2-tridecanone were the dominant compounds. Our study revealed that L33a has substantial potential as an effective biocontrol agent, offering valuable insights into postharvest fruit biocontrol.

Abstract 139: Modeling and genomic profiling of osteosarcoma development in murine chondrocyte-origin models

Abstract Osteosarcoma (OS) manifests during puberty, coinciding with rapid bone growth, a feature requiring exploration. We selectively deleted p53 or both p53 and Rb1 alleles in growth-dependent chondrocyte-origin models, utilizing Col10a1-Cre or Agc-CreERT2, incorporating with ROSA-Tomato for tracing and isolating the mutant cell collection. Col10p53flfl(tm) and Col10p53flflRbfl+(tm) mutants exhibited 100% penetrance in osteoblastic OS, with the latter displaying shortened latencies (mean: 335 vs 265 days) and increased metastases. Tamoxifen-induced AgcCreERT2 models highlighted growth stage-dependent OS development, correlating penetrance inversely and latencies positively with mouse age at tamoxifen treatment. Tumors exclusively occurred in endochondral bones, near growth plates. Whole-genome profiling of tm+ tumor cells revealed genetic variations, including SNPs, Indels, CNVs, and SVs. CNVs involved genes altered in human OS, emphasizing losses in Dmd and gains in Myc, Vegfa, Foxm1, Akt1, Aurkb, and Ccen1. A common CNV (losses, Chr. 10: 34,158,531-34,443,999, Log ratio ←0.05) occurred in all sequenced primary and metastatic samples, with male specimens sharing a loss in the Y chr. p region (approximately 725,200-799,409). Orthotopic transplantation of tm+ p53− stromal cells from 4-month-old Col10p53flfltm mice gave rise to bone tumors and lung migration in wild-type recipients, explicitly establishing the transplanted cells as the direct origin of the cancer precursors within early tm+ p53− stromal populations. Whole-genome sequencing unveiled a discernible progression of genomic alterations from 1 to 4-month-old tm+ p53− stromal cells, culminating in donor-derived tumors. Notably, only these specific donor-derived tumors, and not the early tm+ p53− stromal cells, exhibited the Chr. 10 CNV observed in autochthonous malignancies. Micro-metastases, identified by fluorescence imaging, were prevalent, alongside macro-metastases (~10% in Col10p53flflRbfl+tm and ~25% in AgcCreERT2p53flflRbfl+tm cohorts). Notably, amputation of primary OS sites in mice surviving longer revealed larger and more abundant tm+ cell clusters. Some micro-metastases not only expressed signature genes typical of macro-lung metastases but also shared genomic variations with corresponding primaries, including the prevalent loss in Chr. 10. Enhanced immune cell infiltration was evident in lungs hosting micro-metastases, suggesting a potential immune response triggered by pre-tumor early seeding cells. Murine OS models faithfully replicate human OS features, underscoring chondrocytes as significant contributors to OS cell sources. The inclusion of a tomato reporter enables precise tracking, rendering these models invaluable for studying OS etiology, particularly in unraveling pre-tumor molecular events in both primary and metastatic settings. Citation Format: Xin Zhou, Hannah Beird, Yanhua Yi, Zhaohui Xu, Zhongting Zhang, Harjeet Singh, Wendong Zhang, Yifei Wang, Sylvester Jusu, Michael Roth, Jonatha Gill, Richard Gorlick. Modeling and genomic profiling of osteosarcoma development in murine chondrocyte-origin models [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 139.

Whole-Genome DNA Methylation Profiling of Intrahepatic Cholangiocarcinoma Reveals Prognostic Subtypes with Distinct Biological Drivers.

Characterization of the DNA methylome of intrahepatic cholangiocarcinoma integrated with genomic, transcriptomic, and proteomic analyses uncovers molecular mechanisms affected by genome-wide DNA methylation alterations, providing a resource for identifying potential therapeutic targets.

Rare variant of large pediatric glioneuronal tumor with novel MYO5A::NTRK3 fusion: illustrative case.

Glioneuronal tumors (GNTs) comprise a rare class of central nervous system (CNS) neoplasms with varying degrees of neuronal and glial differentiation that predominately affect children and young adults. Within the current 2021 World Health Organization (WHO) classification of CNS tumors, GNTs encompass 14 distinct tumor types. Recently, the use of whole-genome DNA methylation profiling has allowed more precise classification of this tumor group. A 3-year-old male presented with a 3-month history of increasing head circumference, regression of developmental milestones, and speech delay. Magnetic resonance imaging of the brain was notable for a large left hemispheric multiseptated mass with significant mass effect and midline shift that was treated with near-total resection. Histological and molecular assessment demonstrated a glioneuronal tumor harboring an MYO5A::NTRK3 fusion. By DNA methylation profiling, this tumor matched to a provisional methylation class known as "glioneuronal tumor kinase-fused" (GNT kinase-fused). The patient was later started on targeted therapy with larotrectinib. This is the first report of an MYO5A::NTRK3 fusion in a pediatric GNT. GNT kinase-fused is a provisional methylation class not currently included in the WHO classification of CNS tumors. This case highlights the impact of thorough molecular characterization of CNS tumors, especially with the increasing availability of novel gene targeting therapies.

Lacticaseibacillus paracasei AD22 Stress Response in Brined White Cheese Matrix: In Vitro Probiotic Profiles and Molecular Characterization.

Functionalizing foods involve discovering and integrating new candidate health-promoting bacteria into the food matrix. This study aimed (i) to reveal the probiotic potential of autochthonous Lacticaseibacillus paracasei AD22 by a series of in vitro tests and molecular characterization and (ii) to evaluate its application to the matrix of brined white cheese, which is the most common cheese in Türkiye, in terms of survival and stress response. To evaluate in vitro probiotic characteristics, L. paracasei AD22 was exposed to functional, technological, and safety tests. Pilot scale production was conducted to integrate L. paracasei AD22 into the brined white cheese matrix. The expression levels of stress-related genes (dnaK, groES, ftsH, argH, and hsp20) were detected by reverse-transcriptase polymerase chain reaction to determine the transcriptional stress response during ripening. The presence of genes encoding stress-related proteins was determined by whole-genome sequence analysis using a subsystem approach; the presence of antibiotic resistance and virulence genes was determined by ResFinder4.1 and VirulenceFinder 2.0 databases. The BAGEL4 database determined the presence of bacteriocin clusters. L. paracasei AD22 was found to survive in pH 2 and medium with 12% NaCl and did not cause hemolysis. Adhesion of the strain to Caco2 cells was 76.26 ± 4.81% and it had coaggregation/autoaggregation properties. It was determined that L. paracasei AD22 exceeded 7 log cfu/g in the cheese matrix at the end of the ripening period. Total mesophilic aerobes decreased in the cheese inoculated with L. paracasei AD22 after the 45th day of ripening. While hsp20 and groES genes were downregulated during ripening, argH was upregulated. Both downregulation and upregulation were observed in dnaK and ftsH. Fold changes indicating the expression levels of dnaK, groES, ftsH, argH, and hsp20 genes were not statistically significant during ripening (p > 0.05). Whole-genome sequence profiles revealed that the strain did not contain antibiotic and virulence genes but bacteriocin clusters encoding Enterolysin A (Class III bacteriocin), Carnosine CP52 (class II bacteriocin), Enterocin X beta chain (Class IIc bacteriocin), and the LanT region. Subsystems approach manifested that the most functional part of the genomic distribution belonged to metabolism, protein processing, and stress response functions. The study findings highlight that L. paracasei AD22 will provide biotechnological innovation as a probiotic adjunct because it contains tolerance factors and probiotic characteristics to produce new functional foods.