Transposon DNA sequences facilitate the tissue-specific gene transfer of circulating tumor DNA between human cells.

The exchange of genes between cells is known to play an important physiological and pathological role in many organisms. We show that circulating tumor DNA (ctDNA) facilitates cell-specific gene transfer between human cancer cells and explain part of the mechanisms behind this phenomenon. As ctDNA migrates into the nucleus, genetic information is transferred. Cell targeting and ctDNA integration require ERVL, SINE or LINE DNA sequences. Chemically manufactured AluSp and MER11C sequences replicated multiple myeloma (MM) ctDNA cell targeting and integration. Additionally, we found that ctDNA may alter the treatment response of MM and pancreatic cancer models. This study shows that retrotransposon DNA sequences promote cancer gene transfer. However, because cell-free DNA has been detected in physiological and other pathological conditions, our findings have a broader impact than just cancer. Furthermore, the discovery that transposon DNA sequences mediate tissue-specific targeting will open up a new avenue for the delivery of genes and therapies.

Pan-cancer analysis of telomere maintenance mechanisms.

Telomeres, protective caps at chromosome ends, maintain genomic stability and control cell lifespan. Dysregulated telomere maintenance mechanisms (TMMs) are cancer hallmarks, enabling unchecked cell proliferation. We conducted a pan-cancer evaluation of TMM using RNA sequencing data from The Cancer Genome Atlas for 33 different cancer types and analyzed the activities of telomerase-dependent (TEL) and alternative lengthening of telomeres (ALT) TMM pathways in detail. To further characterize the TMM profiles, we categorized the tumors based on their ALT and TEL TMM pathway activities into five major phenotypes: ALT high TEL low, ALT low TEL low, ALT middle TEL middle, ALT high TEL high, and ALT low TEL high. These phenotypes refer to variations in telomere maintenance strategies, shedding light on the heterogeneous nature of telomere regulation in cancer. Moreover, we investigated the clinical implications of TMM phenotypes by examining their associations with clinical characteristics and patient outcomes. Specific TMM profiles were linked to specific survival patterns, emphasizing the potential of TMM profiling as a prognostic indicator and aiding in personalized cancer treatment strategies. Gene ontology analysis of the TMM phenotypes unveiled enriched biological processes associated with cell cycle regulation (both TEL and ALT), DNA replication (TEL), and chromosome dynamics (ALT) showing that telomere maintenance is tightly intertwined with cellular processes governing proliferation and genomic stability. Overall, our study provides an overview of the complexity of transcriptional regulation of telomere maintenance mechanisms in cancer.

Generation of iPSCs from a Patient with the M694V Mutation in the MEFV Gene Associated with Familial Mediterranean Fever and Their Differentiation into Macrophages.

Familial Mediterranean fever (FMF) is a systemic autoinflammatory disorder caused by inherited mutations in the MEFV (Mediterranean FeVer) gene, located on chromosome 16 (16p13.3) and encoding the pyrin protein. Despite the existing data on MEFV mutations, the exact mechanism of their effect on the development of the pathological processes leading to the spontaneous and recurrent autoinflammatory attacks observed in FMF, remains unclear. Induced pluripotent stem cells (iPSCs) are considered an important tool to study the molecular genetic mechanisms of various diseases due to their ability to differentiate into any cell type, including macrophages, which contribute to the development of FMF. In this study, we developed iPSCs from an Armenian patient with FMF carrying the M694V, p.(Met694Val) (c.2080A>G, rs61752717) pathogenic mutation in exon 10 of the MEFV gene. As a result of direct differentiation, macrophages expressing CD14 and CD45 surface markers were obtained. We found that the morphology of macrophages derived from iPSCs of a patient with the MEFV mutation significantly differed from that of macrophages derived from iPSCs of a healthy donor carrying the wild-type MEFV gene.

Molecular Epidemiology and Virulence of Non-Typhoidal Salmonella in Armenia.

In this work, we analysed human isolates of nontyphoidal Salmonella enterica subsp. enterica (NTS), which were collected from salmonellosis cases in Armenia from 1996 to 2019. This disease became a leading food-borne bacterial infection in the region, with the younger age groups especially affected. The isolates were characterised by serotyping, Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR) typing, and whole genome sequencing (WGS). The main serotypes were S. Typhimurium, S. Enteritidis, and S. Arizonae. ERIC-PCR indicated a high degree of clonality among S. Typhimurium strains, which were also multidrug-resistant and produced extended spectrum beta-lactamases. During the study period, the frequency of S. Typhimurium and S. Arizonae isolations decreased, but with the increase in S. Enteritidis and other NTS. A total of 42 NTS isolates were subjected to WGS and explored for virulence-related traits and the corresponding genetic elements. Some virulence and genetic factors were shared by all NTS serotypes, while the main differences were attributed to the serotype-specific diversity of virulence genes, SPIs, virulence plasmids, and phages. The results indicated the variability and dynamics in the epidemiology of salmonellosis and a high virulence potential of human NTS isolates circulating in the region.

The Effect of Low-Energy Laser-Driven Ultrashort Pulsed Electron Beam Irradiation on Erythropoiesis and Oxidative Stress in Rats.

Anemia is a commonly observed consequence of whole-body exposure to a dose of X-ray or gamma irradiation of the order of the mean lethal dose in mammals, and it is an important factor for the determination of the survival of animals. The aim of this study was to unravel the effect of laser-driven ultrashort pulsed electron beam (UPEB) irradiation on the process of erythropoiesis and the redox state in the organism. Wistar rats were exposed to laser-driven UPEB irradiation, after which the level of oxidative stress and the activities of different antioxidant enzymes, as well as blood smears, bone marrow imprints and sections, erythroblastic islets, hemoglobin and hematocrit, hepatic iron, DNA, and erythropoietin levels, were assessed on the 1st, 3rd, 7th, 14th, and 28th days after irradiation. Despite the fact that laser-driven UPEB irradiation requires quite low doses and repetition rates to achieve the LD50 in rats, our findings suggest that whole-body exposure with this new type of irradiation causes relatively mild anemia in rats, with subsequent fast recovery up to the 28th day. Moreover, this novel type of irradiation causes highly intense processes of oxidative stress, which, despite being relatively extinguished, did not reach the physiologically stable level even at the 28th day after irradiation due to the violations in the antioxidant system of the organism.

A Transcriptome-Wide Isoform Landscape of Melanocytic Nevi and Primary Melanomas Identifies Gene Isoforms Associated with Malignancy.

Genetic splice variants have become of central interest in recent years, as they play an important role in different cancers. Little is known about splice variants in melanoma. Here, we analyzed a genome-wide transcriptomic dataset of benign melanocytic nevi and primary melanomas (n = 80) for the expression of specific splice variants. Using kallisto, a map for differentially expressed splice variants in melanoma vs. benign melanocytic nevi was generated. Among the top genes with differentially expressed splice variants were Ras-related in brain 6B (RAB6B), a member of the RAS family of GTPases, Macrophage Scavenger Receptor 1 (MSR1), Collagen Type XI Alpha 2 Chain (COLL11A2), and LY6/PLAUR Domain Containing 1 (LYPD1). The Gene Ontology terms of differentially expressed splice variants showed no enrichment for functional gene sets of melanoma vs. nevus lesions, but between type 1 (pigmentation type) and type 2 (immune response type) melanocytic lesions. A number of genes such as Checkpoint Kinase 1 (CHEK1) showed an association of mutational patterns and occurrence of splice variants in melanoma. Moreover, mutations in genes of the splicing machinery were common in both benign nevi and melanomas, suggesting a common mechanism starting early in melanoma development. Mutations in some of these genes of the splicing machinery, such as Serine and Arginine Rich Splicing Factor A3 and B3 (SF3A3, SF3B3), were significantly enriched in melanomas as compared to benign nevi. Taken together, a map of splice variants in melanoma is presented that shows a multitude of differentially expressed splice genes between benign nevi and primary melanomas. The underlying mechanisms may involve mutations in genes of the splicing machinery.

Transcriptome Patterns of BRCA1- and BRCA2- Mutated Breast and Ovarian Cancers.

Mutations in the BRCA1 and BRCA2 genes are known risk factors and drivers of breast and ovarian cancers. So far, few studies have been focused on understanding the differences in transcriptome and functional landscapes associated with the disease (breast vs. ovarian cancers), gene (BRCA1 vs. BRCA2), and mutation type (germline vs. somatic). In this study, we were aimed at systemic evaluation of the association of BRCA1 and BRCA2 germline and somatic mutations with gene expression, disease clinical features, outcome, and treatment. We performed BRCA1/2 mutation centered RNA-seq data analysis of breast and ovarian cancers from the TCGA repository using transcriptome and phenotype "portrayal" with multi-layer self-organizing maps and functional annotation. The results revealed considerable differences in BRCA1- and BRCA2-dependent transcriptome landscapes in the studied cancers. Furthermore, our data indicated that somatic and germline mutations for both genes are characterized by deregulation of different biological functions and differential associations with phenotype characteristics and poly(ADP-ribose) polymerase (PARP)-inhibitor gene signatures. Overall, this study demonstrates considerable variation in transcriptomic landscapes of breast and ovarian cancers associated with the affected gene (BRCA1 vs. BRCA2), as well as the mutation type (somatic vs. germline). These results warrant further investigations with larger groups of mutation carriers aimed at refining the understanding of molecular mechanisms of breast and ovarian cancers.

Involvement of polymorphisms of the nerve growth factor and its receptor encoding genes in the etiopathogenesis of ischemic stroke.

BACKGROUND: Despite the important role of the nerve growth factor in the survival and maintenance of neurons in ischemic stroke, data regarding the relationships between variations in the encoding gene and stroke are lacking. In the present study, we evaluated the association of the functional polymorphisms in NGF (rs6330) and NGFR (rs2072446 and rs734194) genes with ischemic stroke in an Armenian population. METHODS: In total, 370 unrelated individuals of Armenian nationality were enrolled in this study. Genomic DNA samples of patients and healthy controls were genotyped using polymerase chain reaction with sequence-specific primers. RESULTS: The results obtained indicate that the minor allele of rs6330 (P corr = 2.4E-10) and rs2072446 (P corr = 0.02) are significantly overrepresented in stroke group, while the minor allele of rs734194 (P corr = 8.5E-10) was underrepresented in diseased subjects. Single nucleotide polymorphisms in NGF gene (rs6330) and NGFR gene (rs2072446 and rs734194) are associated with the disease. Furthermore, it was shown that the carriage of the NGF rs6330*T minor allele is associated with increased infarct volume and higher risk of recurrent stroke. CONCLUSIONS: In conclusion, our findings suggest that the NGF rs6330*T and NGFR rs2072446*T minor alleles might be nominated as a risk factor for developing ischemic stroke and NGFR rs734194*G minor allele as a protective against this disease at least in Armenian population.

Genomic and transcriptomic heterogeneity of colorectal tumours arising in Lynch syndrome.

Colorectal cancer (CRC) arising in Lynch syndrome (LS) comprises tumours with constitutional mutations in DNA mismatch repair genes. There is still a lack of whole-genome and transcriptome studies of LS-CRC to address questions about similarities and differences in mutation and gene expression characteristics between LS-CRC and sporadic CRC, about the molecular heterogeneity of LS-CRC, and about specific mechanisms of LS-CRC genesis linked to dysfunctional mismatch repair in LS colonic mucosa and the possible role of immune editing. Here, we provide a first molecular characterization of LS tumours and of matched tumour-distant reference colonic mucosa based on whole-genome DNA-sequencing and RNA-sequencing analyses. Our data support two subgroups of LS-CRCs, G1 and G2, whereby G1 tumours show a higher number of somatic mutations, a higher amount of microsatellite slippage, and a different mutation spectrum. The gene expression phenotypes support this difference. Reference mucosa of G1 shows a strong immune response associated with the expression of HLA and immune checkpoint genes and the invasion of CD4+ T cells. Such an immune response is not observed in LS tumours, G2 reference and normal (non-Lynch) mucosa, and sporadic CRC. We hypothesize that G1 tumours are edited for escape from a highly immunogenic microenvironment via loss of HLA presentation and T-cell exhaustion. In contrast, G2 tumours seem to develop in a less immunogenic microenvironment where tumour-promoting inflammation parallels tumourigenesis. Larger studies on non-neoplastic mucosa tissue of mutation carriers are required to better understand the early phases of emerging tumours. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Genetic variability of interleukin-1 beta as prospective factor from developing post-traumatic stress disorder.

Individual susceptibility to post-traumatic stress disorder (PTSD) is conditioned by genetic factors, and association between this disorder and polymorphisms of several genes have been shown. The aim of this study was to explore a potential association between single nucleotide polymorphisms (SNP) of the IL-1ß gene (IL1B) and PTSD. In genomic DNA samples of PTSD-affected and healthy subjects, the rs16944, rs1143634, rs2853550, rs1143643, and rs1143633 SNPs of IL1B gene have been genotyped. The results obtained demonstrated that IL1B rs1143633*C and rs16944*A minor allele frequency were significantly lower in patients than in controls. Our results confirm that IL1B rs1143633 and rs16944 SNPs are negatively associated with PTSD which allows us to consider them as protective variants for PTSD. IL1B rs1143633*C and rs16944*A minor allele frequencies and carriage rates are significantly lower in the PTSD patients as compared to the controls. These results may provide a base to conclude that above-mentioned alleles can be protective against PTSD, and IL1B gene can be involved in the pathogenesis of this disorder.

Quantitative trait association study for mean telomere length in the South Asian genomes.

MOTIVATION: Mean telomere length (MTL) is associated with cancers and age-related diseases, which necessitates identification of genomic and environmental factors that impact telomere length dynamics. Here, we present a pilot genome wide association (GWA) study for MTL in South Asian population using publicly available next generation whole genome sequences (WGS), both for MTL and genotype calculations. RESULTS: MTL in the studied population was not correlated with age, which is in accordance with previous reports. Further, we identified that individuals with Sikh religion had longer telomeres, which may be the result of complex interaction between genetic background and environmental factors. Finally, we identified 51 MTL-associated SNPs residing in five loci. The top ones were located in ADARB2 gene, which has previously been implicated with extreme old age. CONCLUSION: Our results show that WGS data can be used in telomere length studies. In addition, we introduce novel loci implicated in MTL that may be worth considering in further telomere studies. CONTACT: aarakelyan@sci.am SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Time-course human urine proteomics in space-flight simulation experiments.

BACKGROUND: Long-term space travel simulation experiments enabled to discover different aspects of human metabolism such as the complexity of NaCl salt balance. Detailed proteomics data were collected during the Mars105 isolation experiment enabling a deeper insight into the molecular processes involved. RESULTS: We studied the abundance of about two thousand proteins extracted from urine samples of six volunteers collected weekly during a 105-day isolation experiment under controlled dietary conditions including progressive reduction of salt consumption. Machine learning using Self Organizing maps (SOM) in combination with different analysis tools was applied to describe the time trajectories of protein abundance in urine. The method enables a personalized and intuitive view on the physiological state of the volunteers. The abundance of more than one half of the proteins measured clearly changes in the course of the experiment. The trajectory splits roughly into three time ranges, an early (week 1-6), an intermediate (week 7-11) and a late one (week 12-15). Regulatory modes associated with distinct biological processes were identified using previous knowledge by applying enrichment and pathway flow analysis. Early protein activation modes can be related to immune response and inflammatory processes, activation at intermediate times to developmental and proliferative processes and late activations to stress and responses to chemicals. CONCLUSIONS: The protein abundance profiles support previous results about alternative mechanisms of salt storage in an osmotically inactive form. We hypothesize that reduced NaCl consumption of about 6 g/day presumably will reduce or even prevent the activation of inflammatory processes observed in the early time range of isolation. SOM machine learning in combination with analysis methods of class discovery and functional annotation enable the straightforward analysis of complex proteomics data sets generated by means of mass spectrometry.

KEGGParser: parsing and editing KEGG pathway maps in Matlab.

SUMMARY: KEGG pathway database is a collection of manually drawn pathway maps accompanied with KGML format files intended for use in automatic analysis. KGML files, however, do not contain the required information for complete reproduction of all the events indicated in the static image of a pathway map. Several parsers and editors of KEGG pathways exist for processing KGML files. We introduce KEGGParser-a MATLAB based tool for KEGG pathway parsing, semiautomatic fixing, editing, visualization and analysis in MATLAB environment. It also works with Scilab. AVAILABILITY AND IMPLEMENTATION: The source code is available at http://www.mathworks.com/matlabcentral/fileexchange/37561.

Temporal changes of gene expression in health, schizophrenia, bipolar disorder, and major depressive disorder.

The molecular events underlying the development, manifestation, and course of schizophrenia, bipolar disorder, and major depressive disorder span from embryonic life to advanced age. However, little is known about the early dynamics of gene expression in these disorders due to their relatively late manifestation. To address this, we conducted a secondary analysis of post-mortem prefrontal cortex datasets using bioinformatics and machine learning techniques to identify differentially expressed gene modules associated with aging and the diseases, determine their time-perturbation points, and assess enrichment with expression quantitative trait loci (eQTL) genes. Our findings revealed early, mid, and late deregulation of expression of functional gene modules involved in neurodevelopment, plasticity, homeostasis, and immune response. This supports the hypothesis that multiple hits throughout life contribute to disease manifestation rather than a single early-life event. Moreover, the time-perturbed functional gene modules were associated with genetic loci affecting gene expression, highlighting the role of genetic factors in gene expression dynamics and the development of disease phenotypes. Our findings emphasize the importance of investigating time-dependent perturbations in gene expression before the age of onset in elucidating the molecular mechanisms of psychiatric disorders.

Development of an optimized, non-stem cell line for intranasal delivery of therapeutic cargo to the central nervous system.

Neural stem cells (NSCs) are considered to be valuable candidates for delivering a variety of anti-cancer agents, including oncolytic viruses, to brain tumors. However, owing to the previously reported tumorigenic potential of NSC cell lines after intranasal administration (INA), here we identified the human hepatic stellate cell line LX-2 as a cell type capable of longer resistance to replication of oncolytic adenoviruses (OAVs) as a therapeutic cargo, and that is non-tumorigenic after INA. Our data show that LX-2 cells can longer withstand the OAV XVir-N-31 replication and oncolysis than NSCs. By selecting the highly migratory cell population out of LX-2, an offspring cell line with a higher and more stable capability to migrate was generated. Additionally, as a safety backup, we applied genomic herpes simplex virus thymidine kinase (HSV-TK) integration into LX-2, leading to high vulnerability to ganciclovir (GCV). Histopathological analyses confirmed the absence of neoplasia in the respiratory tracts and brains of immuno-compromised mice 3 months after INA of LX-2 cells. Our data suggest that LX-2 is a novel, robust, and safe cell line for delivering anti-cancer and other therapeutic agents to the brain.

PSF toolkit: an R package for pathway curation and topology-aware analysis.

Most high throughput genomic data analysis pipelines currently rely on over-representation or gene set enrichment analysis (ORA/GSEA) approaches for functional analysis. In contrast, topology-based pathway analysis methods, which offer a more biologically informed perspective by incorporating interaction and topology information, have remained underutilized and inaccessible due to various limiting factors. These methods heavily rely on the quality of pathway topologies and often utilize predefined topologies from databases without assessing their correctness. To address these issues and make topology-aware pathway analysis more accessible and flexible, we introduce the PSF (Pathway Signal Flow) toolkit R package. Our toolkit integrates pathway curation and topology-based analysis, providing interactive and command-line tools that facilitate pathway importation, correction, and modification from diverse sources. This enables users to perform topology-based pathway signal flow analysis in both interactive and command-line modes. To showcase the toolkit's usability, we curated 36 KEGG signaling pathways and conducted several use-case studies, comparing our method with ORA and the topology-based signaling pathway impact analysis (SPIA) method. The results demonstrate that the algorithm can effectively identify ORA enriched pathways while providing more detailed branch-level information. Moreover, in contrast to the SPIA method, it offers the advantage of being cut-off free and less susceptible to the variability caused by selection thresholds. By combining pathway curation and topology-based analysis, the PSF toolkit enhances the quality, flexibility, and accessibility of topology-aware pathway analysis. Researchers can now easily import pathways from various sources, correct and modify them as needed, and perform detailed topology-based pathway signal flow analysis. In summary, our PSF toolkit offers an integrated solution that addresses the limitations of current topology-based pathway analysis methods. By providing interactive and command-line tools for pathway curation and topology-based analysis, we empower researchers to conduct comprehensive pathway analyses across a wide range of applications.

Long-term environmental metal exposure is associated with hypomethylation of CpG sites in NFKB1 and other genes related to oncogenesis.

BACKGROUND: Long-term environmental exposure to metals leads to epigenetic changes and may increase risks to human health. The relationship between the type and level of metal exposure and epigenetic changes in subjects exposed to high concentrations of metals in the environment is not yet clear. The aim of our study is to find the possible association of environmental long-term exposure to metals with DNA methylation changes of genes related to immune response and carcinogenesis. We investigated the association of plasma levels of 21 essential and non-essential metals detected by ICP-MS and the methylation level of 654 CpG sites located on NFKB1, CDKN2A, ESR1, APOA5, IGF2 and H19 genes assessed by targeted bisulfite sequencing in a cohort of 40 subjects living near metal mining area and 40 unexposed subjects. Linear regression was conducted to find differentially methylated positions with adjustment for gender, age, BMI class, smoking and metal concentration. RESULTS: In the metal-exposed group, five CpGs in the NFKB1 promoter region were hypomethylated compared to unexposed group. Four differentially methylated positions (DMPs) were associated with multiple metals, two of them are located on NFKB1 gene, and one each on CDKN2A gene and ESR1 gene. Two DMPs located on NFKB1 (chr4:102500951, associated with Be) and IGF2 (chr11:2134198, associated with U) are associated with specific metal levels. The methylation status of the seven CpGs located on NFKB1 (3), ESR1 (2) and CDKN2A (2) positively correlated with plasma levels of seven metals (As, Sb, Zn, Ni, U, I and Mn). CONCLUSIONS: Our study revealed methylation changes in NFKB1, CDKN2A, IGF2 and ESR1 genes in individuals with long-term human exposure to metals. Further studies are needed to clarify the effect of environmental metal exposure on epigenetic mechanisms and pathways involved.

Wild grapes of Armenia: unexplored source of genetic diversity and disease resistance.

The present study is the first in-depth research evaluating the genetic diversity and potential resistance of Armenian wild grapes utilizing DNA-based markers to understand the genetic signature of this unexplored germplasm. In the proposed research, five geographical regions with known viticultural history were explored. A total of 148 unique wild genotypes were collected and included in the study with 48 wild individuals previously collected as seed. A total of 24 nSSR markers were utilized to establish a fingerprint database to infer information on the population genetic diversity and structure. Three nSSR markers linked to the Ren1 locus were analyzed to identify potential resistance against powdery mildew. According to molecular fingerprinting data, the Armenian V. sylvestris gene pool conserves a high genetic diversity, displaying 292 different alleles with 12.167 allele per loci. The clustering analyses and diversity parameters supported eight genetic groups with 5.6% admixed proportion. The study of genetic polymorphism at the Ren1 locus revealed that 28 wild genotypes carried three R-alleles and 34 wild genotypes carried two R-alleles associated with PM resistance among analyzed 107 wild individuals. This gene pool richness represents an immense reservoir of under-explored genetic diversity and breeding potential. Therefore, continued survey and research efforts are crucial for the conservation, sustainable management, and utilization of Armenian wild grape resources in the face of emerging challenges in viticulture.

Generation of three induced pluripotent stem cell lines (RAUi001-A, RAUi001-B and RAUi001-C) from peripheral blood mononuclear cells of a healthy Armenian individual.

The study of pathological processes in cells carrying mutations should be carried out in comparison with a healthy control group. Familial Mediterranean fever (FMF), which is caused by a mutation in the MEFV gene, is predominantly found in people of Armenian nationality with the prevalence of 14-100 per 10000. We have obtained induced pluripotent stem cells (iPSCs) from Armenian healthy patient, which will be included as a control group in the study of this disease. iPSCs rapidly proliferate in colonies of cells with a typical pluripotent-like morphology, have a normal karyotype (46,XX). iPSCs express pluripotency markers (OCT4, SOX2, TRA-1-60, NANOG) and are able to give derivatives of three germ layers.

Transcriptomic Maps of Colorectal Liver Metastasis: Machine Learning of Gene Activation Patterns and Epigenetic Trajectories in Support of Precision Medicine.

The molecular mechanisms of the liver metastasis of colorectal cancer (CRLM) remain poorly understood. Here, we applied machine learning and bioinformatics trajectory inference to analyze a gene expression dataset of CRLM. We studied the co-regulation patterns at the gene level, the potential paths of tumor development, their functional context, and their prognostic relevance. Our analysis confirmed the subtyping of five liver metastasis subtypes (LMS). We provide gene-marker signatures for each LMS, and a comprehensive functional characterization that considers both the hallmarks of cancer and the tumor microenvironment. The ordering of CRLMs along a pseudotime-tree revealed a continuous shift in expression programs, suggesting a developmental relationship between the subtypes. Notably, trajectory inference and personalized analysis discovered a range of epigenetic states that shape and guide metastasis progression. By constructing prognostic maps that divided the expression landscape into regions associated with favorable and unfavorable prognoses, we derived a prognostic expression score. This was associated with critical processes such as epithelial-mesenchymal transition, treatment resistance, and immune evasion. These factors were associated with responses to neoadjuvant treatment and the formation of an immuno-suppressive, mesenchymal state. Our machine learning-based molecular profiling provides an in-depth characterization of CRLM heterogeneity with possible implications for treatment and personalized diagnostics.