Gene Copy Number Research Articles

Siderophore-harboring gut bacteria and fecal siderophore genes for predicting the responsiveness of fecal microbiota transplantation for active ulcerative colitis

BackgroundPredictive markers for fecal microbiota transplantation (FMT) outcomes in patients with active ulcerative colitis (UC) are poorly defined. We aimed to investigate changes in gut microbiota pre- and post-FMT and to assess the potential value in determining the total copy number of fecal bacterial siderophore genes in predicting FMT responsiveness.MethodsPatients with active UC (Mayo score ≥ 3) who had undergone two FMT procedures were enrolled. Fecal samples were collected before and 8 weeks after each FMT session. Patients were classified into clinical response and non-response groups, based on their Mayo scores. The fecal microbiota profile was accessed using metagenomic sequencing, and the total siderophore genes copy number via quantitative real-time polymerase chain reaction. Additionally, we examined the association between the total siderophore genes copy number and FMT efficacy.ResultsSeventy patients with UC had undergone FMT. The clinical response and remission rates were 50% and 10% after the first FMT procedure, increasing to 72.41% and 27.59% after the second FMT. The cumulative clinical response and clinical remission rates were 72.86% and 25.71%. Compared with baseline, the response group showed a significant increase in Faecalibacterium, and decrease in Enterobacteriaceae, consisted with the changes of the total bacterial siderophore genes copy number after the second FMT (1889.14 vs. 98.73 copies/ng, P < 0.01). Virulence factor analysis showed an enriched iron uptake system, especially bacterial siderophores, in the pre-FMT response group, with a greater contribution from Escherichia coli. The total baseline copy number was significantly higher in the response group than non-response group (1889.14 vs. 94.86 copies/ng, P < 0.01). A total baseline copy number cutoff value of 755.88 copies/ng showed 94.7% specificity and 72.5% sensitivity in predicting FMT responsiveness.ConclusionsA significant increase in Faecalibacterium, and decrease in Enterobacteriaceae and the total fecal siderophore genes copy number were observed in responders after FMT. The siderophore genes and its encoding bacteria may be of predictive value for the clinical responsiveness of FMT to active ulcerative colitis.

ISAba1-mediated intrinsic chromosomal oxacillinase amplification confers carbapenem resistance in Acinetobacter baumannii

Tandem amplification of carbapenemase genes increases gene copy number and enhances carbapenem resistance. These amplifications are often heterogeneous, transient, and located on plasmids, which also contribute to heteroresistance. Amplification of encoding genes is especially important for enzymes with low hydrolysis activity, which are often overlooked. Here, we reported an intrinsic oxacillinase oxaAb amplification flanked by ISAba1. The amplification is in the chromosome and contains up to 25 repeats. We provided genomic, transcriptomic, and proteomic evidence that the amplification resulted in oxacillinase overproduction. Notably, no point mutations of oxaAb were found during the amplification process. Strains of Acinetobacter baumannii with intrinsic amplified or external transformed ISAba1-oxaAb exhibited higher meropenem hydrolysis activity. Furthermore, the number of repeats in the amplification decreased gradually over a period of 21 d cultured with carbapenem withdrawal. However, upon re-exposure to meropenem, the ISAba1 flanked oxaAb responded rapidly, with repeat numbers reaching or exceeding pre-carbapenem withdrawal levels within 24 h. Taken together, these findings suggest that ISAba1-mediated gene amplification and overproduction of intrinsic low-activity oxacillinase oxaAb resulted in carbapenem resistance.

Significance of concurrent evaluation of HER2 gene amplification and p53 and Ki67 expression in gastric cancer tissues.

The primary objective of this study is to explore the significance of concurrent evaluation of HER2 gene amplification and p53 and Ki67 expression in gastric cancer tissues. Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) methodologies were used to detect HER2 gene amplification, as well as the expression levels of HER2, p53, and Ki67 proteins, across a group of 78 gastric cancer cases. The expression rate of the HER2 protein was determined to be 43.6% (34/78), with 17.9% (14/78) categorized as HER2 protein 3 + , 14.1% (11/78) as HER2 protein 2 + , and 11.5% (9/78) as HER2 protein 1 + . Using FISH technology, the HER2 gene amplification rate was identified as 19.2% (15/78), including 3 cases of HER2 gene cluster amplification, 5 cases of large granular amplification, 4 cases of punctate amplification, and 3 cases of high polysomy. The positive rate of p53 in gastric cancer cells was 52.6% (41/78), with 62.8% (49/78) of patients exhibiting a ki67 proliferation index ≤ 30, and 37.2% (29/78) accounting for a ki67 proliferation index > 30. The expression rates of the HER2 gene, p53, and ki67 in gastric cancer tissues were significantly associated with both gastric cancer staging and lymph node metastasis (P < 0.05). The HER2 gene amplification rate and gene copy number exhibit a positive correlation with the expression rates of p53 and ki67. Combining these assessments can provide crucial insights into the assessment of metastatic potential, disease progression, and prognosis of gastric tumor cells. This holds paramount importance in steering the formulation of individualized treatment strategies.

Rapid inference of antibiotic resistance and susceptibility for Klebsiella pneumoniae by clinical shotgun metagenomic sequencing

ObjectivesThe study aimed to develop a genotypic antimicrobial resistance testing method for Klebsiella pneumoniae using metagenomic sequencing data. MethodsWe utilized Lasso regression on assembled genomes to identify genetic resistance determinants for six antibiotics (Gentamicin, Tobramycin, Imipenem, Meropenem, Ceftazidime, Trimethoprim/Sulfamethoxazole). The genetic features were weighted, grouped into clusters to establish classifier models. Origin species of detected antibiotic resistant gene (ARG) was determined by novel strategy integrating “possible species,” “gene copy number calculation” and “species-specific kmers.” The performance of the method was evaluated on retrospective case studies. ResultsOur study employed machine learning on 3928 K. pneumoniae isolates, yielding stable models with AUCs > 0.9 for various antibiotics. GenseqAMR, a read-based software, exhibited high accuracy (AUC 0.926–0.956) for short-read datasets. The integration of a species-specific kmer strategy significantly improved ARG-species attribution to an average accuracy of 96.67%. In a retrospective study of 191 K. pneumoniae-positive clinical specimens (0.68–93.39% genome coverage), GenseqAMR predicted 84.23% of AST results on average. It demonstrated 88.76–96.26% accuracy for resistance prediction, offering genotypic AST results with a shorter turnaround time (mean ± SD: 18.34 ± 0.87 hours) than traditional culture-based AST (60.15 ± 21.58 hours). Furthermore, a retrospective clinical case study involving 63 cases showed that GenseqAMR could lead to changes in clinical treatment for 24 (38.10%) cases, with 95.83% (23/24) of these changes deemed beneficial. ConclusionsIn conclusion, GenseqAMR is a promising tool for quick and accurate AMR prediction in Klebsiella pneumoniae, with the potential to improve patient outcomes through timely adjustments in antibiotic treatment.

Deep learning for predicting 16S rRNA gene copy number

Culture-independent 16S rRNA gene metabarcoding is a commonly used method for microbiome profiling. To achieve more quantitative cell fraction estimates, it is important to account for the 16S rRNA gene copy number (hereafter 16S GCN) of different community members. Currently, there are several bioinformatic tools available to estimate the 16S GCN values, either based on taxonomy assignment or phylogeny. Here we present a novel approach ANNA16, Artificial Neural Network Approximator for 16S rRNA gene copy number, a deep learning-based method that estimates the 16S GCN values directly from the 16S gene sequence strings. Based on 27,579 16S rRNA gene sequences and gene copy number data from the rrnDB database, we show that ANNA16 outperforms the commonly used 16S GCN prediction algorithms. Interestingly, Shapley Additive exPlanations (SHAP) shows that ANNA16 can identify unexpected informative positions in 16S rRNA gene sequences without any prior phylogenetic knowledge, which suggests potential applications beyond 16S GCN prediction.

Inoculum source determines the stress resistance of electroactive functional taxa in biofilms: A metagenomic perspective

The inoculum has a crucial impact on bioreactor initialization and performance. However, there is currently a lack of guidance on selecting appropriate inocula for applications in environmental biotechnology. In this study, we applied microbial electrolysis cells (MECs) as models to investigate the differences in the functional potential of electroactive microorganisms (EAMs) within anodic biofilms developed from four different inocula (natural or artificial), using shotgun metagenomic techniques. We specifically focused on extracellular electron transfer (EET) function and stress resistance, which affect the performance and stability of MECs. Community profiling revealed that the family Geobacteraceae was the key EAM taxon in all biofilms, with Geobacter as the dominant genus. The c-type cytochrome gene imcH showed universal importance for Geobacteraceae EET and was utilized as a marker gene to evaluate the EET potential of EAMs. Additionally, stress response functional genes were used to assess the stress resistance potential of Geobacter species. Comparative analysis of imcH gene abundance revealed that EAMs with comparable overall EET potential could be enriched from artificial and natural inocula (P > 0.05). However, quantification of stress response gene copy numbers in the genomes demonstrated that EAMs originating from natural inocula possessed superior stress resistance potential (196 vs. 163). Overall, this study provides novel perspectives on the inoculum effect in bioreactors and offers theoretical guidance for selecting inoculum in environmental engineering applications.

Genomic and transcriptomic analyses highlight evolutionary and functional characteristics of WRKY genes in Asterids

WRKY transcription factors regulate development, dormancy, germination, senescence, and reactions to abiotic and biotic stress. Asterids, a major group of eudicots, are commercially and pharmacologically important. However, large-scale WRKY gene family analyses in Asterids are lacking. We performed genome-wide re-annotation and downstream analyses of WRKY genes in nine Asterid species in addition to Arabidopsis thaliana and Beta vulgaris. We identified a total of 1,013 WRKY genes, 91 of which were newly annotated in our analysis. These were divided into six subgroups based on domain structure, phylogeny, and motif composition. Duplication analysis revealed increased copy numbers of WRKY genes belonging to specific subgroups in Helianthus annuus (sunflower), Actinidia chinensis (kiwifruit), and Olea europaea var. sylvestris (wild olive). Both H. annuus and A. chinensis displayed recent duplications of the WRKY gene after their divergence, whereas O. europaea var. sylvestris showed ancient duplication of WRKY genes. Transcriptome analyses suggested the potential function of WRKY genes in responses to abiotic and biotic stresses in H. annuus and A. chinensis, respectively. Our data shed light on the evolutionary and functional characteristics of WRKY genes, especially among Asterid plant species.

Comprehensive analysis of EphA2 in pan-cancer: A prognostic biomarker associated with cancer immunity.

Several studies have reported a significant relationship between Ephrin receptor A2 (EphA2) and malignant progression in numerous cancers. However, there is a lack of comprehensive pan-cancer analysis on the prognostic value, mutation status, methylation landscape, and potential immunological function of EphA2. Using The Cancer Genome Atlas, Genotype Tissue Expression Database and GEO data, we analysed the differences in EphA2 expression between normal and tumour tissues and the effects of EphA2 on the prognosis of different tumours. Furthermore, using GSCALite, cBioPortal, TISDB, ULCLAN and TIMER 2.0 databases or platforms, we comprehensively analysed the potential oncogenic mechanisms or manifestations of EphA2 in 33 different tumour types, including tumour mutation status, DNA methylation status and immune cell infiltration. The correlation of EphA2 with immune checkpoints, tumour mutational burden, DNA microsatellite instability and DNA repair genes was also calculated. Finally, the effects of EphA2 inhibitors on the proliferation of human glioma and lung cancer cells were verified in cellular experiments. EphA2 is differentially expressed in different tumours, and patients with overexpression have poorer overall survival. In addition, gene mutations, gene copy number variation and DNA/RNA methylation of EphA2 have been identified in various tumours. Moreover, EphA2 is positively associated with immune infiltration involving macrophages and CD8+ T cells. Further, EphA2 mRNA expression is significantly associated with immune checkpoint in various cancers, especially programmed death-ligand 1. Finally, the EphA2 inhibitor ALW-II-41-27 shows potent anti-tumour activity. Our first pan-cancer study of EphA2 provides insight into the prognostic and immunological roles of EphA2 in different tumours, suggesting that EphA2 might be a potential biomarker for poor prognosis and immune infiltration in cancer.

Origin, evolution and diversity of SINA E3 ubiquitin ligases in plants

SINA (Seven in absentia) E3 ligases are critical components of the ubiquitin-proteasome system (UPS). They are responsible for ubiquitination and are involved in numerous cellular processes. The functional mechanisms of SINAs have been extensively studied in a few angiosperms. However, our understanding of the origin and evolution of plant SINA genes remain limited. Here we performed a large-scale comprehensive analysis of SINA proteins from various plant lineages. The plant SINA family genes likely originated from a common ancestral gene prior to the divergence of bryophyte and gave rise to two clades. Within clade I, subsequent parallel innovations in lycophytes and ferns resulted in the formation of two branches. All SINA proteins contain an N-terminal cysteine-rich really interesting new gene (RING) domain for ubiquitination, two zinc-finger motifs, and a C-terminal domain required for substrate-binding and dimerization. The SINA genes gains and losses occurred in angiosperms, resulting in an increase in the number of gene copies in eudicots and monocots. The Gossypium genus SINAs showed a tendency for expansion via whole-genome duplication and polyploidy. Finally, global expression patterns revealed the functional diversification of SINA genes in developmental stage and response to hormones and abiotic stresses in Arabidopsis and rice. These findings provide an insight into the evolution and diversification of SINA E3 ligases in plants and enhance our understanding of the role they play in determining substrate specificity and environment stress adaptations in angiosperms.

Genome-scale models of metabolism and expression predict the metabolic burden of recombinant protein expression

The production of recombinant proteins in a host using synthetic constructs such as plasmids comes at the cost of detrimental effects such as reduced growth, energetic inefficiencies, and other stress responses, collectively known as metabolic burden. Increasing the number of copies of the foreign gene increases the metabolic load but increases the expression of the foreign protein. Thus, there is a trade-off between biomass and product yield in response to changes in heterologous gene copy number. This work proposes a computational method, rETFL (recombinant Expression and Thermodynamic Flux), for analyzing and predicting the responses of recombinant organisms to the introduction of synthetic constructs. rETFL is an extension to the ETFL formulations designed to reconstruct models of metabolism and expression (ME-models). We have illustrated the capabilities of the method in four studies to (i) capture the growth reduction in plasmid-containing E. coli and recombinant protein production; (ii) explore the trade-off between biomass and product yield as plasmid copy number is varied; (iii) predict the emergence of overflow metabolism in recombinant E. coli in agreement with experimental data; and (iv) investigate the individual pathways and enzymes affected by the presence of the plasmid. We anticipate that rETFL will serve as a comprehensive platform for integrating available omics data for recombinant organisms and making context-specific predictions that can help optimize recombinant expression systems for biopharmaceutical production and gene therapy.

1187-P: Increased C4B Gene Copy Number Is Associated with Increased Inflammation in Adolescent Type 1 Diabetes

1187-P: Increased C4B Gene Copy Number Is Associated with Increased Inflammation in Adolescent Type 1 Diabetes

Single-cell RNA sequencing and bulk sequencing reveal the immunosuppressive role of malignant cells in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and the limiting efficacy is achieved for TNBC patients with immunotherapy. The role of malignant cells in the tumor microenvironment (TME) is still complicated. Here, we firstly identified and characterized the malignant cells in the TME of TNBC based on single-cell and bulk RNA sequencing. Then a high score of gene copy number variation (CNV) was found in the malignant cells. And 2507 up-regulated genes and 830 down-regulated genes were obtained based on a difference analysis between the malignant cells and other epithelial cells. Furthermore, KLF3-targeted genes were identified as being associated with the development of breast cancer. Finally, an immunosuppressive role of the malignant cells was identified in the TME of TNBC as the strong interactions between tumor cells and CD8-positive exhausted T cells. Together, these findings provide new insights into the malignant cells of TME and a new idea to develop therapeutic strategy for immunotherapy of TNBC.

Comparative Genomics and the Salivary Transcriptome of the Redbanded Stink Bug Shed Light on Its High Damage Potential to Soybean.

The redbanded stink bug, Piezodorus guildinii (Westwood) (Hemiptera: Pentatomidae), is a significant soybean pest in the Americas, which inflicts more physical damage on soybean than other native stink bugs. Studies suggest that its heightened impact is attributed to the aggressive digestive properties of its saliva. Despite its agricultural importance, the factors driving its greater ability to degrade plant tissues have remained unexplored in a genomic evolutionary context. In this study, we hypothesized that lineage-specific gene family expansions have increased the copy number of digestive genes expressed in the salivary glands. To investigate this, we annotated a previously published genome assembly of the redbanded stink bug, performed a comparative genomic analysis on 11 hemipteran species, and reconstructed patterns of gene duplication, gain, and loss in the redbanded stink bug. We also performed RNA-seq on the redbanded stink bug's salivary tissues, along with the rest of the body without salivary glands. We identified hundreds of differentially expressed salivary genes, including a subset lost in other stink bug lineages, but retained and expressed in the redbanded stink bug's salivary glands. These genes were significantly enriched with protein families involved in proteolysis, potentially explaining the redbanded stink bug's heightened damage to soybeans. Contrary to our hypothesis, we found no support for an enrichment of duplicated digestive genes that are also differentially expressed in the salivary glands of the redbanded stink bug. Nonetheless, these results provide insight into the evolution of this important crop pest, establishing a link between its genomic history and its agriculturally important physiology.

Copy number variations of CCND1 gene and chromosome 11 centromere in acral melanoma

Objective: To study the correlation between the copy number variations of CCND1 gene and chromosome 11 and their associations with clinicopathologic features in acral melanoma. Methods: Thirty-three acral melanoma cases diagnosed at the Department of Pathology of Peking University Third Hospital, Beijing, China from January 2018 to August 2021 were collected. Fluorescence in situ hybridization (FISH) was used to detect the copy number of CCND1 gene and centromere of chromosome 11. The relationship between the copy numbers of CCND1 and chromosome 11 centromere, and the correlation between CCND1 copy number and clinicopathologic characteristics were analyzed. Results: There were 15 male and 18 female patients, with an age ranging from 22-86 years. 63.6% (21/33) of the patients had an increased CCND1 gene copy number. 21.2% (7/33) of patients with increased CCND1 copy number had an accompanying chromosome 11 centromere copy number increase. 27.3% (9/33) of the cases had a low copy number of CCND1 gene, and 4 of them (4/33, 12.1%) were accompanied by chromosome 11 centromere copy number increase. 36.4% (12/33) of the cases had a high copy number of CCND1 gene, and 3 (3/33, 9.1%) of them were accompanied by chromosome 11 centromere copy number increase. No cases with CCND1 low copy number increase showed CCND1/CEP11 ratio greater than 2.00. The 11 cases with CCND1 high copy number increase showed CCND1/CEP11 ratio greater than or equal to 2.00. However, there was no significant correlation between CCND1 copy number increase and any of the examined clinicopathologic features such as age, sex, histological type, Breslow thickness, ulcer and Clark level. Conclusions: CCND1 copy number increase is a significant molecular alteration in acral melanoma. In some cases, CCND1 copy number increase may be accompanied by the copy number increase of chromosome 11. For these cases the copy number increase in CCND1 gene may be a result of the copy number change of chromosome 11.

Identification of a conserved G-quadruplex within the E165R of African swine fever virus (ASFV) as a potential antiviral target

African swine fever virus (ASFV) is a double-stranded DNA arbovirus with high transmissibility and mortality rates. It has caused immense economic losses to the global pig industry. Currently, no effective vaccines or medications are to combat ASFV infection. G-quadruplex (G4) structures have attracted increasing interest because of their regulatory role in vital biological processes. In this study, we identified a conserved G-rich sequence within the E165R gene of ASFV. Subsequently, using various methods, we verified that this sequence could fold into a parallel G-quadruplex (G4). In addition, the G4-stabilizers pyridostatin (PDS) and 5,10,15,20-tetrakis-(N-methyl-4-pyridyl) porphin (TMPyP4) can bind and stabilize this G4 structure, thereby inhibiting E165R gene expression, and the inhibitory effect is associated with G4 formation. Moreover, the G4 ligand PDS substantially impeded ASFV proliferation in Vero cells by reducing gene copy number and viral protein expression. These compelling findings suggest that G4 structures may represent a promising and novel antiviral target against ASFV.

Characterization of Taxonomic and Functional Dynamics Associated with Harmful Algal Bloom Formation in Recreational Water Ecosystems.

Harmful algal bloom (HAB) formation leads to the eutrophication of water ecosystems and may render recreational lakes unsuitable for human use. We evaluated the applicability and comparison of metabarcoding, metagenomics, qPCR, and ELISA-based methods for cyanobacteria/cyanotoxin detection in bloom and non-bloom sites for the Great Lakes region. DNA sequencing-based methods robustly identified differences between bloom and non-bloom samples (e.g., the relative prominence of Anabaena and Planktothrix). Shotgun sequencing strategies also identified the enrichment of metabolic genes typical of cyanobacteria in bloom samples, though toxin genes were not detected, suggesting deeper sequencing or PCR methods may be needed to detect low-abundance toxin genes. PCR and ELISA indicated microcystin levels and microcystin gene copies were significantly more abundant in bloom sites. However, not all bloom samples were positive for microcystin, possibly due to bloom development by non-toxin-producing species. Additionally, microcystin levels were significantly correlated (positively) with microcystin gene copy number but not with total cyanobacterial 16S gene copies. In summary, next-generation sequencing-based methods can identify specific taxonomic and functional targets, which can be used for absolute quantification methods (qPCR and ELISA) to augment conventional water monitoring strategies.

Clinicopathologic Characteristics of MYC Copy Number Amplification in Breast Cancer.

Introduction. MYC overexpression is a known phenomenon in breast cancer. This study investigates the correlation of MYC gene copy number amplification and MYC protein overexpression with coexisting genetic abnormalities and associated clinicopathologic features in breast cancer patients. Methods. The study analyzed data from 81 patients with localized or metastatic breast cancers using targeted next-generation sequencing and MYC immunohistochemical studies, along with pathological and clinical data. Results. Applying the criteria of MYC/chromosome 8 ratio ≥5, MYC copy number amplified tumors (n = 11, 14%) were associated with invasive ductal carcinoma (91% vs 68%, P = .048), poorly differentiated (grade 3, 64% vs 30%, P = .032), mitotically active (Nottingham mitotic score 3, 71% vs 20%, P = .004), estrogen receptor (ER)-negative (45% vs 12%, P = .008), and triple-negative (56% vs 12%, P = .013) compared to MYC non-amplified tumors. Among MYC-amplified breast cancer patients, those with triple-negative status showed significantly shorter disease-free survival time than non-triple negative MYC-amplified patients (median survival month: 25.5 vs 127.6, P = .049). MYC amplification is significantly associated with TP53 mutation (P = .007). The majority (10 of 11; 91%) of MYC-amplified tumors showed positive c-MYC immunostaining. Conclusion. Breast cancers with MYC copy number amplication display distinct clinicopathologic characteristics indicative of more aggressive behavior.

Genome evolution of the ancient hexaploid Platanus × acerifolia (London planetree)

Whole-genome duplication (WGD; i.e., polyploidy) and chromosomal rearrangement (i.e., genome shuffling) significantly influence genome structure and organization. Many polyploids show extensive genome shuffling relative to their pre-WGD ancestors. No reference genome is currently available for Platanaceae (Proteales), one of the sister groups to the core eudicots. Moreover, Platanus × acerifolia (London planetree; Platanaceae) is a widely used street tree. Given the pivotal phylogenetic position of Platanus and its 2-y flowering transition, understanding its flowering-time regulatory mechanism has significant evolutionary implications; however, the impact of Platanus genome evolution on flowering-time genes remains unknown. Here, we assembled a high-quality, chromosome-level reference genome for P. × acerifolia using a phylogeny-based subgenome phasing method. Comparative genomic analyses revealed that P. × acerifolia (2n = 42) is an ancient hexaploid with three subgenomes resulting from two sequential WGD events; Platanus does not seem to share any WGD with other Proteales or with core eudicots. Each P. × acerifolia subgenome is highly similar in structure and content to the reconstructed pre-WGD ancestral eudicot genome without chromosomal rearrangements. The P. × acerifolia genome exhibits karyotypic stasis and gene sub-/neo-functionalization and lacks subgenome dominance. The copy number of flowering-time genes in P. × acerifolia has undergone an expansion compared to other noncore eudicots, mainly via the WGD events. Sub-/neo-functionalization of duplicated genes provided the genetic basis underlying the unique flowering-time regulation in P. × acerifolia. The P. × acerifolia reference genome will greatly expand understanding of the evolution of genome organization, genetic diversity, and flowering-time regulation in angiosperms.

Integrated analysis of -omic landscapes in breast cancer subtypes

The subtypes of breast cancer exhibit diverse histology, molecular features, therapeutic response, aggressiveness, and patient outcomes. Multi-omics high-throughput technologies, which are widely used in cancer research, generated waste amounts of multimodal omic datasets calling for new approaches of integrated analyses to uncover patterns of transcriptomic, genomic, and epigenetic changes in breast cancer subtypes and connect them to disease clinical characteristics. Here, we applied multi-layer self-organizing map (ml-SOM) algorithms to PAM50-classified TCGA breast cancer samples to disentangle the diversity of the effects of gene expression, methylation, copy number, and somatic single nucleotide variation in the disease subtypes. Furthermore, we studied the association of perturbed gene modules with survival, prognosis, and other clinical characteristics. Our findings highlight the power of multi-omic analyses to offer a better understanding of the molecular diversity of breast cancer subtypes compared to single-omic analyses. Moreover, they highlight the complex subtype-characteristic associations between gene expression and epigenetic/genomic factors and their implications for survival and clinical outcomes.

Analytical validation of the Labcorp Plasma Complete test to enable precision oncology through solid tumor liquid biopsy comprehensive genomic profiling.

3063 Background: To help guide treatment decisions and enable clinical trial matching for cancer patients, tumor genomic profiling is an essential precision oncology tool. Liquid biopsy may be used as a complementary approach to assess tumor-specific DNA alterations circulating in the blood. The Labcorp Plasma Complete test is a next-generation-sequencing (NGS), cell-free DNA (cfDNA) comprehensive genomic profiling test that identifies actionable and clinically relevant variants in advanced and metastatic solid cancers across 521 genes. Methods: The Labcorp Plasma Complete test is a hybrid capture based, targeted NGS test, optimized for 25 ng of cfDNA input and is intended for variant detection in all solid cancer indications. The test interrogates all coding exons of 521 genes to comprehensively detect single nucleotide variants (SNVs) and insertion and deletions (indels). The test also reports copy number amplifications (CNAs) and gene translocations in 12 genes, and microsatellite instability (MSI). In total, 631 cancer patient plasma samples were evaluated by the test including lung, breast, colorectal, head and neck, pancreatic, gynecologic, and prostate cancers among other indications. Well-characterized reference samples (Genome in a Bottle) were utilized to assess analytical specificity. Contrived cell lines and clinical samples were utilized to establish analytical sensitivity, accuracy, as well as precision, reproducibility, and repeatability (PRR). Results: The test yielded results for 98.3% (620/631) of cases and identified variants in 94.7% (587/620) with a median of 6 variants per case. Clinically actionable variants were detected in 42.1% of clinical samples. Analytical specificity was ≥99.9999% for SNVs and 100% for indels, CNAs, translocations, and MSI. Analytical sensitivity (limit of detection, 95%) was verified for each variant type, with a median variant allele frequency (VAF) of 1.42% for SNVs and 1.43% for indels, 1.7-fold for amplifications, 0.35% fusion read fraction for translocations, and 0.82% VAF for MSI. The intra-laboratory PRR study resulted in 94.9% average percent agreement (APA) and 99.9% average negative agreement (ANA) for sequence variants (SNVs and indels) and 100% APA and ANA for CNAs, translocations, and MSI. Orthogonal assays including other NGS tests and digital PCR were utilized to assess Labcorp Plasma Complete accuracy which demonstrated an aggregate analytical concordance of 96.28% positive percent agreement and &gt;99.9999% negative percent agreement for all variants. Conclusions: The analytical validation of the Labcorp Plasma Complete test demonstrates that the liquid biopsy approach is highly sensitive, specific, accurate, reproducible, and robust for comprehensive genomic profiling to complement tissue-based testing and inform clinical decision making.