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Genome Biology Jun 2024Neuroblastoma is a common pediatric cancer, where preclinical studies suggest that a mesenchymal-like gene expression program contributes to chemotherapy resistance....
BACKGROUND
Neuroblastoma is a common pediatric cancer, where preclinical studies suggest that a mesenchymal-like gene expression program contributes to chemotherapy resistance. However, clinical outcomes remain poor, implying we need a better understanding of the relationship between patient tumor heterogeneity and preclinical models.
RESULTS
Here, we generate single-cell RNA-seq maps of neuroblastoma cell lines, patient-derived xenograft models (PDX), and a genetically engineered mouse model (GEMM). We develop an unsupervised machine learning approach ("automatic consensus nonnegative matrix factorization" (acNMF)) to compare the gene expression programs found in preclinical models to a large cohort of patient tumors. We confirm a weakly expressed, mesenchymal-like program in otherwise adrenergic cancer cells in some pre-treated high-risk patient tumors, but this appears distinct from the presumptive drug-resistance mesenchymal programs evident in cell lines. Surprisingly, however, this weak-mesenchymal-like program is maintained in PDX and could be chemotherapy-induced in our GEMM after only 24 h, suggesting an uncharacterized therapy-escape mechanism.
CONCLUSIONS
Collectively, our findings improve the understanding of how neuroblastoma patient tumor heterogeneity is reflected in preclinical models, provides a comprehensive integrated resource, and a generalizable set of computational methodologies for the joint analysis of clinical and pre-clinical single-cell RNA-seq datasets.
Topics: Neuroblastoma; Humans; Animals; Single-Cell Analysis; Mice; RNA-Seq; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Drug Resistance, Neoplasm; Transcriptome; Single-Cell Gene Expression Analysis
PubMed: 38898465
DOI: 10.1186/s13059-024-03309-4 -
Scientific Reports Jun 2024This study probes the utility of biomarkers for microsatellite instability (MSI) detection and elucidates the molecular dynamics propelling colorectal cancer (CRC)...
This study probes the utility of biomarkers for microsatellite instability (MSI) detection and elucidates the molecular dynamics propelling colorectal cancer (CRC) progression. We synthesized a primer panel targeting 725 MSI loci, informed by The Cancer Genome Atlas (TCGA) and ancillary databases, to construct an amplicon library for next-generation sequencing (NGS). K-means clustering facilitated the distillation of 8 prime MSI loci, including activin A receptor type 2A (ACVR2A). Subsequently, we explored ACVR2A's influence on CRC advancement through in vivo tumor experiments and hematoxylin-eosin (HE) staining. Transwell assays gauged ACVR2A's role in CRC cell migration and invasion, while colony formation assays appraised cell proliferation. Western blotting illuminated the impact of ACVR2A suppression on CRC's PI3K/AKT/mTOR pathway protein expressions under hypoxia. Additionally, ACVR2A's influence on CRC-induced angiogenesis was quantified via angiogenesis assays. K-means clustering of NGS data pinpointed 32 MSI loci specific to tumor and DNA mismatch repair deficiency (dMMR) tissues. ACVR2A emerged as a pivotal biomarker, discerning MSI-H tissues with 90.97% sensitivity. A curated 8-loci set demonstrated 100% sensitivity and specificity for MSI-H detection in CRC. In vitro analyses corroborated ACVR2A's critical role, revealing its suppression of CRC proliferation, migration, and invasion. Moreover, ACVR2A inhibition under CRC-induced hypoxia markedly escalated MMP3, CyclinA, CyclinD1, and HIF1α protein expressions, alongside angiogenesis, by triggering the PI3K/AKT/mTOR cascade. The 8-loci ensemble stands as the optimal marker for MSI-H identification in CRC. ACVR2A, a central element within this group, deters CRC progression, while its suppression amplifies PI3K/AKT/mTOR signaling and angiogenesis under hypoxic stress.
Topics: Colorectal Neoplasms; Humans; Microsatellite Instability; Activin Receptors, Type II; Disease Progression; Animals; Cell Movement; Mice; Cell Line, Tumor; Cell Proliferation; Biomarkers, Tumor; Signal Transduction; Male; High-Throughput Nucleotide Sequencing; Female; Proto-Oncogene Proteins c-akt
PubMed: 38898042
DOI: 10.1038/s41598-024-62753-1 -
Nature Communications Jun 2024Single-cell RNA sequencing (scRNA-seq) has been widely used to characterize cell types based on their average gene expression profiles. However, most studies do not...
Single-cell RNA sequencing (scRNA-seq) has been widely used to characterize cell types based on their average gene expression profiles. However, most studies do not consider cell type-specific variation across donors. Modelling this cell type-specific inter-individual variation could help elucidate cell type-specific biology and inform genes and cell types underlying complex traits. We therefore develop a new model to detect and quantify cell type-specific variation across individuals called CTMM (Cell Type-specific linear Mixed Model). We use extensive simulations to show that CTMM is powerful and unbiased in realistic settings. We also derive calibrated tests for cell type-specific interindividual variation, which is challenging given the modest sample sizes in scRNA-seq. We apply CTMM to scRNA-seq data from human induced pluripotent stem cells to characterize the transcriptomic variation across donors as cells differentiate into endoderm. We find that almost 100% of transcriptome-wide variability between donors is differentiation stage-specific. CTMM also identifies individual genes with statistically significant stage-specific variability across samples, including 85 genes that do not have significant stage-specific mean expression. Finally, we extend CTMM to partition interindividual covariance between stages, which recapitulates the overall differentiation trajectory. Overall, CTMM is a powerful tool to illuminate cell type-specific biology in scRNA-seq.
Topics: Humans; Single-Cell Analysis; Sequence Analysis, RNA; Induced Pluripotent Stem Cells; Transcriptome; Cell Differentiation; Gene Expression Profiling; RNA-Seq; Endoderm
PubMed: 38898015
DOI: 10.1038/s41467-024-49242-9 -
Bioinformatics (Oxford, England) Jun 2024Ribosome profiling is a widely-used technique for measuring ribosome occupancy at nucleotide resolution. However, the need to analyze this data at nucleotide resolution...
MOTIVATION
Ribosome profiling is a widely-used technique for measuring ribosome occupancy at nucleotide resolution. However, the need to analyze this data at nucleotide resolution introduces unique challenges in data visualization and analyses.
RESULTS
In this study, we introduce RiboGraph, a dedicated visualization tool designed to work with .ribo files, a specialized and efficient format for ribosome occupancy data. Unlike existing solutions that rely on large alignment files and time-consuming preprocessing steps, RiboGraph operates on a purpose designed compact file type. This efficiency allows for interactive, real-time visualization at ribosome-protected fragment length resolution. By providing an integrated toolset, RiboGraph empowers researchers to conduct comprehensive visual analysis of ribosome occupancy data.
AVAILABILITY AND IMPLEMENTATION
Source code, step-by-step installation instructions and links to documentation are available on GitHub: https://github.com/ribosomeprofiling/ribograph. On the same page, we provide test files and a step-by-step tutorial highlighting the key features of RiboGraph.
Topics: Ribosomes; Software; Computational Biology; Ribosome Profiling
PubMed: 38897662
DOI: 10.1093/bioinformatics/btae369 -
Cancer Medicine Jun 2024Breast cancer is a heterogeneous disease categorized based on molecular characteristics, including hormone receptor (HR) and human epidermal growth factor receptor 2...
BACKGROUND
Breast cancer is a heterogeneous disease categorized based on molecular characteristics, including hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) expression levels. The emergence of profiling technology has revealed multiple driver genomic alterations within each breast cancer subtype, serving as biomarkers to predict treatment outcomes. This study aimed to explore the genomic landscape of breast cancer in the Taiwanese population through comprehensive genomic profiling (CGP) and identify diagnostic and predictive biomarkers.
METHODS
Targeted next-generation sequencing-based CGP was performed on 116 archived Taiwanese breast cancer specimens, assessing genomic alterations (GAs), including single nucleotide variants, copy number variants, fusion genes, tumor mutation burden (TMB), and microsatellite instability (MSI) status. Predictive variants for FDA-approved therapies were evaluated within each subtype.
RESULTS
In the cohort, frequent mutations included PIK3CA (39.7%), TP53 (36.2%), KMT2C (9.5%), GATA3 (8.6%), and SF3B1 (6.9%). All subtypes had low TMB, with no MSI-H tumors. Among HR + HER2- patients, 42% (27/65) harbored activating PIK3CA mutations, implying potential sensitivity to PI3K inhibitors and resistance to endocrine therapies. HR + HER2- patients exhibited intrinsic hormonal resistance via FGFR1 gene gain/amplification (15%), exclusive of PI3K/AKT pathway alterations. Aberrations in the PI3K/AKT/mTOR and FGFR pathways were implicated in chemoresistance, with a 52.9% involvement in triple-negative breast cancer. In HER2+ tumors, 50% harbored GAs potentially conferring resistance to anti-HER2 therapies, including PIK3CA mutations (32%), MAP3K1 (2.9%), NF1 (2.9%), and copy number gain/amplification of FGFR1 (18%), FGFR3 (2.9%), EGFR (2.9%), and AKT2 (2.9%).
CONCLUSION
This study presents CGP findings for treatment-naïve Taiwanese breast cancer, emphasizing its value in routine breast cancer management, disease classification, and treatment selection.
Topics: Humans; Female; Taiwan; Breast Neoplasms; Middle Aged; Mutation; Biomarkers, Tumor; Adult; Aged; High-Throughput Nucleotide Sequencing; DNA Copy Number Variations; Genomics; Class I Phosphatidylinositol 3-Kinases; Microsatellite Instability; Receptor, ErbB-2; Gene Expression Profiling
PubMed: 38895905
DOI: 10.1002/cam4.7384 -
Novel Transcriptional and DNA Methylation Abnormalities of SORT1 Gene in Non-Small Cell Lung Cancer.Cancers Jun 2024Sortilin is an important regulator with potential tumour-suppressor function by limiting EGFR signalling. In this study, we undertook a comprehensive expression analysis...
Sortilin is an important regulator with potential tumour-suppressor function by limiting EGFR signalling. In this study, we undertook a comprehensive expression analysis of sortilin transcript variants and the DNA methylation status of their corresponding promoters in human non-small cell carcinomas (NSCLCs). RNA/DNA was extracted from 81 NSCLC samples and paired normal tissue. mRNA expression was measured by qPCR and DNA methylation determined by pyrosequencing. BigDye-terminator sequencing was used to confirm exon-8 alternative splicing. Results demonstrated that both SORT1A and SORT1B variants were downregulated in lung tumours. The SORT1A/SORT1B expression ratio was higher in tumours compared to normal tissue. SORT1B promoter hypermethylation was detected in lung tumours compared to normal lung (median difference 14%, Mann-Whitney test = 10). Interestingly, SORT1B is hypermethylated in white blood cells, but a small and very consistent drop in methylation (6%, = 10) was observed in the lung cancer cases compared to control subjects. We demonstrate that the SORT1B exon-8 splice variation, reported in sequence databases, is also a feature of SORT1A. The significantly altered quantitative and qualitative characteristics of sortilin mRNA in NSCLC indicate a significant involvement in tumour pathogenesis and may have significant impact for its utility as a predictive marker in lung cancer management.
PubMed: 38893272
DOI: 10.3390/cancers16112154 -
Cancers Jun 2024: In cancer care, the MLH1 gene is crucial for DNA mismatch repair (MMR), serving as a vital tumor suppressor. Evaluating MLH1 protein expression status, followed by...
: In cancer care, the MLH1 gene is crucial for DNA mismatch repair (MMR), serving as a vital tumor suppressor. Evaluating MLH1 protein expression status, followed by analysis of MLH1 promoter methylation, has become a key diagnostic and prognostic approach. Our study investigates the complex link between MLH1 methylation and prognosis in endometrial adenocarcinoma (EA) patients. : MLH1 methylation status was accessed by a Pyrosequencing (PSQ) assay. Qualitative positivity for methylation was established if it exceeded the 11% cut-off; as well, a quantitative methylation analysis was conducted to establish correlations with clinicopathological data, relapse-free survival, and disease-free survival. Our study revealed that 33.3% of patients without MLH1 methylation experienced relapses, surpassing the 23.3% in patients with methylation. Furthermore, 16.7% of patients without methylation succumbed to death, with a slightly higher rate of 17.6% in methylated patients. Qualitative comparisons highlighted that the mean methylation rate in patients experiencing relapse was 35.8%, whereas in those without relapse, it was 42.2%. This pattern persisted in disease-specific survival (DSS), where deceased patients exhibited a higher mean methylation level of 49.1% compared to living patients with 38.8%. : Our findings emphasize the efficacy of PSQ for evaluating MLH1 methylation. While unmethylation appears to be associated with a higher relapse rate, the survival rate does not seem to be influenced by methylation. Quantitative percentages suggest that elevated MLH1 methylation is linked to relapse and mortality, though a study with a larger sample size would be essential for statistically significant results.
PubMed: 38893238
DOI: 10.3390/cancers16112119 -
International Journal of Molecular... Jun 2024For patients with hereditary breast and ovarian cancer, the probability of carrying two pathogenic variants (PVs) in dominant cancer-predisposing genes is rare. Using...
For patients with hereditary breast and ovarian cancer, the probability of carrying two pathogenic variants (PVs) in dominant cancer-predisposing genes is rare. Using targeted next-generation sequencing (NGS), we investigated a 49-year-old Caucasian woman who developed a highly aggressive breast tumor. Our analyses identified an intragenic germline heterozygous duplication in with an additional likely PV in the gene. The variant was confirmed by multiplex ligation probe amplification (MLPA), and genomic breakpoints were characterized at the nucleotide level (c.135-2578_442-1104dup). mRNA extracted from lymphocytes was amplified by RT-PCR and then Sanger sequenced, revealing a tandem duplication r.135_441dup; p.(Gln148Ilefs*20). This duplication results in the synthesis of a truncated and, most likely, nonfunctional protein. Following functional studies, the exon 5 c.472C > T; p.(Arg158Cys) missense variant was classified as likely pathogenic by the Li-Fraumeni Syndrome (LFS) working group. This type of unexpected association will be increasingly identified in the future, with the switch from targeted BRCA sequencing to hereditary breast and ovarian cancer (HBOC) panel sequencing, raising the question of how these patients should be managed. It is therefore important to record and investigate these rare double-heterozygous genotypes.
Topics: Humans; Female; Middle Aged; Tumor Suppressor Protein p53; BRCA1 Protein; Triple Negative Breast Neoplasms; Gene Duplication; Genetic Predisposition to Disease; Germ-Line Mutation; High-Throughput Nucleotide Sequencing
PubMed: 38892462
DOI: 10.3390/ijms25116274 -
International Journal of Molecular... Jun 2024RNA sequencing (RNA-Seq) is a powerful technique and is increasingly being used in clinical research and drug development. Currently, several RNA-Seq methods have been... (Comparative Study)
Comparative Study
RNA sequencing (RNA-Seq) is a powerful technique and is increasingly being used in clinical research and drug development. Currently, several RNA-Seq methods have been developed. However, the relative advantage of each method for degraded RNA and low-input RNA, such as RNA samples collected in the field of clinical setting, has remained unknown. The Standard method of RNA-Seq captures mRNA by poly(A) capturing using Oligo dT beads, which is not suitable for degraded RNA. Here, we used three commercially available RNA-Seq library preparation kits (SMART-Seq, xGen Broad-range, and RamDA-Seq) using random primer instead of Oligo dT beads. To evaluate the performance of these methods, we compared the correlation, the number of detected expressing genes, and the expression levels with the Standard RNA-Seq method. Although the performance of RamDA-Seq was similar to that of Standard RNA-Seq, the performance for low-input RNA and degraded RNA has decreased. The performance of SMART-Seq was better than xGen and RamDA-Seq in low-input RNA and degraded RNA. Furthermore, the depletion of ribosomal RNA (rRNA) improved the performance of SMART-Seq and xGen due to increased expression levels. SMART-Seq with rRNA depletion has relative advantages for RNA-Seq using low-input and degraded RNA.
Topics: Sequence Analysis, RNA; Humans; RNA Stability; Gene Expression Profiling; High-Throughput Nucleotide Sequencing; RNA; RNA, Ribosomal; RNA, Messenger; RNA-Seq
PubMed: 38892331
DOI: 10.3390/ijms25116143 -
International Journal of Molecular... Jun 2024The microbiome of the ocular surface has been characterised, but only limited information is available on a possible silent intraocular microbial colonisation in normal...
The microbiome of the ocular surface has been characterised, but only limited information is available on a possible silent intraocular microbial colonisation in normal eyes. Therefore, we performed next-generation sequencing (NGS) of 16S rDNA genes in the aqueous humour. The aqueous humour was sampled from three patients during cataract surgery. Air swabs, conjunctival swabs from patients as well as from healthy donors served as controls. Following DNA extraction, the V3 and V4 hypervariable regions of the 16S rDNA gene were amplified and sequenced followed by denoising. The resulting Amplicon Sequence Variants were matched to a subset of the Ribosomal Database Project 16S database. The deduced bacterial community was then statistically analysed. The DNA content in all samples was low (0-1.49 ng/µL) but sufficient for analysis. The main phyla in the samples were (48%), (26%), (14%), (8%), and (2%). Patients' conjunctival control samples and anterior chamber fluid showed similar patterns of bacterial species containing many waterborne species. Non-disinfected samples showed a different bacterial spectrum than the air swab samples. The data confirm the existence of an ocular surface microbiome. Meanwhile, a distinct intraocular microbiome was not discernible from the background, suggesting the absence of an intraocular microbiome in normal eyes.
Topics: Humans; Aqueous Humor; Microbiota; High-Throughput Nucleotide Sequencing; RNA, Ribosomal, 16S; Bacteria; DNA, Bacterial; Male; Female; Aged; Middle Aged; Sequence Analysis, DNA
PubMed: 38892316
DOI: 10.3390/ijms25116128