Clinically relevant morphological structures in breast cancer represent transcriptionally distinct tumor cell populations with varied degrees of epithelial-mesenchymal transition and CD44+CD24- stemness

Intratumor morphological heterogeneity in breast cancer is represented by different morphological structures (tubular, alveolar, solid, trabecular, and discrete) and contributes to poor prognosis; however, the mechanisms involved remain unclear. In this study, we performed 3D imaging, laser microdissection-assisted array comparative genomic hybridization and gene expression microarray analysis of different morphological structures and examined their association with the standard immunohistochemistry scorings and CD44+CD24- cancer stem cells. We found that the intratumor morphological heterogeneity is not associated with chromosomal aberrations. By contrast, morphological structures were characterized by specific gene expression profiles and signaling pathways and significantly differed in progesterone receptor and Ki-67 expression. Most importantly, we observed significant differences between structures in the number of expressed genes of the epithelial and mesenchymal phenotypes and the association with cancer invasion pathways. Tubular (tube-shaped) and alveolar (spheroid-shaped) structures were transcriptionally similar and demonstrated co-expression of epithelial and mesenchymal markers. Solid (large shapeless) structures retained epithelial features but demonstrated an increase in mesenchymal traits and collective cell migration hallmarks. Mesenchymal genes and cancer invasion pathways, as well as Ki-67 expression, were enriched in trabecular (one/two rows of tumor cells) and discrete groups (single cells and/or arrangements of 2-5 cells). Surprisingly, the number of CD44+CD24- cells was found to be the lowest in discrete groups and the highest in alveolar and solid structures. Overall, our findings indicate the association of intratumor morphological heterogeneity in breast cancer with the epithelial-mesenchymal transition and CD44+CD24- stemness and the appeal of this heterogeneity as a model for the study of cancer invasion

Application of Long-Read Nanopore Sequencing to the Search for Mutations in Hypertrophic Cardiomyopathy

Increasing evidence suggests that both coding and non-coding regions of sarcomeric protein genes can contribute to hypertrophic cardiomyopathy (HCM). Here, we introduce an experimental workflow (tested on four patients) for complete sequencing of the most common HCM genes (MYBPC3, MYH7, TPM1, TNNT2, and TNNI3) via long-range PCR, Oxford Nanopore Technology (ONT) sequencing, and bioinformatic analysis. We applied Illumina and Sanger sequencing to validate the results, FastQC, Qualimap, and MultiQC for quality evaluations, MiniMap2 to align data, Clair3 to call and phase variants, and Annovar’s tools and CADD to assess pathogenicity of variants. We could not amplify the region encompassing exons 6–12 of MYBPC3. A higher sequencing error rate was observed with ONT (6.86–6.92%) than with Illumina technology (1.14–1.35%), mostly for small indels. Pathogenic variant p.Gln1233Ter and benign polymorphism p.Arg326Gln in MYBPC3 in a heterozygous state were found in one patient. We demonstrated the ability of ONT to phase single-nucleotide variants, enabling direct haplotype determination for genes TNNT2 and TPM1. These findings highlight the importance of long-range PCR efficiency, as well as lower accuracy of variant calling by ONT than by Illumina technology; these differences should be clarified prior to clinical application of the ONT method

A Comparison of Genome-Wide DNA Methylation Patterns between Different Vascular Tissues from Patients with Coronary Heart Disease

<div><p>Epigenetic mechanisms of gene regulation in context of cardiovascular diseases are of considerable interest. So far, our current knowledge of the DNA methylation profiles for atherosclerosis affected and healthy human vascular tissues is still limited. Using the Illumina Infinium Human Methylation27 BeadChip, we performed a genome-wide analysis of DNA methylation in right coronary artery in the area of advanced atherosclerotic plaques, atherosclerotic-resistant internal mammary arteries, and great saphenous veins obtained from same patients with coronary heart disease. The resulting DNA methylation patterns were markedly different between all the vascular tissues. The genes hypomethylated in athero-prone arteries to compare with atherosclerotic-resistant arteries were predominately involved in regulation of inflammation and immune processes, as well as development. The great saphenous veins exhibited an increase of the DNA methylation age in comparison to the internal mammary arteries. Gene ontology analysis for genes harboring hypermethylated CpG-sites in veins revealed the enrichment for biological processes associated with the development. Four CpG-sites located within the <i>MIR10B</i> gene sequence and about 1 kb upstream of the <i>HOXD4</i> gene were also confirmed as hypomethylated in the independent dataset of the right coronary arteries in the area of advanced atherosclerotic plaques in comparison with the other vascular tissues. The DNA methylation differences observed in vascular tissues of patients with coronary heart disease can provide new insights into the mechanisms underlying the development of pathology and explanation for the difference in graft patency after coronary artery bypass grafting surgery.</p></div

Flow chart describing experiment and analysis.

<p>Flow chart describing experiment and analysis.</p

Volcano plot of - log10 (P-value) against delta beta value, representing the methylation difference between right coronary arteries in the area of advanced atherosclerotic plaques (CAP) and internal mammary arteries (IMA).

<p>A total of 194 CpG-sites hypermethylated in CAP with a delta beta ≥ 0.2 and FDR-adjusted p<0.05 are shown in red. A total of 164 CpG-sites hypomethylated in CAP with a delta beta ≤ -0.2 and FDR-adjusted p<0.05 are shown in blue. CpG-sites that exhibited a methylation level difference less 20% are shown in grey. Light red and light blue colors indicate highly differentially methylated CpG-sites (delta beta ≥ 0.40 or delta beta ≤ -0.40 with FDR-adjusted p<0.05). Dashed lines indicate cut-offs for significance.</p

Methylation level (mean ±SD) at the promoter of <i>HOXD4</i> gene in paired vascular tissues from twenty one patients with atherosclerosis.

<p>CAP indicates right coronary arteries in the area of advanced atherosclerotic plaques; IMA, internal mammary arteries; GSV, great saphenous veins.</p><p>Methylation level (mean ±SD) at the promoter of <i>HOXD4</i> gene in paired vascular tissues from twenty one patients with atherosclerosis.</p

Volcano plot of - log10 (P-value) against delta beta value, representing the methylation difference between great saphenous veins (GSV) and internal mammary arteries (IMA).

<p>A total of 200 CpG-sites hypermethylated in GSV with a delta beta ≥ 0.2 and FDR-adjusted p<0.05 are shown in red. A total of 135 CpG-sites hypomethylated in GSV with a delta beta ≤ -0.2 and FDR-adjusted p<0.05 are shown in blue. CpG-sites that exhibited a methylation level difference less 20% are shown in grey. Light red and light blue colors indicate highly differentially methylated CpG-sites (delta beta ≥ 0.40 or delta beta ≤ -0.40 with FDR-adjusted p<0.05). Dashed lines indicate cut-offs for significance.</p

Heatmap analysis of 22 highly differentially methylated CpG-sites (delta beta ≥ 0.40 or delta beta ≤ -0.40 with FDR-adjusted p<0.05) between right coronary arteries in the area of advanced atherosclerotic plaques (CAP) and internal mammary arteries (IMA).

<p>Regions shaded blue in the heat map represent hypomethylated regions, regions shaded red represent hypermethylated regions. The top black rectangles shows columns representing CpG-sites located inside CpG-island. Gene symbols and CpG-site IDs are shown on the bottom. Sample IDs are on the right.</p

Comparison of β-values (average ± s.d.) in paired vascular tissues from six patients with atherosclerosis.

<p>CAP indicates right coronary arteries in the area of advanced atherosclerotic plaques; IMA, internal mammary arteries; GSV, great saphenous veins.</p><p>Comparison of β-values (average ± s.d.) in paired vascular tissues from six patients with atherosclerosis.</p