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The large-scale heterogeneity of genetic diseases necessitated the deeper examination of nucleotide sequence alterations enhancing the discovery of new targeted drug attack points. The appearance of new sequencing techniques was essential to get more interpretable genomic data. In contrast to the previous short-reads, longer lengths can provide a better insight into the potential health threatening genetic abnormalities. Long-reads offer more accurate variant identification and genome assembly methods, indicating advances in nucleotide deflect-related studies. In this review, we introduce the historical background of sequencing technologies and show their benefits and limits, as well. Furthermore, we highlight the differences between short- and long-read approaches, including their unique advances and difficulties in methodologies and evaluation. Additionally, we provide a detailed description of the corresponding bioinformatics and the current applications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Szakállas, Barták, Valcz, Nagy, Takács and Molnár.)

In recent years, the evolution of the molecular biological technical background led to the widespread application of single-cell sequencing, a versatile tool particularly useful in the investigation of tumor heterogeneity. Even 10 years ago the comprehensive characterization of colorectal cancers by The Cancer Genome Atlas was based on measurements of bulk samples. Nowadays, with single-cell approaches, tumor heterogeneity, the tumor microenvironment, and the interplay between tumor cells and their surroundings can be described in unprecedented detail. In this review article we aimed to emphasize the importance of single-cell analyses by presenting tumor heterogeneity and the limitations of conventional investigational approaches, followed by an overview of the whole single-cell analytic workflow from sample isolation to amplification, sequencing and bioinformatic analysis and a review of recent literature regarding the single-cell analysis of colorectal cancers., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kothalawala, Barták, Nagy, Zsigrai, Szigeti, Valcz, Takács, Kalmár and Molnár.)

Colorectal sessile serrated adenomas (SSA) are hypothesized to be precursor lesions of an alternative, serrated pathway of colorectal cancer, abundant in genes with aberrant promoter DNA hypermethylation. In our present pilot study, we explored DNA methylation profiles and examined selected gene mutations in SSA. Biopsy samples from patients undergoing screening colonoscopy were obtained during endoscopic examination. After DNA isolation and quality analysis, SSAs (n = 4) and healthy controls (n = 5) were chosen for further analysis. DNA methylation status of 96 candidate genes was screened by q(RT)PCR using Methyl-Profiler PCR array system. Amplicons for 12 gene mutations were sequenced by GS Junior Instrument using ligated and barcoded adaptors. Analysis of DNA methylation revealed 9 hypermethylated genes in both normal and SSA samples. 12 genes (CALCA, DKK2, GALR2, OPCML, PCDH10, SFRP1, SFRP2, SLIT3, SST, TAC1, VIM, WIF1) were hypermethylated in all SSAs and 2 additional genes (BNC1 and PDLIM4) were hypermethylated in 3 out of 4 SSAs, but in none of the normal samples. 2 SSAs exhibited BRAF mutation and synchronous MLH1 hypermethylation and were microsatellite instable by immunohistochemical analysis. Our combined mutation and DNA methylation analysis revealed that there is a common DNA methylation signature present in pre-neoplastic SSAs. This study advocates for the use of DNA methylation as a potential biomarker for the detection of SSA; however, further investigation is needed to better characterize the molecular background of these newly recognized colorectal lesions.

Our aim was to examine cell transition events by detecting the frequency of intrapithelial α-smooth muscle actin (SMA)(+)/cytokeratin (CK)(+) cells during colorectal adenoma-carcinoma sequence, in relation to E-cadherin expression. Our further aim was to determine the proliferative activity of intraepithelial α-SMA(+) cells. Histologically healthy, adenoma, and colorectal cancer (CRC) biopsy samples were taken during routine colonoscopy and were included into tissue microarrays (TMAs). Slides immunostained for Ki-67, α-SMA, E-cadherin and pan-cytokeratin were digitalized and analyzed by using a digital microscope software. The proportion of α-SMA(+)/CK(+) cells was significantly higher in CRC samples (3.34 ± 1.01%) compared to healthy (1.94 ± 0.69%) or adenoma (1.62 ± 0.78%) samples (p < 0.01). E-cadherin expression negatively correlated with the number of α-SMA(+) cells. The majority of intraepithelial α-SMA(+) cells were in the proliferative phase. During tumor progression, the appearance of dot-like α-SMA staining in CK positive cells may indicate the initial phase of the epithelial-to-mesenchymal transition (EMT). The high proportion of intraepithelial α-SMA(+) proliferating cells may refer to their increased plasticity compared to differentiated cells. The negative correlation between E-cadherin and intraepithelial α-SMA expression suggests that EMT is facilitated by a loss of epithelial cell contact.

Colorectal cancer is one of the leading death causes in the world. Specificity and sensitivity of the present screening methods are unsuitable and their compliance is too low. Nowadays the most effective method is the colonoscopy, because it gives not only macroscopic diagnosis but therapeutic possibility as well, however the compliance of the patients is very low. Hence development of new diagnostic methods is needed. Altered expression of septin 9 was found in several tumor types including colorectal cancer. The aim of this study was to detect the methylation related mRNA and protein expression changes of septin 9 in colorectal adenoma-dysplasia-carcinoma sequence and to analyze its reversibility by demethylation treatment. Septin 9 protein expression showed significant difference between normal and colorectal cancer (CRC) samples (p < 0,001). According to biopsy microarray results, septin 9 mRNA expression decreased in the progression of colon neoplastic disease (p < 0,001). In laser microdissected epithelial cells, septin 9 significantly underexpressed in CRC compared to healthy controls (p < 0,001). The expression of septin9&#95;v1 region was higher in the healthy samples, while septin9&#95;v2, v4, v4*, v5 overexpression were detected in cancer epithelial cells compared to normal. The septin 9 mRNA and protein levels of HT29 cells increased after demethylation treatment. The increasing methylation of septin 9 gene during colorectal adenoma-dysplasia-carcinoma sequence progression is reflected in the decreasing mRNA and protein expression, especially in the epithelium. These changes can be reversed by demethylation agents converting this screening marker gene into therapeutic target.

Bone marrow derived mesenchymal stem cells (BM-MSCs) take part in the colonic mucosal regeneration. They are multipotent cells, which can be identified with both negative (i.e. CD13, CD 14, CD45, c-Kit, major histocompatibility complex /MHC class I and II) and positive (i.e. CD54 (ICAM1), CD133, CD146 (MCAM), CD166, Flk-1, Sca-1, Thy-1, stage-specific antigen I /SSEA-I and Musashi-1, HLA class I) markers. These cells can repopulate the gastrointestinal mucosa as they may differentiate into stromal- (i.e. myofi-broblast) or epithelial-like (Paneth-, epithel-, goblet or enteroendocrin) cells without proliferation. During the mesenchymal to epithelial transition (MET) stem cells enter the epithelial layer and take up epithelial cell-like properties. Rarely BM-MSCs may retain their stem cell characteristics and are capable of producing progeny. The isolated lymphoid aggregates may serve as a platform from where BM-MSCs migrate to the nearby crypts as mediated by several chemoattractant proteins, which are expressed in injured tissue. The number of BM-MSCs is influenced by the degree of inflammation. In this review we summarize the current information about the role of BM-MSCs in the repair progress of injured colonic epithelium and their potential clinical applications.

Colorectal cancer progression is characterized by altered epithelial proliferation and apoptosis and by changed expression of tumor development regulators. Our aims were to determine the proliferative/apoptotic epithelial cell ratio (PAR) in the adenoma-dysplasia-carcinoma sequence (ADCS), and to examine its association with osteopontin (OPN), a previously identified protein product related to cancer development. One mm diameter cores from 13 healthy colons, 13 adenomas and 13 colon carcinoma samples were included into a tissue microarray (TMA) block. TUNEL reaction and Ki-67 immunohistochemistry were applied to determine the PAR. The osteopontin protein was also immunodetected. Stained slides were semiquantitatively evaluated using digital microscope and statistically analyzed with logistic regression and Fisher's exact test. The PAR continuously increased along the ADCS. It was significantly (p < 0.001) higher in cancer epithelium (8.84 ± 7.01) than in adenomas (1.40 ± 0.78) and in normal controls (0.89 ± 0.21) (p < 0.001). Also, significant positive correlation was observed between elevated PAR and the expression of osteopontin. Cytoplasmic OPN expression was weak in healthy samples. In contrast, cytoplasmic immunoreaction was moderately intensive in adenomas, while in colon cancer strong, diffuse cytoplasmic immune staining was detected. Increasing PAR and OPN expression along ADCS may help monitoring colorectal cancer progression. The significantly elevated OPN protein levels we found during normal epithelium to carcinoma progression may contribute to the increased fibroblast-myofibroblast transition determining stem cell niche in colorectal cancer.