The feasibility of population screening for colorectal cancer has been demonstrated in several studies. Most of these studies have considered individual characteristics, diagnostic approaches, epidemiological data, and socioeconomic factors. In this article, we comment on an editorial by Metaxas et al published in the recent issue of the journal. The authors emphasized the need to raise public awareness through health education programs and the possibility of using easily accessible non-invasive screening methods. Here, we focus on non-invasive molecular genetic approaches that can aid in colorectal cancer screening. On the one hand, we highlighted the use of tumor DNA/RNA markers directly for screening and, on the other hand, underline the use of polygenic risk assessment and hereditary predisposition to select individuals for more thorough cancer screening.

The dynamic interaction between genomic DNA, epigenetic modifications, and phenotypic traits was examined in identical twins. Environmental perturbations can induce epigenetic changes in DNA methylation, influencing gene expression and phenotypes. Although DNA methylation mediates gene-environment correlations, the quantitative effects of external factors on DNA methylation remain underexplored. This study aimed to quantify these effects using a novel approach.

A cohort study was conducted on healthy monozygotic twins to evaluate the influence of environmental stimuli on DNA methylation. We developed the Environmental Factor Index (EFI) to identify methylation sites showing statistically significant changes in response to environmental stimuli. We analyzed the identified sites for associations with disorders, DNA methylation markers, and CpG islands.

The EFI identified methylation sites that exhibited significant associations with genes linked to various disorders, particularly cancer. These sites were overrepresented on CpG islands compared to other genomic features, highlighting their regulatory importance.

The EFI is a valuable tool for understanding the molecular mechanisms underlying disease pathogenesis. It provides insights into the development of preventive and therapeutic strategies and offers a new perspective on the role of environmental factors in epigenetic regulation.

Since the mid-1970s, increasingly innovative methods to detect DNA methylation provided detailed information about its distribution, functions, and dynamics. As a result, new concepts were formulated and older ones were revised, transforming our understanding of the associated biology and catalyzing unprecedented advances in biomedical research, drug development, anthropology, and evolutionary biology. In this review, we discuss a few of the most notable advances, which are intimately intertwined with the study of DNA methylation, with a particular emphasis on the past three decades. Examples of these strides include elucidating the intricacies of 5-methylcytosine (5-mC) oxidation, which are at the core of the reversibility of this epigenetic modification; the three-dimensional structural characterization of eukaryotic DNA methyltransferases, which offered insights into the mechanisms that explain several disease-associated mutations; a more in-depth understanding of DNA methylation in development and disease; the possibility to learn about the biology of extinct species; the development of epigenetic clocks and their use to interrogate aging and disease; and the emergence of epigenetic biomarkers and therapies.

To explore the medical significance of methylated septin9 (mSEPT9) in monitoring recurrence and prognostic assessment in individuals with surgically treated colorectal cancer (CRC).

To investigate the role of Septin9 in colorectal cancer, we utilized the TIMER2.0 database to analyze its differential expression between tumor tissues and adjacent normal tissues. Colorectal cancer RNA-seq data from the TCGA database was downloaded and curated. The clinical relevance of mSEPT9 in colorectal cancer was explored by examining the correlation between Septin9 methylation levels and clinical characteristics using UALCAN and MethSurv software. Peripheral blood samples were obtained from 130 CRC subjects who underwent surgery for the detection of mSEPT9 and carcinoembryonic antigen (CEA) expression, along with collection of clinicopathological data such as age, gender, tumor site, TNM stage, and tumor differentiation. Patients were followed up for at least 3 years post-surgery until the death or final follow-up dates (31/12/2022). Additionally, peripheral blood samples were collected from 30 colorectal cancer surgery patients for mSEPT9 detection before and 7 days after surgery.

Through bioinformatic database analysis, we identified higher expression levels of SEPT9 mRNA in most tumor tissues compared to normal tissues. Similarly, both paired and unpaired CRC tissues exhibited elevated expression of Septin9 when compared to normal tissues. Following GO and KEGG analysis of Septin9 target genes, we discovered their significant associations with ncRNA metabolic processes, ribonucleoprotein complex biogenesis, spliceosomes, and viral carcinogenesis. Furthermore, the overexpression of mSeptin9 was observed in CRC tissues, and it demonstrated a correlation with colon cancer staging and histologic classification. In our clinical sample study, The positive rate of mSEPT9 in CRC patients 7 days after surgery was 43.44% lower than that of preoperative. The differences in TNM stage, tumor differentiation degree, and preoperative CEA expression level between the preoperative mSEPT9 positive and negative groups of CRC were statistically significant (P < 0.05). Recurrence free survival (RFS) and overall survival (OS) were shorter in the preoperative mSEPT9-positive group, meaning preoperative mSEPT9 status was a risk factor for CRC recurrence and prognosis (P < 0.05). The sensitivity, specificity, and AUC value of preoperative mSEPT9 and CEA levels for predicting postoperative recurrence in CRC patients were 88% vs. 72%, 56.19% vs. 55.24%, and 0.721 vs. 0.636 respectively, well the AUC value of the combined prediction of postoperative recurrence was 0.758.

The detection of mSEPT9 combined with CEA in preoperative plasma helps predict recurrence in colorectal cancer patients.

Numerous studies have demonstrated a link between epigenetics and CRC. However, there has been no systematic analysis or visualization of relevant publications using bibliometrics.

839 publications obtained from the Web of Science Core (WoSCC) were systematically analyzed using CiteSpace and VOSviewer software.

The results show that the countries, institutions, and authors with the most published articles are the United States, Harvard University, and Ogino and Shuji, respectively. SEPT9 is a blood test for the early detection of colorectal cancer. Vitamin D and gut microbiota mediate colorectal cancer and epigenetics, and probiotics may reduce colorectal cancer-related symptoms. We summarize the specific epigenetic mechanisms of CRC and the current existence and potential epigenetic drugs associated with these mechanisms. It is closely integrated with clinical practice, and the possible research directions and challenges in the future are proposed.

This study reviews the current research trends and hotspots in CRC and epigenetics, which can promote the development of this field and provide references for researchers in this field.

Human life expectancy is significantly impacted by cancer, with liquid biopsy emerging as an advantageous method for cancer detection because of its noninvasive nature, high accuracy, ease of sampling, and cost-effectiveness compared with conventional tissue biopsy techniques. Liquid biopsy shows promise in early cancer detection, real-time monitoring, and personalized treatment for various cancers, including lung, cervical, and prostate cancers, and offers innovative approaches for cancer diagnosis and management. By utilizing circulating tumor DNA, circulating tumor cells, and exosomes as biomarkers, liquid biopsy enables the tracking of cancer progression. Various techniques commonly used in life sciences research, such as polymerase chain reaction (PCR), next-generation sequencing (NGS), and droplet digital PCR, are employed to assess cancer progression on the basis of different indicators. This review examines the latest advancements in liquid biopsy markers-circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), and exosomes-for cancer diagnosis over the past three years, with a focus on their detection methodologies and clinical applications. It encapsulates the pivotal aims of liquid biopsy, including early detection, therapy response prediction, treatment monitoring, prognostication, and its relevance in minimal residual disease, while also addressing the challenges facing routine clinical adoption. By combining the latest research advancements and practical clinical experiences, this work focuses on discussing the clinical significance of DNA methylation biomarkers and their applications in tumor screening, auxiliary diagnosis, companion diagnosis, and recurrence monitoring. These discussions may help enhance the application of liquid biopsy throughout the entire process of tumor diagnosis and treatment, thereby providing patients with more precise and effective treatment plans.

Colorectal cancer (CRC) has the highest mortality rate among men and is the second highest among women under fifty, with incidence and mortality rates rising in younger populations. Studies indicate that up to one-third of patients diagnosed before fifty have a family history or genetic factors, highlighting the need for earlier screening. Contrariwise, diagnosis in healthy subjects through screening strategies enables early-stage detection of the tumor and better clinical outcomes. In recent years, mortality rates of CRC in Western countries have been on a steady decline, which is largely attributed to widespread screening programs and advancements in treatment modalities. Indeed, early detection through screening significantly improves prognosis, with stark differences in survival rates between localized and metastatic disease. This article aims to provide a comprehensive review of the existing literature, delving into the performance and efficacy of various CRC screening strategies. It navigates through available screening tools, evaluating their efficacy and cost-effectiveness. The discussion extends to delineating target populations for screening, emphasizing the importance of tailored approaches for individuals at heightened risk.

Cancer continues to be one the leading causes of death worldwide, primarily due to the late detection of the disease. Cancers detected at early stages may enable more effective intervention of the disease. However, most cancers lack well-established screening procedures except for cancers with an established early asymptomatic phase and clinically validated screening tests. There is a critical need to identify and develop assays/tools in conjunction with imaging approaches for precise screening and detection of the aggressive disease at an early stage. New developments in molecular cancer screening and early detection include germline testing, synthetic biomarkers, and liquid biopsy approaches.

(1) Background: The detection of methylated SEPT9 (mSEPT9) in plasma is a promising approach to non-invasive colorectal cancer (CRC) screening. Traditional approaches have limitations in sensitivity and cost-effectiveness, particularly in resource-limited settings. (2) Methods: We developed a semi-nested realtime PCR assay utilizing extendable blocking probes (ExBP) to enhance the detection of low-level mSEPT9 based on DNA melting. This assay allows for the discrimination of mSEPT9 in the presence of high concentrations of non-methylated SEPT9 (up to 100,000 times higher). (3) Results: The assay demonstrated a sensitivity of 73.91% and specificity of 80%, showcasing its ability to detect very low levels of methylated DNA effectively. The innovative use of ExBP without costly modified probes simplifies the assay setup and reduces the overall costs, enhancing its applicability in diverse clinical settings. (4) Conclusions: This novel assay significantly improves the detection of mSEPT9, offering a potential advance in CRC screening and monitoring. Its cost-efficiency and high sensitivity make it particularly suitable for the early detection and management of CRC, especially in settings with limited resources. Future studies are encouraged to validate this assay in larger populations to establish its clinical benefits and practical utility.

Methylated circulating tumour DNA (ctDNA) blood tests for BCAT1/IKZF1 (COLVERA) and SEPT9 (Epi proColon) are used to detect colorectal cancer (CRC). However, there are no ctDNA assays approved for other gastrointestinal adenocarcinomas. We aimed to characterize BCAT1, IKZF1 and SEPT9 methylation in different gastrointestinal adenocarcinoma and non-gastrointestinal tumours to determine if these validated CRC biomarkers might be useful for pan-gastrointestinal adenocarcinoma detection.

Tissue DNA methylation data from colorectal (COAD, READ), gastroesophageal (ESCA, STAD), pancreatic (PAAD) and cholangiocarcinoma (CHOL) adenocarcinoma cohorts within The Cancer Genome Atlas were used for differential methylation analyses. Clinicodemographic predictors of BCAT1, IKZF1 and SEPT9 methylation, and the selectivity of hypermethylated BCAT1, IKZF1 and SEPT9 for colorectal adenocarcinomas in comparison to other cancers were each explored with beta regression.

Hypermethylated BCAT1, IKZF1 and SEPT9 were each differentially methylated in colorectal and gastroesophageal adenocarcinomas. IKZF1 was differentially methylated in pancreatic adenocarcinoma. Hypermethylated DNA biomarkers BCAT1, IKZF1 and SEPT9 were largely stable across different stages of disease and were highly selective for gastrointestinal adenocarcinomas relative to other cancer types.

Existing CRC methylated ctDNA blood tests for BCAT1/IKZF1 and SEPT9 might be usefully repurposed for use in other gastrointestinal adenocarcinomas and warrant further prospective ctDNA studies.

Epigenetic changes are heritable changes in gene expression without changes in the nucleotide sequence of genes. Epigenetic changes play an important role in the development of cancer and in the process of malignancy metastasis. Previous studies have shown that abnormal epigenetic changes can be used as biomarkers for disease status and disease prediction. The reversibility and controllability of epigenetic modification changes also provide new strategies for early disease prevention and treatment. In addition, corresponding drug development has also reached the clinical stage. In this paper, we will discuss the recent progress and application status of tumor epigenetic biomarkers from three perspectives: DNA methylation, non-coding RNA, and histone modification, in order to provide new opportunities for additional tumor research and applications.

Colorectal cancer (CRC) ranks third in cancer incidence and stands as the second leading cause of cancer-related deaths globally. CRC tumorigenesis results from a cumulative set of genetic and epigenetic alterations, disrupting cancer-regulatory processes like cell proliferation, metabolism, angiogenesis, cell death, invasion, and metastasis. Key epigenetic modifications observed in cancers encompass abnormal DNA methylation, atypical histone modifications, and irregularities in noncoding RNAs, such as microRNAs and long noncoding RNAs. The advancement in genomic technologies has positioned these genetic and epigenetic shifts as potential clinical biomarkers for CRC patients. This review concisely covers the fundamental principles of CRC-associated epigenetic changes, and examines in detail their emerging role as biomarkers for early detection, prognosis, and treatment response prediction.

Gene therapy has revolutionized the treatment of genetic diseases. Spearheading this revolution are sophisticated genome editing methods such as TALENs, ZFNs, and CRISPR-Cas, which trace their origins back to prokaryotic immune systems. Prokaryotes have developed various antiviral defense systems to combat viral attacks and the invasion of genetic elements. The comprehension of these defense mechanisms has paved the way for the development of indispensable tools in molecular biology. Among them, restriction endonuclease originates from the innate immune system of bacteria. The CRISPR-Cas system, a widely applied genome editing technology, is derived from the prokaryotic adaptive immune system. Single-base editing is a precise editing tool based on CRISPR-Cas system that involves deamination of target base. It is worth noting that prokaryotes possess deamination enzymes as part of their defense arsenal over foreign genetic material. Furthermore, prokaryotic Argonauts (pAgo) proteins, also function in anti-phage defense, play an important role in complementing the CRISPR-Cas system by addressing certain limitations it may have. Recent studies have also shed light on the significance of Retron, a reverse transcription transposon previously showed potential in genome editing, has also come to light in the realm of prokaryotic immunity. These noteworthy findings highlight the importance of studying prokaryotic immune system for advancing genome editing techniques. Here, both the origin of prokaryotic immunity underlying aforementioned genome editing tools, and potential applications of deaminase, pAgo protein and reverse transcriptase in genome editing among prokaryotes were introduced, thus emphasizing the fundamental mechanism and significance of prokaryotic immunity.

Colorectal cancer (CRC) is a multifaceted disease influenced by the interplay of genetic and environmental factors. The clinical heterogeneity of CRC cannot be attributed exclusively to genetic diversity and environmental exposures, and epigenetic markers, especially DNA methylation, play a critical role as key molecular markers of cancer. This review compiles a comprehensive body of evidence underscoring the significant involvement of DNA methylation modifications in the pathogenesis of CRC. Moreover, this review explores the potential utility of DNA methylation in cancer diagnosis, prognostics, assessment of disease activity, and prediction of drug responses. Recognizing the impact of DNA methylation will enhance the ability to identify distinct CRC subtypes, paving the way for personalized treatment strategies and advancing precision medicine in the management of CRC.

Colorectal cancer (CRC) is one of the most common malignancies, and early detection plays a crucial role in enhancing curative outcomes. While colonoscopy is considered the gold standard for CRC diagnosis, noninvasive screening methods of DNA methylation biomarkers can improve the early detection of CRC and precancerous lesions.

Bioinformatics and machine learning methods were used to evaluate CRC-related genes within the TCGA database. By identifying the overlapped genes, potential biomarkers were selected for further validation. Methylation-specific PCR (MSP) was utilized to identify the associated genes as biomarkers. Subsequently, a real-time PCR assay for detecting the presence of neoplasia or cancer of the colon or rectum was established. This screening approach involved the recruitment of 978 participants from five cohorts.

The genes with the highest specificity and sensitivity were Septin9, AXL4, and SDC2. A total of 940 participants were involved in the establishment of the final PCR system and the subsequent performance evaluation test. A multiplex TaqMan real-time PCR system has been illustrated to greatly enhance the ability to detect precancerous lesions and achieved an accuracy of 87.8% (95% CI 82.9-91.5), a sensitivity of 82.7% (95% CI 71.8-90.1), and a specificity of 90.1% (95% CI 84.3-93.9). Moreover, the detection rate of precancerous lesions of this assay reached 55.0% (95% CI 38.7-70.4).

The combined detection of the methylation status of SEPT9, SDC2, and ALX4 in plasma holds the potential to further enhance the sensitivity of CRC detection.

An effective blood-based method for the diagnosis of colorectal cancer has not yet been developed. Molecular alterations of immune cells occur early in tumorigenesis, providing the theoretical underpinning for early cancer diagnosis based on immune cell profiling. Therefore, we aimed to develop an effective detection method based on peripheral blood mononuclear cells (PBMC) to improve the diagnosis of colorectal cancer. Analysis of the genome-wide methylation landscape of PBMCs from patients with colorectal cancer and healthy controls by microarray, pyrosequencing, and targeted bisulfite sequencing revealed five DNA methylation markers for colorectal cancer diagnosis, especially early-stage colorectal cancer. A single-tube multiple methylation-specific quantitative PCR assay (multi-msqPCR) for simultaneous detection of five methylation markers was established, which allowed quantitative analysis of samples with as little as 0.1% PBMC DNA and had better discriminative performance than single-molecule detection. Then, a colorectal cancer diagnostic model (CDM) based on methylation markers and the multi-msqPCR method was constructed that achieved high accuracy for early-stage colorectal cancer (AUC = 0.91; sensitivity = 81.18%; specificity = 89.39%), which was improved compared with CEA (AUC = 0.79). The CDM also enabled a high degree of discrimination for advanced adenoma cases (AUC = 0.85; sensitivity = 63.04%). Follow-up data also demonstrated that the CDM could identify colorectal cancer potential up to 2 years before currently used diagnostic methods. In conclusion, the approach constructed in this study based on PBMC-derived DNA methylation markers and a multi-msqPCR method is a promising and easily implementable diagnostic method for early-stage colorectal cancer.

Development of a diagnostic model for early colorectal cancer based on epigenetic analysis of PBMCs supports the utility of altered DNA methylation in immune cells for cancer diagnosis.

Colorectal cancer (CRC) signifies a significant healthcare challenge in Southeast Asia. Despite advancements in screening approaches and treatment modalities, significant medical gaps remain, ranging from prevention and early diagnosis to determining targeted therapy and establishing personalized approaches to managing CRC. There is a need to expand more validated biomarkers in clinical practice. An advanced technique incorporating high-throughput mass spectrometry as a liquid biopsy to unravel a repertoire of glycoproteins and glycans would potentially drive the development of clinical tools for CRC screening, diagnosis and monitoring, and it can be further adapted to the existing standard-of-care procedure. Therefore this review offers a perspective on glycoproteomics-driven liquid biopsy and its potential integration into the clinical care of CRC in the southeast Asia region.

The generally accepted method of quantifying hypermethylated DNA by qPCR using methylation-specific primers has the risk of underestimating DNA methylation and requires data normalization. This makes the analysis complicated and less reliable.

The end-point PCR method, called qDMA-HP (for quantitative DNA Melting Analysis with hybridization probes), which excludes the normalization procedure, is multiplexed and quantitative, has been proposed. qDMA-HP is characterized by the following features: (i) asymmetric PCR with methylation-independent primers; (ii) fluorescent dual-labeled, self-quenched probes (commonly known as TaqMan probes) covering several interrogated CpGs; (iii) post-PCR melting analysis of amplicon/probe hybrids; (iv) quantitation of unmethylated and methylated DNA alleles by measuring the areas under the corresponding melt peaks.

qDMA-HP was tested in liquid biopsy of colorectal cancer by evaluating SEPT9 and HIST1H4F methylations simultaneously in the single-tube reaction. Differences in the methylation levels in healthy donors versus cancer patients were statistically significant (p < 0.0001), AUCROC values were 0.795-0.921 for various marker combinations.

This proof-of-concept study shows that qDMA-HP is a simple, normalization-independent, quantitative, multiplex and "closed tube" method easily adapted to clinical settings. It is demonstrated, for the first time, that HIST1H4F is a perspective marker for liquid biopsy of colorectal cancer.

Over the past two decades, liquid biopsy has been increasingly used as a supplement, or even, a replacement to the traditional biopsy in clinical oncological practice, due to its noninvasive and early detectable properties. The detections can be based on a variety of features extracted from tumor‑derived entities, such as quantitative alterations, genetic changes, and epigenetic aberrations, and so on. So far, the clinical applications of cancer liquid biopsy mainly aimed at two aspects, prediction (early diagnosis, prognosis and recurrent evaluation, therapeutic response monitoring, etc.) and intervention. In spite of the rapid development and great contributions achieved, cancer liquid biopsy is still a field under investigation and deserves more clinical practice. To better open up future work, here we systematically reviewed and compared the latest progress of the most widely recognized circulating components, including circulating tumor cells, cell-free circulating DNA, noncoding RNA, and nucleosomes, from their discovery histories to clinical values. According to the features applied, we particularly divided the contents into two parts, beyond epigenetics and epigenetic-based. The latter was considered as the highlight along with a brief overview of the advances in both experimental and bioinformatic approaches, due to its unique advantages and relatively lack of documentation.

Colorectal cancer (CRC) screening can help to reduce its incidence and mortality. Noninvasive strategies, such as plasma analysis of epigenetic alterations, can constitute important biomarkers of CRC detection.

This study aimed to evaluate the plasma methylation status of SEPT9 and BMP3 promoters as biomarkers for detection of CRC and its precursor lesions in a Brazilian population.

Plasma samples from 262 participants of the CRC screening program of Barretos Cancer Hospital who had a positive fecal occult blood test and underwent colonoscopy and cancer patients were analyzed. Participants were grouped according to the worst lesion detected in the colonoscopy. Cell-free circulating DNA (cfDNA) was bisulfite treated followed by the analysis of SEPT9 and BMP3 methylation status using a droplet digital PCR system (ddPCR). The best methylation cutoff value for group discrimination was calculated by receiver operating characteristic (ROC) curve analysis.

Among the 262 participants, 38 were diagnosed with CRC, 46 with advanced adenomas 119 with nonadvanced adenomas, three with sessile serrated lesions, and 13 with hyperplastic polyps. In 43 participants, no lesion was detected in the colonoscopy and were used as controls. The CRC group showed the highest cfDNA concentration (10.4 ng/mL). For the SEPT9 gene, a cutoff of 2.5% (AUC = 0.681) that discriminates between CRC and the control group resulted in CRC sensitivity and specificity of 50% and 90%, respectively. Concerning the BMP3 gene, a cutoff of 2.3% (AUC = 0.576) showed 40% and 90% of sensitivity and specificity for CRC detection, respectively. Combining SEPT9, BMP3 status, and age over 60 years resulted in a better performance for detecting CRC (AUC = 0.845) than the individual gene models, yielding 80% and 81% of sensitivity and specificity, respectively.

The present study suggests that a combination of SEPT9 and BMP3 plasma methylation, along with age over 60 years, showed the highest performance in detecting CRC in a Brazilian population. These noninvasive biomarkers can potentially serve as useful tools for CRC screening programs.

Anti-angiogenic therapy has long been considered a promising strategy for solid cancers. Intrinsic resistance to hypoxia is a major cause for the failure of anti-angiogenic therapy, but the underlying mechanism remains unclear. Here, it is revealed that N4-acetylcytidine (ac4C), a newly identified mRNA modification, enhances hypoxia tolerance in gastric cancer (GC) cells by promoting glycolysis addiction. Specifically, acetyltransferase NAT10 transcription is regulated by HIF-1α, a key transcription factor of the cellular response to hypoxia. Further, acRIP-sequencing, Ribosome profiling sequencing, RNA-sequencing, and functional studies confirm that NAT10 in turn activates the HIF-1 pathway and subsequent glucose metabolism reprogramming by mediating SEPT9 mRNA ac4C modification. The formation of the NAT10/SEPT9/HIF-1α positive feedback loop leads to excessive activation of the HIF-1 pathway and induces glycolysis addiction. Combined anti-angiogenesis and ac4C inhibition attenuate hypoxia tolerance and inhibit tumor progression in vivo. This study highlights the critical roles of ac4C in the regulation of glycolysis addiction and proposes a promising strategy to overcome resistance to anti-angiogenic therapy by combining apatinib with ac4C inhibition.

Colorectal cancer (CRC) can develop through several dysregulated molecular pathways, including the serrated pathway, characterized by CpG island methylator (CIMP) phenotype. Although the tumor tissue is a commonly tested material, sample types such as stool or plasma, bring a new, non-invasive approach. Several cancer-related methylated genes have been identified in CRC patients, including gene GRIA4, showing promising diagnostic potential. The aim of our study was to develop a sensitive droplet digital PCR (ddPCR) assay to examine GRIA4 hypermethylation status in CRC patients and evaluate its diagnostic potential in tissue and liquid biopsy samples.

In total, 23 patients participated in this study, 7 patients with primary CRC and 16 patients with liver metastasis of clinically known CRC. We obtained tumor and non-tumor tissues (N=17), blood samples pre- and post-surgery (N=22), and blood of five volunteers without a personal cancer history. We have developed and optimized a ddPCR assay for GRIA4 hypermethylation detection, from tissue and plasma samples.

We detected significantly increased GRIA4 methylation in tumor tissues compared to their adjacent non-tumor tissue, p<0.0001. Receiver operating characteristic (ROC) analysis defined cutoff values to separate primary tumors and metastases from non-tumor colon/rectum, specifically 36.85% for primary tumors and 34.81% for metastases. All primary tumors were above this threshold. When comparing the methylation levels of metastatic vs. non-tumor tissue, a smaller increase was observed in liver metastasis versus colon tissue (3.6× gain; p=0.001), then in liver metastasis versus adjacent liver tissue (17.4× gain; p<0.0001). On average, GRIA4 hypermethylation in primary tumor plasma was 2.8-fold higher (p=0.39), and in metastatic plasma, 16.4-fold higher (p=0.0011) compared to healthy individuals. Hypermethylation in metastatic plasma was on average 5.9 times higher (p=0.051) than in primary tumor plasma. After tumor removal surgery, average hypermethylation decrease in plasma was 1.6× for primary (p=0.037) and 4.5× for metastatic patients (p=0.023).

Based on our data, it can be inferred that GRIA4 serves as a tissue specific biomarker for the colon/rectum tissue, thus is suitable for cancer classification. This biomarker showed the potential to be an attractive target for early non-invasive detection of metastases of clinically known CRC, although additional analysis has to be performed.

Methylation of cytosine to 5-methylcytosine on CpG dinucleotides is the most frequently studied epigenetic modification involved in the regulation of gene expression. In normal tissues, tissue-specific CpG methylation patterns are established during development. In contrast, alterations in methylation patterns have been observed in abnormal cells, such as cancer cells. Cancer type-specific CpG methylation patterns have been identified and used as biomarkers for cancer diagnosis. In this study, we developed a hybridization-based CpG methylation level sensing system using a methyl-CpG-binding domain (MBD)-fused fluorescent protein. In this system, the target DNA is captured by a complementary methylated probe DNA. When the target DNA is methylated, a symmetrically methylated CpG is formed in the double-stranded DNA. MBD specifically recognizes symmetrical methyl-CpG on double-stranded DNA; therefore, the methylation level is quantified by measuring the fluorescence intensity of the bound MBD-fused fluorescent protein. We prepared MBD-fused AcGFP1 and quantified the CpG methylation levels of the target DNA against SEPT9, BRCA1, and long interspersed nuclear element-1 (LINE-1) using MBD-AcGFP1. This detection principle can be applied to the simultaneous and genome-wide modified base detection systems using microarrays coupled with modified base binding proteins fused to fluorescent proteins.

Colorectal cancer (CRC) is a major health issue, being responsible for nearly 10% of all cancer-related deaths. Since CRC is often an asymptomatic or paucisymptomatic disease until it reaches advanced stages, screening is crucial for the diagnosis of preneoplastic lesions or early CRC.

The aim of this review is to summarize the literature evidence on currently available CRC screening tools, with their pros and cons, focusing on the level of accuracy reached by each test over time. We also provide an overview of novel technologies and scientific advances that are currently being investigated and that in the future may represent real game-changers in the field of CRC screening.

We suggest that best screening modalities are annual or biennial FIT and colonoscopy every 10 years. We believe that the introduction of artificial intelligence (AI)-tools in the CRC screening field could lead to a significant improvement of the screening efficacy in reducing CRC incidence and mortality in the future. More resources should be put into implementing CRC programmes and support research project to further increase accuracy of CRC screening tests and strategies.

Over the last four decades, methodological innovations have continuously changed transcriptome profiling. It is now feasible to sequence and quantify the transcriptional outputs of individual cells or thousands of samples using RNA sequencing (RNA-seq). These transcriptomes serve as a connection between cellular behaviors and their underlying molecular mechanisms, such as mutations. This relationship, in the context of cancer, provides a chance to unravel tumor complexity and heterogeneity and uncover novel biomarkers or treatment options. Since colon cancer is one of the most frequent malignancies, its prognosis and diagnosis seem to be critical. The transcriptome technology is developing for an earlier and more accurate diagnosis of cancer which can provide better protectivity and prognostic utility to medical teams and patients. A transcriptome is a whole set of expressed coding and non-coding RNAs in an individual or cell population. The cancer transcriptome includes RNA-based changes. The combined genome and transcriptome of a patient may provide a comprehensive picture of their cancer, and this information is beginning to affect treatment decision-making in real-time. A full assessment of the transcriptome of colon (colorectal) cancer has been assessed in this review paper based on risk factors such as age, obesity, gender, alcohol use, race, and also different stages of cancer, as well as non-coding RNAs like circRNAs, miRNAs, lncRNAs, and siRNAs. Similarly, they have been examined independently in the transcriptome study of colon cancer.

Colorectal cancer (CRC) is a significant cause of morbidity and mortality worldwide. National screening guidelines have been implemented to identify and remove precancerous polyps before they become cancer. Routine CRC screening is advised for people with average risk starting at age 45 because it is a common and preventable malignancy. Various screening modalities are currently in use, ranging from stool-based tests (fecal occult blood test (FOBT), fecal immunochemical test (FIT), and FIT-DNA test), radiologic tests (computed tomographic colonography (CTC), double contrast barium enema), and visual endoscopic examinations (flexible sigmoidoscopy (FS), colonoscopy, and colon capsule endoscopy (CCE)) with their varying sensitivity and specificity. Biomarkers also play a vital role in assessing the recurrence of CRC. This review offers a summary of the current screening options, including biomarkers available to detect CRC, highlighting the benefits and challenges encompassing each screening modality.

Hypermethylation of tumor-suppressor genes and global hypomethylation, which is related to methylation level at the retroelement, have been recognized as features of the cancer genome. In this study, we developed a hybridization-based CpG methylation level detection method using methyl-CpG-binding domain-fused firefly luciferase (MBD-Fluc). In this method, methylated probe oligonucleotides were used to capture target oligonucleotides. Fully methylated and hemimethylated double-stranded DNA (dsDNA) was formed by hybridization of the methylated captured oligonucleotides with methylated or unmethylated target oligonucleotides, respectively. MBD-Fluc specifically binds to fully methylated dsDNA but not to hemimethylated dsDNA; therefore, methylated target oligonucleotides can be detected by measuring the luciferase activity of the bound MBD-Fluc. Using the corresponding methylated probe oligonucleotides, the CpG methylation levels of SEPT9, BRCA1, and long interspersed nuclear element-1 (LINE-1) oligonucleotides were quantified. Moreover, we demonstrated that the emission detection signal was not affected by the methylation state of the overhang region of the target oligonucleotide, which was not hybridized to the probe oligonucleotide, indicating that methylated CpG of the target region could be accurately detected. Unmethylated-CpG-binding domain-fused luciferases and 5-hydroxymethyl-CpG-binding domain-fused luciferases have been constructed, suggesting that other modified bases can be detected by the same platform.

Colorectal cancer is a common malignant tumor of the digestive tract. Despite advances in diagnostic techniques and medications. Its prognosis remains challenging. DNA methylation-driven related circulating tumor cells have attracted enormous interest in diagnosing owing to their non-invasive nature and early recognition properties. However, the mechanism through which risk biomarkers act remains elusive. Here, we designed a risk model based on differentially expressed genes, DNA methylation, robust, and survival-related factors in the framework of Cox regression. The model has satisfactory performance and is independently verified by an external and isolated dataset in terms of C-index value, ROC, and tROC. The model was applied to Colorectal cancer patients who were subsequently divided into high- and low-risk groups. Functional annotations, genomic alterations, tumor immune environment, and drug sensitivity were analyzed. We observed that up-regulated genes are associated with epithelial cell differentiation and MAPK signaling pathways. The down-regulated genes are related to IL-7 signaling and apoptosis-induced DNA fragmentation. Interestingly, the immune system was inhibited in high-risk groups. High-frequency mutation genes tend to co-occur. High-risk score patients are related to copy number amplification events. To address the challenges, we suggested eleven and twenty-one drugs that are sensitive to low- and high-risk patients. Finally, an artificial neural network was provided to evaluate the immunotherapeutic efficiency. Taken together, the findings demonstrated that our risk score model is robust and reliable for evaluating the prognosis with novel diagnostic and treatment targets. It also yields benefits for the treatment and provides unique insights into developing therapeutic strategies.

(1) Background: Circulating tumor DNA (ctDNA) has emerged as a promising biomarker for many kinds of tumors. However, whether ctDNA could be an accurate diagnostic biomarker in colorectal cancer (CRC) remains to be clarified. The aim of this study was to evaluate the diagnostic accuracy of ctDNA in CRC. (2) Methods: PubMed, Web of Science, and Cochrane databases were searched to identify studies reporting the use of ctDNA to screen and diagnose CRC, and all relevant studies published until October 2022 were enrolled for our analysis. These studies were divided into three primer subgroups: the subgroup of quantitative or qualitative analysis of ctDNA and the subgroup of septin9 (SEPT9) methylation assay. (3) Results: A total of 79 qualified articles with 25,240 subjects were incorporated into our meta-analysis. For quantitative studies, the combined sensitivity (SEN), specificity (SPE), and diagnostic odds ratio (DOR) were 0.723 (95% CI: 0.623-0.803), 0.920 (95% CI: 0.827-0.966), and 23.305 (95% CI: 9.378-57.906), respectively, yielding an AUC of 0.860. The corresponding values for qualitative studies were 0.610 (95% CI: 0.566-0.651), 0.891 (95% CI: 0.878-0.909), 12.569 (95% CI: 9.969-15.848), and 0.823, respectively. Detection of SEPT9 methylation depicted an AUC of 0.879, with an SEN of 0.679 (95% CI: 0.622-0.732), an SPE of 0.903 (95% CI: 0.878-0.923), and a DOR of 20.121 (95% CI:14.404-28.106), respectively. (4) Conclusion: Blood-based ctDNA assay would be a potential novel biomarker for CRC screening and diagnosis. Specifically, quantitative analysis of ctDNA or qualitative analysis of SEPT9 methylation exhibited satisfying diagnostic efficiency. Larger sample studies are needed to further confirm our conclusions and to make the ctDNA approach more sensitive and specific.

There are no robust tools for the diagnosis of synchronous colorectal cancer (SyCRC). Herein, we developed the first methylation signature to identify and characterise patients with SyCRC.

For biomarker discovery, we analysed the genome-wide methylation profiles of 16 SyCRC and 18 solitary colorectal cancer (SoCRC) specimens. We thereafter established a methylation signature risk-scoring model to identify SyCRC in an independent cohort of 38 SyCRC and 42 SoCRC patients. In addition, we evaluated the prognostic value of the identified methylation profile.

We identified six differentially methylated CpG probes/sites that distinguished SyCRC from SoCRC. In the validation cohort, we developed a methylation panel that identified patients with SyCRC from not only larger tumour (AUC = 0.91) but also the paired remaining tumour (AUC = 0.93). Moreover, high risk scores of our panel were associated with the development of metachronous CRC among patients with SyCRC (AUC = 0.87) and emerged as an independent predictor for relapse-free survival (hazard ratio = 2.72; 95% CI = 1.12-6.61). Furthermore, the risk stratification model which combined with clinical risk factors was a diagnostic predictor of recurrence (AUC = 0.90).

Our novel six-gene methylation panel robustly identifies patients with SyCRC, which has the clinical potential to improve the diagnosis and management of patients with CRC.

Background: The gonads of Chrysemys picta, a turtle with temperature-dependent sex determination (TSD), exhibit differential DNA methylation between males and females, but whether the same is true in somatic tissues remains unknown. Such differential DNA methylation in the soma would provide a non-lethal sex diagnostic for TSD turtle hatchings who lack visually detectable sexual dimorphism when young. Methods: Here, we tested multiple approaches to study DNA methylation in tail clips of Chrysemys picta hatchlings, to identify differentially methylated candidate regions/sites that could serve as molecular sex markers To detect global differential methylation in the tails we used methylation-sensitive ELISA, and to test for differential local methylation we developed a novel hybrid method by sequencing immunoprecipitated and bisulfite converted DNA (MeDIP-BS-seq) followed by PCR validation of candidate regions/sites after digestion with a methylation-sensitive restriction enzyme. Results: We detected no global differences in methylation between males and females via ELISA. While we detected inter-individual variation in DNA methylation in the tails, this variation was not sexually dimorphic, in contrast with hatchling gonads. Conclusions: Results highlight that differential DNA methylation is tissue-specific and plays a key role in gonadal formation (primary sexual development) and maintenance post-hatching, but not in the somatic tail tissue.

Aims: Early detection of colorectal cancer (CRC) provides substantially better survival rates. This study aimed to develop a blood-based screening assay named SPOT-MAS ('screen for the presence of tumor by DNA methylation and size') for early CRC detection with high accuracy. Methods: Plasma cell-free DNA samples from 159 patients with nonmetastatic CRC and 158 healthy controls were simultaneously analyzed for fragment length and methylation profiles. We then employed a deep neural network with fragment length and methylation signatures to build a classification model. Results: The model achieved an area under the curve of 0.989 and a sensitivity of 96.8% at 97% specificity in detecting CRC. External validation of our model showed comparable performance, with an area under the curve of 0.96. Conclusion: SPOT-MAS based on integration of cancer-specific methylation and fragmentomic signatures could provide high accuracy for early-stage CRC detection.

In fragmented DNA, PCR-based methods quantify the number of intact regions at a specific amplicon length. However, the relationship between the population of DNA fragments within a sample and the likelihood they will amplify has not been fully described. To address this, we have derived a mathematical equation that relates the distribution profile of a stochastically fragmented DNA sample to the probability that a DNA fragment within that sample can be amplified by any PCR assay of arbitrary length. Two panels of multiplex PCR assays for quantifying fragmented DNA were then developed: a four-plex panel that can be applied to any human DNA sample and used to estimate the percentage of regions that are intact at any length; and a two-plex panel optimized for quantifying circulating cell-free DNA (cfDNA). For these assays, regions of the human genome least affected by copy number aberration were identified and selected; within these copy-neutral regions, each PCR assay was designed to amplify both genomic and bisulfite-converted DNA; and all assays were validated for use in both conventional qPCR and droplet-digital PCR. Finally, using the cfDNA-optimized assays we find evidence of universally conserved nucleosome positioning among individuals.

Molecular markers for the detection of lymph node micrometastases of malignant tumors have been extensively investigated. However, epigenetic signatures have rarely been reported for identification of metastatic lymph nodes and disease relapse. Septin 9 is the most frequently reported hypermethylated gene in colorectal cancer (CRC). This study aimed to assess the clinical relevance of Septin 9 methylation in regional lymph nodes in recurrence/metastases of CRC.

We analyzed Septin 9 methylation of DNA from resected lymph nodes in 75 CRC patients with or without tumor recurrence using quantitative methylation-sensitive PCR (qMS-PCR).

Of the 30 histologically negative lymph node CRC patients without recurrence (group 1), methylated Septin 9 was detected in 3 (10 %) cases. The positivity rate of methylated Septin 9 in group 2 containing 30 histologically node-negative CRC patients with recurrence was 30 % (9/30). For group 3, lymphatic invasion as well as tumor recurrence, 11 (73 %) out of 15 subjects had Septin 9 methylation-positive lymph nodes. Moreover, patients in group 3 had a higher level of methylated Septin 9 compared to subjects in group 1 and group 2 (p < 0.05). In addition, CRC patients with Septin 9 methylation in lymph nodes had significantly reduced survival (Log-rank P < 0.0001).

Our data support the predictive role of Septin 9 methylation analysis of lymph node micrometastases for tumor relapse after surgery.

Colorectal cancer (CRC) is one of the most common malignant tumors of the digestive tract worldwide and is a serious threat to human life and health. CRC occurs and develops in a multi-step, multi-stage, and multi-gene process, in which abnormal gene expression plays an important role. CRC is currently diagnosed via endoscopy combined with tissue biopsy. Compared with tissue biopsy, liquid biopsy technology has received increasingly more attention and applications in the field of molecular detection due to its non-invasive, safe, comprehensive, and real-time dynamic nature. This review article discusses the application and limitations of current liquid biopsy analytes in the diagnosis, treatment, and prognosis of CRC, as well as directions for their future development.

Methylated SDC2 has been proved as a diagnostic marker for human colorectal cancer (CRC), noninvasive stool DNA-based methylation testing also emerges as a novel approach for detecting CRC. The aim of this study was to evaluate the clinical performance of stool DNA-based SDC2 methylation test by a new qPCR detection reagent for early detection of CRC.

A new qPCR detection reagent contained two differentially methylated regions in SDC2 CpG islands for the detection of CRC was used in this study. Performance of the SDC2 methylation detection reagent was evaluated by analyzing limit of detection, precision, and specificity. The effect of interfering substances on assay performance was also tested. 339 subjects (102 CRC patients, 50 patients with advanced adenomas, 39 patients with non-advanced adenomas, 18 colitis patients and 130 normal individuals) from the China-Japan Friendship Hospital were evaluated. Approximately 2.5 g of stool sample was collected from each participant. Stool DNA was extracted and bisulfite-converted, followed by qPCR assay, which contained two pairs of primers for the methylation detection of two fragments of the SDC2 gene (named SDC2-A and SDC2-B). The diagnostic value of this test in CRC was evaluated by calculating receiver operating characteristic (ROC) curve, and value of the area under the curve (AUC).

The test kit was able to detect methylated SDC2 in stool DNA samples with concentrations as low as 90 copies/μL in 100% of replicates. The sensitivity for detecting CRC by methylated SDC2-A alone was 85.29% (95% CI 77.03-91.00%) with a specificity of 96.15% (95% CI 91.08-98.58%). The sensitivity by methylated SDC2-B alone was 83.33% (95% CI 74.82-89.42%) with a specificity of 97.69% (95% CI 93.14-99.51%). However, when methylated SDC2-A and methylated SDC2-B were combined, the sensitivity for CRC detection improved to 87.25% (95% CI 79.27-92.53%) with a specificity of 94.62% (95% CI 89.11-97.56%). Further, the detection reagent achieved ROC-AUC 0.874 (95% CI 0.822-0.927) for SDC2-A, 0.906 (95% CI 0.859-0.952) for SDC2-B, and 0.939 (95% CI 0.902-0.977) for SDC2-Combine A&B.

This study validated the capability of stool DNA-based SDC2 methylation test for early screening of CRC, and combined detection of two fragments of SDC2 gene could improve detection sensitivity.

DNA methylation is an epigenetic mechanism regulating gene expression. Changes in DNA methylation were suggested to be useful biomarkers for diagnosis, and for the determination of prognosis and treatment response. Here, we provide an overview of methylation-based biomarkers in colorectal cancer. First, we start with the two methylation-based diagnostic biomarkers already approved for colorectal cancer, SEPT9 and the combination of NDRG4 and BMP3. Then, we provide a list-based overview of new biomarker candidates depending on the sample source including plasma, stool, urine, and surgically removed tumor tissues. The most often identified markers like SDC2, VIM, APC, MGMT, SFRP1, SFRP2, and NDRG4 have distinct functions previously linked to tumor progression. Although numerous studies have identified tumor-specific methylation changes, most of these alterations were observed in a single study only. The lack of validation in independent samples means low reproducibility and is a major limitation. The genome-wide determination of methylation status (methylome) can provide data to solve these issues. In the third section of the review, methylome studies focusing on different aspects related to CRC, including precancerous lesions, CRC-specific changes, molecular subtypes, aging, and chemotherapy response are summarized. Notably, techniques simultaneously analyzing a large set of regions can also uncover epigenetic regulation of genes which have not yet been associated with tumorigenesis previously. A remaining constraint of studies published to date is the low patient number utilized in these preventing the identification of clinically valuable biomarker candidates. Either future large-scale studies or the integration of already available methylome-level data will be necessary to uncover biomarkers sufficiently robust for clinical application.