Genes In Colorectal Cancer Patients Research Articles

The association of the killer cell immunoglobulin-like receptor (KIR) genotype distribution and HLA-C ligands in colorectal cancer in the eastern region of Saudi Arabia

Colorectal cancer (CRC) is the most common cause of cancer-related mortality globally. It ranks as the most common cancer among men and the third most prevalent among women in Saudi Arabia, especially in the eastern region. The immune system's response to cancer is significantly influenced by natural killer cells through their killer-cell immunoglobulin-like receptors (KIRs) interaction with human leukocyte antigen (HLA) molecules. Our research aimed to target a homogeneous ethnic group in the eastern region of Saudi Arabia, analyzing the distribution of 16 KIR genes and HLA-C1/C2 allotypes in 50 CRC patients and 63 healthy controls using the sequence-specific PCR amplification method. Findings revealed significantly lower frequencies of the 2DL2 and 3DL1 genes in CRC patients compared to controls (OR = 2.21; P = 0.04) and (OR = 7.46; P = 0.00009), respectively. Additionally, a higher prevalence of HLA-C2 was noted in CRC patients (84 %) versus controls (66.7 %) (OR = 2.26; P = 0.04). Conversely, the HLA-C1C2 combination showed a protective effect against CRC (OR = 0.38; P = 0.04). Our combinatorial analysis further identified specific gene combinations correlating with CRC risk or protection. Notably, the absence of KIR2DL2 combined with the presence of KIR2DS2 emerged as a significant risk factor, while various combinations involving KIR2DL2 and/or KIR2DS1 with HLA-C ligands were associated with either increased susceptibility or protection. The study underscores the protective role of KIR2DL2 against CRC in Saudi Arabia's eastern region and suggests that the absence of KIR2DL2, especially when KIR2DS2 is present, might increase CRC risk. This research contributes to understanding genetic influences on CRC susceptibility, emphasizing the potential of KIR and HLA interactions as biomarkers for CRC risk assessment.

Associations Between Fusobacterium nucleatum and msh2, mlh1, and msh6 Gene Expression in Colorectal Cancer

Background: Colorectal cancer (CRC) is a major global health concern, and the link with Fusobacterium nucleatum has received considerable attention. Objectives: This study aimed to explore the prevalence of F. nucleatum and to assess the expression of the msh2, mlh1, and msh6 genes in CRC patients compared to a control group using real-time PCR. Methods: Forty CRC patients and twenty individuals from a control group participated in this study. Gastroenterologists collected biopsy specimens from which DNA and RNA were extracted using a specialized tissue extraction kit. Complementary DNA (cDNA) was then synthesized. Real-time PCR was employed to evaluate the expression levels of the msh2, mlh1, and msh6 genes and the presence of the F. nucleatum-specific 16srRNA gene to determine the relative prevalence of this bacterium in each group. Results: Results indicated a higher prevalence of the F. nucleatum-specific 16srRNA gene in CRC patients than in the control group. Additionally, expression levels of the msh2, mlh1, and msh6 genes were significantly higher in the cancer group, suggesting their role in CRC pathogenesis. The distribution of F. nucleatum was particularly high in the sigmoid and rectum areas of the colon. Conclusions: This study underscores the significance of F. nucleatum in CRC and provides insights into its association with altered gene expression patterns. Understanding the prevalence of F. nucleatum and its impact on msh2, mlh1, and msh6 genes may aid in developing improved diagnostic and therapeutic strategies for CRC. Further research is necessary to elucidate these relationships more comprehensively.

Molecular analysis to identify novel potential biomarkers as drug targets in colorectal cancer therapy: an integrated bioinformatics analysis

ABSTRACT Colorectal cancer (CRC) is a heterogeneous disease that requires new diagnostic and prognostic markers. Integrated bioinformatics approach to identify novel therapeutic targets associated with CRC. Using GEO2R identified DEGs in CRC, and Funrich software facilitated the visualization of DEGs through Venn diagrams. From a total of 114 enhanced DEGs, potential hub genes were further filtered based on their nodal strength and edges using STRING database. To gain insights into the functional roles of these hub genes, gene ontology and pathway enrichment were conducted thorough g: profiler web server. Subsequently, overall survival plots from GEPIA and oncogenic predictive functions like mRNA expressions for stages and nodal metastasis were employed to identify hub genes in CRC patient samples. Additionally, the cBioPortal and HPA databases also revealed genetic alterations and expression levels in these hub genes in CRC patients, further supporting their involvement in colorectal cancer. Gene expression by RT-PCR shows upregulation of hub genes in HT-29 cells. Finally, our integrated bioinformatic analysis revealed that ABCE1, AURKA, HSPD1, PHKA1, CDK4, and YWHAE as hub genes with potential oncogenic roles in CRC. These genes hold promise as diagnostic and prognostic markers for colorectal tumorigenesis, providing insights into targeted therapies for improved patient outcomes.

KRAS Mutation Status in Colorectal Epithelial Tumors of Colorectal Cancer

Introduction: Colorectal cancer (CRC) is one of the commonest malignancies in industrialized nations with lower incidence in Asia and Africa. Majority of CRCs are non-hypermutated. KRAS is one of the commonest mutated genes in CRC patients. Given the KRAS mutated CRCs are resistant to EGFR targeted chemotherapy, expanded KRAS mutation testing is recommended before starting chemotherapy. The aim of this study was to study the genetic variation of KRAS in group of CRC cancer patients referred to a teaching hospital in Kolkata, India.Methods: Out of 15 cases of colorectal cancer studied, 2 specimens were right hemicolectomies for clinically diagnosed colon carcinomas and 13 specimens were biopsies of colonoscopy done for anaemia, changed bowel habit, occult bleeding. Formalin fixed paraffin embedded (FFPE) tissue sections, stained by routine Hematoxylin and Eosin were examined microscopically. The DNA was extracted from FFPE tissue with Qiagen kit. KRAS Mutation analysis was done by TheraScreen – KRAS Mutation Kit.Results: The age of patients ranged from 28 to 64 years. Nine were male and remaining 6 were female. histopathological examination revealed 10 adenomas and 5 adenocarcinomas. One adenoma, tubulovillous with high grade dysplasia featured positive KRAS mutation (Gly12Asp). Three out of 4 adenocarcinomas with positive KRAS mutation, one showed Gly12Ala and 2 showed Gly12Asp. Conclusion: The frequency of KRAS mutations was very low in the studied sample.

Comprehensive analysis of basement membrane-related gene based on single-cell and bulk RNA sequencing data to predict prognosis and evaluate immune characteristics in colorectal cancer.

Basement membrane-related genes (BMs) participate in regulating cell polarity, invasion, metastasis, and survival across different tumor types. Nevertheless, the specific functions of BMs in colorectal cancer (CRC) remain uncertain. To investigate the clinical relevance of BMs in CRC, we retrieved both gene expression and clinical data from The Cancer Genome Atlas (TCGA) datasets for subsequent analysis. The Kaplan-Meier (K-M) survival curve was employed to evaluate prognosis in high- and low-risk groups. Furthermore, additional analyses, including nomogram construction, functional enrichment, examination of the tumor immune microenvironment, prediction of small-molecule drugs, and more, were conducted to delve into the significance of BM-related signatures in CRC. Single-cell data from seven CRC patients were obtained from the TISCH2 database, and expression validation and cell source exploration of BM-related signatures were performed. Lastly, the expression and function of TIMP1, a key gene in BMs that may play a role in the progression of CRC, was validated in vitro through a series of basic experiments. We constructed a seven BMs-based model to categorize CRC patients into high-risk and low-risk groups. K-M survival analysis indicated a poorer prognosis for high-risk CRC patients. Cox regression analysis further identified the risk score as an independent prognostic factor for CRC patients. The nomogram model exhibited superior discrimination and calibration abilities of CRC patients. Based on the results from GO/KEGG and GSEA, genes in the high-risk subgroup were implicated in immune-related pathways and exhibited a positive correlation with immune checkpoints. In single-cell data, we found that TIMP1 is highly expressed in many cells, especially in malignant tumor cells. We also observed up-regulation of TIMP1 in CRC cell lines, promoting cancer invasion and migration in vitro. Our study has discovered a novel prognostic index derived from BM-related genes in CRC patients. Specifically, the new model enables patient stratification, improving the selection of individuals likely to benefit from immunotherapy.

Exon Sequence Analysis of the ATG5, ATG12, ATG9B Genes in Colorectal Cancer Patients During Radiotherapy

Exon Sequence Analysis of the ATG5, ATG12, ATG9B Genes in Colorectal Cancer Patients During Radiotherapy

The effects of thymidylate synthase 3'UTR genotype on methylation of tumor-specific genes promoter in 22 colorectal cancer patients from southern Iran.

To investigate the effects of thymidylate synthase (TS) 3'UTR genotype on promotor methylation of tumor-related genes in 22 patients with sporadic colorectal cancer (CRC) from southern Iran. We evaluated the correlations of TS 3'UTR genotype with promoter methylation of hTERT, hMLH1, MSH2, MMP2, CDH1, p14, p16, and p21 genes in CRC patients. The polymorphism of TS 3'UTR was evaluated through mutagenically specific PCR. The genes promoter methylation was determined using methylation-specific PCR. For 10 patients, the gene expression profile of epigenetic regulating enzymes, histone deacetylases (HDACs) and DNA methyltransferases (DNMTs), was also examined in both tumor and normal adjacent tissues by quantitative real time PCR. There was a significant association between the hMLH1 methylation and age of patients (P= 0.039) and also between MSH2 methylation and tumor site (P= 0.036). There was insignificant association between gene-specific methylation and TS 3'UTR genotype. However, all polymorphic genotypes of TS were associated with higher methylation of hMLH1 and CDH1 and lower methylation of MSH2. The -6bp/+6bp (heterozygous mutant) and [-6bp/+6bp, +6bp/+6bp] (homozygous mutant) genotypes resulted in higher methylation of p16, and -6bp/+6bp and [-6bp/+6bp, +6bp/+6bp] genotypes were correlated with lower methylation of MMP2. The overexpression of epigenetic enzymes, HDACs and DNMTs, was also demonstrated. There was no association between DNMTs transcript levels and gene-specific hypermethylation. The polymorphic TS genotypes, especially -6bp/+6bp, could affect methylation frequencies of studied genes. Moreover, promoter methylation status was not dependent on DNMTs gene expression. Large sample size studies may contribute to validate these findings.

Molecular characteristics, clinical significance, and immune landscape of extracellular matrix remodeling-associated genes in colorectal cancer.

Extracellular matrix (ECM) remodeling is one of the hallmark events in cancer and has been shown to be closely related to tumor immunity. Immunotherapy has evolved as an important tool to treat various cancers and improve patient prognosis. The positive response to immunotherapy relies on the unique interaction between cancer and the tumor microenvironment (TME). However, the relationship between ECM remodeling and clinical outcomes, immune cell infiltration, and immunotherapy in colorectal cancer (CRC) remains unknown. We systematically evaluated 69 ECM remodeling-associated genes (EAGs) and comprehensively identified interactions between ECM remodeling and prognosis and the immune microenvironment in CRC patients. The EAG_score was used to quantify the subtype of ECM remodeling in patients. We then assessed their value in predicting prognosis and responding to treatment in CRC. After elaborating the molecular characteristics of ECM remodeling-related genes in CRC patients, a model consisting of two ECM remodeling-related genes (MEIS2, SLC2A3) was developed for predicting the prognosis of CRC patients, Receiver Operating Characteristic (ROC) and Kaplan-Meier (K-M) analysis verified its reliable predictive ability. Furthermore, we created a highly reliable nomogram to enhance the clinical feasibility of the EAG_score. Significantly differences in TME and immune function, such as macrophages and CD8+ T cells, were observed between high- and low-risk CRC patients. In addition, drug sensitivity is also strongly related to EAG_score. Overall, we developed a prognostic model associated with ECM remodeling, provided meaningful clinical implications for immunotherapy, and facilitated individualized treatment for CRC patients. Further studies are needed to reveal the underlying mechanisms of ECM remodeling in CRC.

Dual targeting of TGF-β and PD-L1 inhibits tumor growth in TGF-β/PD-L1-driven colorectal carcinoma

Immunosuppressive factors within the tumor microenvironment (TME), such as Transforming growth factor beta (TGF-β), constitute a crucial hindrance to immunotherapeutic approaches in colorectal cancer (CRC). Furthermore, immune checkpoint factors (e.g., programmed death-ligand 1 [PD-L1]) inhibit T-cell proliferation and activation. To cope with the inhibitory effect of immune checkpoints, the therapeutic value of dual targeting PD-L1 and TGF-β pathways via M7824 plus 5-FU in CRC has been evaluated. Integrative-systems biology approaches and RNAseq were used to assess the differential level of genes associated with 88 metastatic-CRC patients. The level of PD-L1 and TGF-β was evaluated in a validation cohort. The anti-proliferative, migratory, and apoptotic effects of PD-L1/TGF-β inhibitor, M7824, were assessed by MTT, wound-healing assay, and flow cytometry. Anti-tumor activity was assessed in a xenograft model, followed by biochemical studies and histological staining, and gene/protein expression analyses by RT-PCR and ELISA/IHC. The result of differentially expressed genes (DEGs) analysis showed 1268 upregulated and 1074 downregulated genes in CRC patients. Among the highest scoring genes and dysregulated pathways associated with CRC, PD-L1, and TGF-β were identified and further validated in 92 CRC patients. Targeting of PD-L1-TGF-β inhibited cell growth and migration, associated with modulation of CyclinD1 and MMP9. Furthermore, M7824 inhibited tumor growth via targeting TGF-β and PD-L1 pathways, resulting in modulation of inflammatory response and fibrosis via TNF-α/IL6/CD4–8 and COL1A1/1A2, respectively. In conclusion, our data illustrated that co-targeting PD-L1 and TGF-β pathways increased the effect of Fluorouracil (5-FU) and reduced the tumor growth in PD-L1/TGF-β expressing tumors, providing a new therapeutic option in the treatment of CRC.

Targeting HDAC2-Mediated Immune Regulation to Overcome Therapeutic Resistance in Mutant Colorectal Cancer

A large body of clinical and experimental evidence indicates that colorectal cancer is one of the most common multifactorial diseases. Although some useful prognostic biomarkers for clinical therapy have already been identified, it is still difficult to characterize a therapeutic signature that is able to define the most appropriate treatment. Gene expression levels of the epigenetic regulator histone deacetylase 2 (HDAC2) are deregulated in colorectal cancer, and this deregulation is tightly associated with immune dysfunction. By interrogating bioinformatic databases, we identified patients who presented simultaneous alterations in HDAC2, class II major histocompatibility complex transactivator (CIITA), and beta-2 microglobulin (B2M) genes based on mutation levels, structural variants, and RNA expression levels. We found that B2M plays an important role in these alterations and that mutations in this gene are potentially oncogenic. The dysregulated mRNA expression levels of HDAC2 were reported in about 5% of the profiled patients, while other specific alterations were described for CIITA. By analyzing immune infiltrates, we then identified correlations among these three genes in colorectal cancer patients and differential infiltration levels of genetic variants, suggesting that HDAC2 may have an indirect immune-related role in specific subgroups of immune infiltrates. Using this approach to carry out extensive immunological signature studies could provide further clinical information that is relevant to more resistant forms of colorectal cancer.

Mutation status of full RAS and BRAF in 169 Moroccan patients with colorectal cancer.

216 Background: Morocco has the highest cancer mortality rate in MENA Countries. Colorectal cancer (CRC) is classified as the first digestive cancer and remains a burden in Morocco. According to the data on GLOBOCAN 2020, there were 4324 new CRC patients and 2374 deaths, accounting for 7.3% and 2.8% of all cancers, respectively. Our study aimed to investigate the frequency of the full RAS ( KRAS, NRAS) and BRAF genes in CRC patients from Morocco and their possible associations with clinico-pathological features. Methods: Archived FFPE of 169 CRC patients were screened for KRAS, NRAS and BRAF mutations by Idylla technology. Results: Full RAS mutations were identified in 46.1% (42% in KRAS, 4.1% in NRAS). In KRAS gene, exon 2 mutations accounted for 84.5% (69% in codon 12, 15.5 % in codon 13). Within codon 12, KRAS G12D and G12C were more frequently detected (29.5% and 16.9%, respectively). Detection of KRAS mutations, and particularly G12D and G12C subtypes, are of large significance for CRC patients, have possible therapeutic implications. Within codon 13, the most frequently observed mutation was G13D (15.5 %). Outside exon 2, the mutation rate was 35.1% (8.4% in exon 3 and 26.7% in exon 4). Concurrent KRAS mutations were identified in 8 cases, which suggests that multiple mutations can occur in the same or different codons. In NRAS gene, the mutation rates of exon 2 and 3 were 71.4% and 57.1% respectively. G13V and Q61K were the most common mutations, accounting for 28.6 % of each. Concurrent KRAS mutations were identified in 2 cases. Of the 169 samples, mutations in the BRAF gene at V600E were detected in 3.5%. Combined mutational analysis of KRAS, NRAS and BRAF was able to identify 49.6% of patients with CRC as likely non-responders to anti-EGFR therapy. There was an association between KRAS mutations and age, which were higher in the age group>50 years old (p=0.022). Tumors in the left colon (36.61%) are more likely to harbor mutations in KRAS than the rectum (19.7%) in both sexes. The adenocarcinoma well-differentiated was the most frequent for patients with KRAS mutations (54.9%). No significant clinicopathologic correlations with NRAS and BRAF mutations were identified. Conclusions: Beside established anti-CRC treatment, better understanding of the causality of CRC can be established by combining epidemiology and genetic/epigenetic on CRC etiology in Morocco. This approach may be able to significantly reduce the burden of disease in the country. Moreover, the Moroccan government should develop policy on CRC prevention and public health programs which may serve as a feasible setting to increase public awareness on lifestyle risk factors.

Expression profiling of anticancer genes in colorectal cancer patients and their in vitro induction by riproximin, a ribosomal inactivating plant protein.

Ectopic expression of anticancer genes (ACGs) imposes antineoplastic effects on transformed cells. Clinically, reduced expression of these genes has been linked with poor prognosis, metastasis and chemo/radiotherapy resistance in cancers. Identifying expression pattern of ACGs is crucial to establish their prognostic and therapeutic relevance in colorectal cancer (CRC). In addition to the clinical perspective, naturally occurring compounds can be explored in parallel for inducing ACGs to achieve cancer cell-specific death. Expression profiles of three ACGs (NOXA, PAR-4, TRAIL) were identified via real-time PCR in CRC clinical isolates. Time lapse-based expression modifications in ACGs were studied in a CRC liver metastasis animal model using microarray methodology. Effects of a purified plant protein (riproximin) on selected ACGs were identified in three primary and metastatic CRC cell lines by real-time PCR. Lastly, importance of the ACGs in a cellular environment was highlighted via bioinformatic analysis. ACGs (except NOXA) were persistently downregulated in clinical isolates when comparing the overall mean expression values with normal mucosa levels. In vivo studies showed a prominent inhibition of NOXA and PAR-4 genes in implanted CRC cells during rat liver colonization. TRAIL showed deviation from this theme while showing marked induction during the early period of liver colonization (days 3 and 6 after CRC cell implantation). Riproximin exhibited substantial potential of inducing ACGs at transcriptome levels in selected CRC cell lines. Bioinformatic analysis showed that vital molecular/functional aspects of a cell are associated with the presence of ACGs. ACGs are downregulated in primary and metastatic phase of CRC. Riproximin effectively induces ACGs in CRC cells and can be exploited for clinical investigations over time.

Mutational profile of KRAS, NRAS, BRAF, PIK3CA, and AKT1 genes in colorectal cancer patients in a tertiary care hospital, Dhaka

BackgroundColorectal carcinoma (CRC) is the third most common cancer in the world and incidences are on the rise in Bangladesh. KRAS, NRAS, BRAF, PIK3CA, and AKTI gene mutations are predictive markers of biological therapies, monoclonal antibodies targeting EGFR. The purpose of this study was to detect KRAS, NRAS, BRAF, PIK3CA, and AKT1 genes mutation in CRC patients by multiplex real-time Polymerase Chain Reaction (PCR) and evaluate the association of the mutations with clinicopathological features. MethodologyThis cross-sectional study was carried out in the Department of Microbiology & Immunology, Bangabandhu Sheikh Mujib Medical University (BSMMU) from March 2019 to January 2020. Tissues from surgically resected colorectal tumors were collected from 44 histopathologically confirmed adult colorectal cancer patients, who were admitted in the Department of Colorectal Surgery, BSMMU. ResultsAmong 44 histopathologically diagnosed colorectal cancer patients, KRAS, BRAF, and PIK3CA gene mutations were identified in 31.5%, 4.85%, and 4.85% tumors, respectively. In this study, no mutation was detected in NRAS and AKT1 genes. Concurrent mutation in KRAS and PIK3CA genes were found in one patient. Among the 14 KRAS mutant cases, most (92.85%) were in exon 2 (codon 12/13) and only 7.15% of the mutations were in exon 3 (codon 61). No mutation was detected in codon 59, 117, and 146. Among the two PIK3CA mutations, one was present in exon 9 and another was in exon 20. KRAS mutations were significantly associated with well and moderately differentiated tumors than poorly differentiated tumors (p < 0.05). Histopathologically there was no significant association of KRAS mutations with age, sex, tumor location, TNM staging, and other parameters. ConclusionA combined evaluation of genetic biomarkers can classify about one-third of colorectal cancer patients as mutant for any one of these KRAS, BRAF, and PIK3CA genes.

Chronic Inflammation and Its Role in Colorectal Cancer Development

Colorectal cancer (CRC) is the third common cancer and fourth cancer related mortality, worldwide. Chronic Inflammation is one of the underlying mechanisms leads to this cancer. The final aim of this research was to assess the expression level of pattern recognition receptor (PRRs)-related genes TLR2, TLR4, NLRP3, NF-kβ, MBL1, GAL1 and oxidative agent NOS2 in colon tissue and blood monocytes of CRC patients to find the relative role of chronic inflammation in the initiation of the stage II CRC development. by this approach, the expression level of mentioned genes was assessed in colon tissues and monocytes of 24 cases (12 CRC and 12 healthy control persons) by 65±11 years old men. In CRC patients’ monocytes, the expression level of TLR2, TLR4, GAL1 and MBL1 genes were significantly less than those of healthy controls (p<0.05). the expression level of NOS2 and NF-kβ genes in CRC patients’ monocytes was significantly higher than those of in healthy controls (p<0.05).

The prevalence of KRAS and BRAF mutation in colorectal cancer patients in Bali

Mutations in the KRAS (Kirsten rat sarcoma viral oncogene homolog gene) and BRAF (v‐Raf murine sarcoma viral oncogene homolog B1) gene play a significant role in primary resistance to colorectal cancer therapy. Around 85‐90% of KRAS mutations in colorectal cancer occur in exon 2 (codon 12 and 13), whereas approximately 96% of BRAF mutations occur in exon 15 codon 600 (V600E). This study aimed to determine the prevalence and mutation characteristics of the KRAS and BRAF genes in colorectal cancer patients in Bali. The DNA was isolated from 44 formalin‐fixed paraffin‐embedded colorectal cancer samples which were stored in the Department of Pathology, Sanglah General Hospital in 2017. Detection of mutation was carried out by polymerase chain reaction (PCR) and direct sequencing. Out of 44 samples, only 27 were successfully amplified and sequenced. Our findings showed six samples (22.2%) with mutated KRAS at codons 12 and 13 (including two samples with G12D, one sample with G12V, and three samples with G13D). Interestingly, we found three samples (11.1%) of BRAF mutation, including two samples with V600E mutation and one with V600L mutation. Taken together, our results showed that KRAS and BRAF mutations were identified and occurred exclusively. Further studies are essential to identify the correlation of these mutations with colorectal cancer prognosis and response to chemotherapy

Integrated Analysis of DNA Repair Genes Identifies SLC6A1 as a New Marker for the Clinical Outcome of Patients with Colorectal Cancer.

Colorectal cancer (CRC) remains an important malignancy worldwide with poor prognosis. It has been known that DNA repair genes are involved in the development and progression of various tumors. Therefore, the purpose of this study was to explore DNA repair gene-based prognostic biomarkers for CRC. In this study, the expressing pattern and prognostic values of DNA repair genes in CRC patients were analyzed using TCGA database. GO and KEGG enrichment analyses were conducted to clarify the functional roles of dysregulated genes. We observed 358 differentially expressed DNA repair genes in CRC specimens, including 84 downregulated genes and 275 upregulated genes. 36 survival-related DNA repair genes were correlated with CRC patients' five-year survival, including 6 low-risk genes and 30 high-risk genes. Among the 10 overlapping genes, we focused on SLC6A1 which was highly expressed in CRC, and multivariate analysis confirmed that SLC6A1 expression as well as age and clinical stage could be regarded as an independent predicting factor for CRC prognosis. KEGG assays revealed that SLC6A1 may influence the clinical progression via regulating TGF-beta and PI3K-Akt signaling pathways. In addition, we observed that SLC6A1 was negatively regulated by SLC6A1 methylation, leading to its low expression in CRC specimens. Overall, SLC6A1 is upexpressed in CRC and can be used as a marker of poor prognosis in CRC patients.

Low Expression of AGPAT5 Is Associated With Clinical Stage and Poor Prognosis in Colorectal Cancer and Contributes to Tumour Progression.

Colorectal cancer (CRC) has a high prevalence and poor prognosis. This study aimed to identify biomarkers related to the clinical stage (I-IV) of CRC. The LinkedOmics database was used as the discovery cohort, and two Gene Expression Omnibus (GEO) databases (GSE41258 and GSE422848) served as validation cohorts. The trend test of genes related to clinical stage (I-IV) of CRC patients was identified by the Jonckheere-Terpstra test. The cBioPortal database, Gene Expression Profiling Interactive Analysis (GEPIA) and PrognoScan databases were used to explore the expression change and prognostic value of clinical stage-related genes in CRC patients. CRC cells overexpressed AGPAT5 were constructed and used for cell counting kit-8 (CCK-8), flow cytometric, and wound healing assays in vitro. We identified four clinical stage-related genes, GSR, AGPAT5, CRLF1, and NPR3, in CRC. The CNA frequencies of GSR, CRLF1, AGPAT5, and NPR3 occurred in 11%, 2.4%, 13%, and 3% of patients, respectively. The expression of GSR and AGPAT5 tended to decrease with CRC stage (I-IV) progression, and the expression of CRLF1 and NPR3 tended to increase with CRC stage (I-IV) progression. Compared with the normal group, AGPAT5 expression was markedly decreased in stage IV CRC. Higher GSR and AGPAT5 expression levels were associated with better overall survival (OS) and disease-free survival (DFS) in CRC patients. Lower CRLF1 and NPR3 expression levels were associated with better OS and DFS in CRC. GSR, CRLF1, AGPAT5, and NPR3 expression were related to CRC progression, microsatellite instability, and tumour purity in CRC. Furthermore, AGPAT5 was downregulated in CRC cell lines, and overexpression of AGPAT5 inhibited cell proliferation and migration and promoted cell apoptosis in CRC cells. Low AGPAT5 expression may serve as a poor prognostic factor and clinical stage biomarker in CRC. In addition, AGPAT5 acts as a tumour suppressor in CRC progression.

Novel diagnostic and prognostic biomarkers of colorectal cancer: Capable to overcome the heterogeneity-specific barrier and valid for global applications.

IntroductionThe heterogeneity-specific nature of the available colorectal cancer (CRC) biomarkers is significantly contributing to the cancer-associated high mortality rate worldwide. Hence, this study was initiated to investigate a system of novel CRC biomarkers that could commonly be employed to the CRC patients and helpful to overcome the heterogenetic-specific barrier.MethodsInitially, CRC-related hub genes were extracted through PubMed based literature mining. A protein-protein interaction (PPI) network of the extracted hub genes was constructed and analyzed to identify few more closely CRC-related hub genes (real hub genes). Later, a comprehensive bioinformatics approach was applied to uncover the diagnostic and prognostic role of the identified real hub genes in CRC patients of various clinicopathological features.ResultsOut of 210 collected hub genes, in total 6 genes (CXCL12, CXCL8, AGT, GNB1, GNG4, and CXCL1) were identified as the real hub genes. We further revealed that all the six real hub genes were significantly dysregulated in colon adenocarcinoma (COAD) patients of various clinicopathological features including different races, cancer stages, genders, age groups, and body weights. Additionally, the dysregulation of real hub genes has shown different abnormal correlations with many other parameters including promoter methylation, overall survival (OS), genetic alterations and copy number variations (CNVs), and CD8+T immune cells level. Finally, we identified a potential miRNA and various chemotherapeutic drugs via miRNA, and real hub genes drug interaction network that could be used in the treatment of CRC by regulating the expression of real hub genes.ConclusionIn conclusion, we have identified six real hub genes as potential biomarkers of CRC patients that could help to overcome the heterogenetic-specific barrier across different clinicopathological features.

Cytoplasmic MSH2 Related to Genomic Deletions in the MSH2/EPCAM Genes in Colorectal Cancer Patients With Suspected Lynch Syndrome.

BackgroundA large proportion of patients with Lynch syndrome (LS) have MSH2 abnormalities, but genotype-phenotype studies of MSH2 mutations in LS are still lacking. The aim of this study was to comprehensively analyze the clinicopathological characteristics and molecular basis of colorectal cancer (CRC) in patients with uncommon MSH2 cytoplasmic expression.MethodsWe retrospectively reviewed 4195 consecutive cases of CRC patients diagnosed between January 2015 and December 2017 at the Cancer Hospital Chinese Academy of Medical Sciences. Of the 4195 patients with CRC, 69 were indicated to have abnormal MSH2 expression through tumor immunohistochemical staining. Genetic tests, such as next-generation sequencing, large genomic rearrangement (LGR) analysis, microsatellite instability status analysis and genomic breakpoint analysis, were performed. Clinicopathological and molecular characteristics and clinical immunotherapy response were analyzed.ResultsForty-five of 69 patients were identified to have LS with pathogenic germline mutations in MSH2 and/or EPCAM. Of these LS patients, 26.7% were confirmed to harbor large genomic rearrangements (LGRs). Of note, three tumors from two unrelated family pedigrees exhibited a rare cytoplasmic MSH2 staining pattern that was found in LS patients with EPCAM/MSH2 deletions. RNA analysis showed that two novel mRNA fusions of EPCAM and MSH2 resulted in the predicted protein fusion with MSH2 cytoplasmic localization. Analyses of genomic breakpoints indicated that two novel deletions of EPCAM and MSH2 originated from Alu repeat-mediated recombination events. Our study also provides clinical evidence for the beneficial effect of the PD-1 inhibitor pembrolizumab for CRC patients that exhibit cytoplasmic MSH2 staining.ConclusionOur study demonstrates that the rare cytoplasmic MSH2 staining pattern should be fully recognized by pathologists and geneticists. Given the specific genotype-phenotype correlation in LS screening, we advocate that all CRC patients with cytoplasmic MSH2 staining in histology should be screened for LGRs of EPCAM and MSH2.

Application of targeted nanopore sequencing for the screening and determination of structural variants in patients with Lynch syndrome.

Lynch syndrome is a hereditary disease characterized by an increased risk of colorectal and other cancers. Germline variants in the mismatch repair (MMR) genes are responsible for this disease. Previously, we screened the MMR genes in colorectal cancer patients who fulfilled modified Amsterdam II criteria, and multiplex ligation-dependent probe amplification (MPLA) identified 11 structural variants (SVs) of MLH1 and MSH2 in 17 patients. In this study, we have tested the efficacy of long read-sequencing coupled with target enrichment for the determination of SVs and their breakpoints. DNA was captured by array probes designed to hybridize with target regions including four MMR genes and then sequenced using MinION, a nanopore sequencing platform. Approximately, 1000-fold coverage was obtained in the target regions compared with other regions. Application of this system to four test cases among the 17 patients correctly mapped the breakpoints. In addition, we newly found a deletion across an 84 kb region of MSH2 in a case without the pathogenic single nucleotide variants. These data suggest that long read-sequencing combined with hybridization-based enrichment is an efficient method to identify both SVs and their breakpoints. This strategy might replace MLPA for the screening of SVs in hereditary diseases.