Identification and validation of a pyroptosis-related prognostic model for colorectal cancer based on bulk and single-cell RNA sequencing data

BACKGROUND Pyroptosis impacts the development of malignant tumors,yet its role in colorectal cancer(CRC)prognosis remains uncertain.AIM To assess the prognostic significance of pyroptosis-related genes and their association with CRC immune infiltration.METHODS Gene expression data were obtained from The Cancer Genome Atlas(TCGA)and single-cell RNA sequencing dataset GSE178341 from the Gene Expression Omnibus(GEO).Pyroptosis-related gene expression in cell clusters was analyzed,and enrichment analysis was conducted.A pyroptosis-related risk model was developed using the LASSO regression algorithm,with prediction accuracy assessed through K-M and receiver operating characteristic analyses.A nomo-gram predicting survival was created,and the correlation between the risk model and immune infiltration was analyzed using CIBERSORTx calculations.Finally,the differential expression of the 8 prognostic genes between CRC and normal samples was verified by analyzing TCGA-COADREAD data from the UCSC database.RESULTS An effective pyroptosis-related risk model was constructed using 8 genes-CHMP2B,SDHB,BST2,UBE2D2,GJA1,AIM2,PDCD6IP,and SEZ6L2(P<0.05).Seven of these genes exhibited differential expression between CRC and normal samples based on TCGA database analysis(P<0.05).Patients with higher risk scores demonstrated increased death risk and reduced overall survival(P<0.05).Significant differences in immune infiltration were observed between low-and high-risk groups,correlating with pyroptosis-related gene expression.CONCLUSION We developed a pyroptosis-related prognostic model for CRC,affirming its correlation with immune infiltration.This model may prove useful for CRC prognostic evaluation.

Action of circulating and infiltrating B cells in the immune microenvironment of colorectal cancer by single-cell sequencing analysis

BACKGROUND The complexity of the immune microenvironment has an impact on the treatment of colorectal cancer(CRC),one of the most prevalent malignancies worldwide.In this study,multi-omics and single-cell sequencing techniques were used to investigate the mechanism of action of circulating and infiltrating B cells in CRC.By revealing the heterogeneity and functional differences of B cells in cancer immunity,we aim to deepen our understanding of immune regulation and provide a scientific basis for the development of more effective cancer treatment strategies.AIM To explore the role of circulating and infiltrating B cell subsets in the immune microenvironment of CRC,explore the potential driving mechanism of B cell development,analyze the interaction between B cells and other immune cells in the immune microenvironment and the functions of communication molecules,and search for possible regulatory pathways to promote the anti-tumor effects of B cells.METHODS A total of 69 paracancer(normal),tumor and peripheral blood samples were collected from 23 patients with CRC from The Cancer Genome Atlas database(https://portal.gdc.cancer.gov/).After the immune cells were sorted by multicolor flow cytometry,the single cell transcriptome and B cell receptor group library were sequenced using the 10X Genomics platform,and the data were analyzed using bioinformatics tools such as Seurat.The differences in the number and function of B cell infiltration between tumor and normal tissue,the interaction between B cell subsets and T cells and myeloid cell subsets,and the transcription factor regulatory network of B cell subsets were explored and analyzed.RESULTS Compared with normal tissue,the infiltrating number of CD20+B cell subsets in tumor tissue increased significantly.Among them,germinal center B cells(GCB)played the most prominent role,with positive clone expansion and heavy chain mutation level increasing,and the trend of differentiation into memory B cells increased.However,the number of plasma cells in the tumor microenvironment decreased significantly,and the plasma cells secreting IgA antibodies decreased most obviously.In addition,compared with the immune microenvironment of normal tissues,GCB cells in tumor tissues became more closely connected with other immune cells such as T cells,and communication molecules that positively regulate immune function were significantly enriched.CONCLUSION The role of GCB in CRC tumor microenvironment is greatly enhanced,and its affinity to tumor antigen is enhanced by its significantly increased heavy chain mutation level.Meanwhile,GCB has enhanced its association with immune cells in the microenvironment,which plays a positive anti-tumor effect.

Special AT-rich sequence-binding protein 1 promotes cell growth and metastasis in colorectal cancer

AIM: To evaluate the expression of special AT-rich sequence-binding protein 1 (SATB1 ) gene in colorectal cancer and its role in colorectal cancer cell proliferation and invasion.METHODS: Immunohistochemistry was used to detect the protein expression of SATB1 in 30 colorectal cancer (CRC) tissue samples and pair-matched adjacent nontumor samples. Cell growth was investigated after enhancing expression of SATB1. Wound-healing assay and Transwell assay were used to investigate the impact of SATB1 on migratory and invasive abilities of SW480 cells in vitro . Nude mice that received subcutaneous implantation or lateral tail vein were used to study the effects of SATB1 on tumor growth or metastasis in vivo . RESULTS: SATB1 was over-expressed in CRC tissues and CRC cell lines. SATB1 promotes cell proliferation and cell cycle progression in CRC SW480 cells. SATB1 over-expression could promote cell growth in vivo . In addition, SATB1 could significantly raise the ability of cell migration and invasion in vitro and promote the ability of tumor metastasis in vivo . SATB1 could up-regulate matrix metalloproteases 2, 9, cyclin D1 and vimentin, meanwhile SATB1 could down-regulate E-cadherin in CRC. CONCLUSION: SATB1 acts as a potential growth and metastasis promoter in CRC. SATB1 may be useful as a therapeutic target for CRC.

Special AT-rich sequence-binding protein 2 acts as a negative regulator of stemness in colorectal cancer cells

AIM To find the mechanisms by which special AT-rich sequence-binding protein 2(SATB2) influences colorectal cancer(CRC) metastasis.METHODS Cell growth assay, colony-forming assay, cell adhesion assay and cell migration assay were used to evaluate the biological characteristics of CRC cells with gain or loss of SATB2. Sphere formation assay was used to detect the self-renewal ability of CRC cells. The m RNA expression of stem cell markers in CRC cells with upregulated or downregulated SATB2 expression was detected by quantitative real-time polymerase chain reaction. Chromatin immunoprecipitation(Ch IP) was used to verify the binding loci of SATB2 on genomic sequences of stem cell markers. The Cancer Genome Atlas(TCGA) database and our clinical samples wereanalyzed to find the correlation between SATB2 and some key stem cell markers.RESULTS Downregulation of SATB2 led to an aggressive phenotype in SW480 and DLD-1 cells, which was characterized by increased migration and invasion abilities. Overexpression of SATB2 suppressed the migration and invasion abilities in SW480 and SW620 cells. Using sequential sphere formation assay to detect the selfrenewal abilities of CRC cells, we found more secondary sphere formation but not primary sphere formation in SW480 and DLD-1 cells after SATB2 expression was knocked down. Moreover, most markers for stem cells such as CD133, CD44, AXIN2, MEIS2 and NANOG were increased in cells with SATB2 knockdown and decreased in cells with SATB2 overexpression. Ch IP assay showed that SATB2 bound to regulatory elements of CD133, CD44, MEIS2 and AXIN2 genes. Using TCGA database and our clinical samples, we found that SATB2 was correlated with some key stem cell markers including CD44 and CD24 in clinical tissues of CRC patients.CONCLUSION SATB2 can directly bind to the regulatory elements in the genetic loci of several stem cell markers and consequently inhibit the progression of CRC by negatively regulating stemness of CRC cells.

Clinical applications of next-generation sequencing in colorectal cancers

Like other solid tumors,colorectal cancer(CRC) is a genomic disorder in which various types of genomic alterations,such as point mutations,genomic rearrangements,gene fusions,or chromosomal copy number alterations,can contribute to the initiation and progression of the disease. The advent of a new DNA sequencing technology known as next-generation sequencing(NGS) has revolutionized the speed and throughput of cataloguing such cancer-related genomic alterations. Now the challenge is how to exploit this advanced technology to better understand the underlying molecular mechanism of colorectal carcinogenesis and to identify clinically relevant genetic biomarkers for diagnosis and personalized therapeutics. In this review,we will introduce NGS-based cancer genomics studies focusing on those of CRC,including a recent large-scale report from the Cancer Genome Atlas. We will mainly discuss how NGS-based exome-,whole genome- and methylome-sequencing have extended our understanding of colorectal carcinogenesis. We will also introduce the unique genomic features of CRC discovered by NGS technologies,such as the relationship with bacterial pathogens and the massive genomic rearrangements of chromothripsis. Finally,we will discuss the necessary steps prior to development of a clinical application of NGS-related findings for the advanced management of patients with CRC.

Gene mutations in stool from gastric and colorectal neoplasia patients by next-generation sequencing

AIM To study cancer hotspot mutations by next-generation sequencing(NGS) in stool DNA from patients with different gastrointestinal tract(GIT) neoplasms. METHODS Stool samples were collected from 87 Finnish patients diagnosed with various gastric and colorectal neoplasms, including benign tumors, and from 14 healthy controls. DNA was isolated from stools by usingthe PSP~? Spin Stool DNA Plus Kit. For each sample, 20 ng of DNA was used to construct sequencing libraries using the Ion AmpliS eq Cancer Hotspot Panel v2 or Ion AmpliS eq Colon and Lung Cancer panel v2. Sequencing was performed on Ion PGM. Torrent Suite Software v.5.2.2 was used for variant calling and data analysis.RESULTS NGS was successful in assaying 72 GIT samples and 13 healthy controls, with success rates of the assay being78% for stomach neoplasia and 87% for colorectal tumors. In stool specimens from patients with gastric neoplasia, five hotspot mutations were found in APC,CDKN2 A and EGFR genes, in addition to seven novel mutations. From colorectal patients, 20 mutations were detected in AKT1, APC, ERBB2, FBXW7, KIT, KRAS,NRAS, SMARCB1, SMO, STK11 and TP53. Healthy controls did not exhibit any hotspot mutations, except for two novel ones. APC and TP53 were the most frequently mutated genes in colorectal neoplasms, with five mutations, followed by KRAS with two mutations.APC was the most commonly mutated gene in stools of patients with premalignant/benign GIT lesions.CONCLUSION Our results show that in addition to colorectal neoplasms,mutations can also be assayed from stool specimens of patients with gastric neoplasms.

Whole exome sequencing and single nucleotide polymorphism array analyses to identify germline alterations in genes associated with testosterone metabolism in a patient with androgen insensitivity syndrome and early-onset colorectal cancer

Background: Androgen insensitivity syndrome(AIS), a disorder of sexual development in 46, XY individuals, is caused by loss-of-function mutations in the androgen receptor(AR) gene. A variety of tumors have been reported in association with AIS, but no cases with colorectal cancer(CRC) have been described.Case presentation: Here, we present a male patient with AIS who developed multiple early-onset CRCs and his pedigree. His first cousin was diagnosed with AIS and harbored the same AR gene mutation, but with no signs of CRC. The difference in clinical management for the two patients was that testosterone treatment was given to the proband for a much longer time compared with the cousin. The CRC family history was negative, and no germline mutations in well-known CRC-related genes were identified. A single nucleotide polymorphism array revealed a microduplication on chromosome 22q11.22 that encompassed a micro RNA potentially related to CRC pathogenesis. In the proband, whole exome sequencing identified a polymorphism in an oncogene and 13 rare loss-of-function variants, of which two were in CRC-related genes and four were in genes associated with other human cancers.Conclusions: By pathway analysis, all inherited germline genetic events were connected in a unique network whose alteration in the proband, together with continuous testosterone stimulation, may have played a role in CRC pathogenesis.

Fecal gene detection based on next generation sequencing for colorectal cancer diagnosis

BACKGROUND Colorectal cancer(CRC)is one of the most common malignancies worldwide.Given its insidious onset,the condition often already progresses to advanced stage when symptoms occur.Thus,early diagnosis is of great significance for timely clinical intervention,efficacy enhancement,and prognostic improvement.Featuring high throughput,fastness,and rich information,next generation sequencing(NGS)can greatly shorten the detection time,which is a widely used detection technique at present.AIM To screen specific genes or gene combinations in fecal DNA that are suitable for diagnosis and prognostic prediction of CRC,and to establish a technological platform for CRC screening,diagnosis,and efficacy monitoring through fecal DNA detection.METHODS NGS was used to sequence the stool DNA of patients with CRC,which were then compared with the genetic testing results of the stool samples of normal controls and patients with benign intestinal disease,as well as the tumor tissues of CRC patients.Specific genes or gene combinations in fecal DNA suitable for diagnosis and prognostic prediction of CRC were screened,and their significances in diagnosing CRC and predicting patients'prognosis were comprehensively evaluated.RESULTS High mutation frequencies of TP53,APC,and KRAS were detected in the stools and tumor tissues of CRC patients prior to surgery.Contrastively,no pathogenic mutations of the above three genes were noted in the postoperative stools,the normal controls,or the benign intestinal disease group.This indicates that tumor-specific DNA was detectable in the preoperative stools of CRC patients.The preoperative fecal expression of tumor-associated genes can reflect the gene mutations in tumor tissues to some extent.Compared to the postoperative stools and the stools in the two control groups,the pathogenic mutation frequencies of TP53 and KRAS were significantly higher for the preoperative stools(χ^(2)=7.328,P<0.05;χ^(2)=4.219,P<0.05),suggesting that fecal TP53 and KRAS genes can be used for CRC screening,diagnosis,and prognostic prediction.No significant difference in the pathogenic mutation frequency of the APC gene was found from the postoperative stools or the two control groups(χ^(2)=0.878,P>0.05),so further analysis with larger sample size is required.Among CRC patients,the pathogenic mutation sites of TP53 occurred in 16 of 27 preoperative stools,with a true positive rate of 59.26%,while the pathogenic mutation sites of KRAS occurred in 10 stools,with a true positive rate of 37.04%.The sensitivity and negative predictive values of the combined genetic testing of TP53 and KRAS were 66.67%(18/27)and 68.97%,respectively,both of which were higher than those of TP53 or KRAS mutation detection alone,suggesting that the combined genetic testing can improve the CRC detection rate.The mutation sites TP53 exon 4 A84G and EGFR exon 20 I821T(mutation start and stop positions were both 7579436 for the former,while 55249164 for the latter)were found in the preoperative stools and tumor tissues.These"undetected"mutation sites may be new types of mutations occurring during the CRC carcinogenesis and progression,which needs to be confirmed through further research.Some mutations of"unknown clinical significance"were found in such genes as TP53,PTEN,KRAS,BRAF,AKT1,and PIK3CA,whose clinical values is worthy of further exploration.CONCLUSION NGS-based fecal genetic testing can be used as a complementary technique for the CRC diagnosis.Fecal TP53 and KRAS can be used as specific genes for the screening,diagnosis,prognostic prediction,and recurrence monitoring of CRC.Moreover,the combined testing of TP53 and KRAS genes can improve the CRC detection rate.

Molecular methods for colorectal cancer screening:Progress with next-generation sequencing evolution

Currently,colorectal cancer(CRC)represents the third most common malignancy and the second most deadly cancer worldwide,with a higher incidence in developed countries.Like other solid tumors,CRC is a heterogeneous genomic disease in which various alterations,such as point mutations,genomic rearrangements,gene fusions or chromosomal copy number alterations,can contribute to the disease development.However,because of its orderly natural history,easily accessible onset location and high lifetime incidence,CRC is ideally suited for preventive intervention,but the many screening efforts of the last decades have been compromised by performance limitations and low penetrance of the standard screening tools.The advent of next-generation sequencing(NGS)has both facilitated the identification of previously unrecognized CRC features such as its relationship with gut microbial pathogens and revolutionized the speed and throughput of cataloguing CRC-related genomic alterations.Hence,in this review,we summarized the several diagnostic tools used for CRC screening in the past and the present,focusing on recent NGS approaches and their revolutionary role in the identification of novel genomic CRC characteristics,the advancement of understanding the CRC carcinogenesis and the screening of clinically actionable targets for personalized medicine.

Sequencing of treatment in metastatic colorectal cancer:Where to fit the target?

Colorectal cancer is a lethal disease if not discovered early.Even though appropriate screening and preventive strategies are in place in many countries,a significant number of patients are still diagnosed at late stages of the disease.The management of metastatic colorectal cancer remains a significant clinical challenge to oncologists worldwide.While cytotoxic regimens constitute the main treatment of choice in this patient population,addition of the five biologics(bevacizumab,cetuximab,aflibercept,panitumumab and regorafenib)to these regimens has improved clinical outcomes.The most commonly used cytotoxic regimens include doublet combinations(FOLFOX/XELOX or FOLFIRI).Many clinical trials have been published and others are underway to compare the biologic agents with one another in order to prove the superiority of one regimen over another.Metastatic colorectal cancer patients have many treatment options;however,the optimal use and sequence of targeted agents remain to be determined.This review entails concise and updated clinical data on the management of metastatic colorectal cancer.The aim of the review is to determine where to fit the five biologic targets into the treatment algorithm of metastatic colorectal cancer patients and to derive treatment sequences that would achieve best clinical outcome based on the current available data.

Comprehensive next-generation sequencing reveals double primary colorectal carcinoma missed by diagnostic imaging: A case report

BACKGROUND Multiple primary colorectal carcinoma(MPCC)is a rare clinical disease,which is challenging to differentiate from metastatic disease using histopathological methods.Next-generation sequencing(NGS)has been employed to identify multiple primary cancers.CASE SUMMARY This study a rare case of a 63-year-old male patient diagnosed with MPCC by targeted NGS,which was initially missed by radiological evaluation.The patient was found to have two tumors located on the surface of the colorectum which had distinct genomic alterations.Based on wild-type KRAS detected in the unresected tumor,the patient benefited from the epidermal growth factor receptor(EGFR)inhibitor cetuximab treatment,but developed novel mutations including KIF5B-RET fusion,which provides a possible resistance mechanism to anti-EGFR therapy.CONCLUSION Our case highlights the necessity of using genetic testing for primary tumor diagnosis and the application of serial plasma circulating tumor DNA profiling for dynamic disease monitoring.

Epithelial toll-like receptor 9 signaling in colorectal inflammation and cancer: Clinico-pathogenic aspects

Toll-like receptors (TLRs) recognize specific motifs which are frequently present in bacteria, fungi, prokaryotes and viruses. Amongst TLRs, TLR9 can be activated by such bacterial or viral DNA fragments, immunoglobulin-DNA complexes or synthetic oligonucleotides, which all contain unmethylated cytosineguanine nucleotide sequences (CpGs). Emerging data indicate that TLR9 signaling has a role in, and may influence, colorectal carcinogenesis and colonic inflammation. CpGs are classified into three groups according to their influence on both the antigen-specific humoraland cellular immunity, and the production of type 1 interferons and proinflammatory cytokines. TLR9 activation via CpGs may serve as a new therapeutic target for several cancerous and various inflammatory conditions. Due to its probable anti-cancer effects, the application possibilities of TLR9-signaling modulation may be extremely diverse even in colorectal tumors. In this review we aimed to summarize the current knowledge about TLR-signaling in the pathogenesis and therapy of inflammatory bowel diseases and colorectal cancer. Due to the species-specific differences in TLR9 expression, however, one must be careful in translating the animal model data into the human system, because of the differences between CpG-oligodeoxynucleotide-responsive cells. TLR9 agonist DNA-based immunomodulatory sequences could also represent a promising therapeutic alternative in systemic inflammatory conditions and chronic colonic inflammations as their side effects are not significant.

Differential mucin phenotypes and their significance in a variation of colorectal carcinoma

AIM: To investigate mucin expression profiles in colorectal carcinoma (CRC) histological subtypes with regard to clinicopathologic variables and prognosis. METHODS: Mucin (MUC)2 and MUC5AC expressions were assessed by immunohistochemistry for a total of 250 CRC cases that underwent surgical resection. CRCs included 63 well-to-moderately differentiated adenocar-cinomas (WMDAs), 91 poorly differentiated adenocarcinomas (PDAs), 81 mucinous adenocarcinoma (MUAs), and 15 signet-ring cell carcinomas (SRCCs). MUC2 and MUC5AC were scored as positive when ≥ 25% and ≥ 1% of cancer cells were stained positive, respectively. The human mutL homolog 1 and human mutS homolog 2 expressions were assessed by immunohistochemistry in PDAs to investigate mismatch-repair (MMR) status.Tumors that did not express either of these two were considered MMR-deficient. Results were analyzed for associations with clinicopathologic variables and the prognosis in individual histological CRC subtypes. RESULTS: MUC2-positive and MUC5AC-positive WMDA percentages were 49.2% and 30.2%, respectively. In contrast, MUC2-positive and MUC5AC-positive PDA percentages were 9.5% and 51.6%, respectively. MUC2 levels tended to decrease and MUC5AC levels tended to increase from WMDA to PDA. In 21 tumors comprising both adenoma and adenocarcinoma components in a single tumor (4 WMDAs, 7 PDAs, and 10 MUAs), MUC2 was significantly downregulated in PDA and MUC5AC was downregulated in PDA and MUA in the adenoma-carcinoma sequence. These results suggested that MUC2 levels might be associated with malignant potential and that MUC5AC expression was an early event in tumorigenesis. Despite worse prognoses than WMDA, high MUC2 expression levels were maintained in MUA (95.1%) and SRCC (71.5%), which suggested a pathogenesis for these subtypes distinct from that of WMDA. No significant associations were found between MUC2 expression and any clinicopathologic variables in any histological subtype. MUC5AC expression in PDA was closely associated with right-sided location (P = 0.017), absence of nodal metastasis (P = 0.010), low tumor node metastasis stage (P = 0.010), and MMR deficiency (P = 0.003). MUC2 expression in WMDA was a marginal prognostic factor for recurrence/metastasis-free survival (RFS) by univariate Cox analysis (P = 0.077) but not by multivariate Cox analysis (P = 0.161). MUC5AC expression in PDA was a significant prognostic factor for RFS by univariate Cox analysis (P = 0.007) but not by multivariate Cox analysis (P = 0.104). Kaplan-Meier curves and log-rank tests revealed that MUC2 expression was marginally associated with a better WMDA prognosis [P = 0.064 for RFS and P = 0.172 for overall survival (OS)] but not for PDA. In contrast, MUC5AC expression was significantly and marginally associated with a better PDA prognosis in terms of RFS and OS, respectively(P = 0.004 for RFS and P = 0.100 for OS), but not for WMDA and MUA. CONCLUSION: Mucin core protein expression profiles and clinical significance differ according to histological CRC subtypes. This may reflect different pathogeneses for these tumors.

Histologic features and genomic alterations of primary colorectal adenocarcinoma predict growth patterns of liver metastasis

BACKGROUND Different histological growth patterns(HGPs)of colorectal carcinoma(CRC)liver metastasis are associated with patients’prognosis and response to antiangiogenic therapy.However,the relationship between HGPs of liver metastasis and clinicopathological and genomic characteristics of primary cancer has not been well established.AIM To assess whether certain clinicopathological and genomic features of primary CRC could predict the HGPs of liver metastasis.METHODS A total of 29 patients with paired resections of both primary CRC and liver metastasis were divided into two groups:A(15 cases with desmoplastic liver metastasis)and B(14 cases with replacement liver metastasis).Clinical information was obtained from patients’charts.Mismatch repair proteins,BRAFV600E,and PD-L1 were evaluated by immunohistochemistry.Five cases were selected randomly from each group for whole exome sequencing(WES)analysis.RESULTS In the primary tumor,expanding growth pattern,low tumor budding score(TBS),and Crohn’s disease-like response(CDR)were associated with desmoplastic liver metastasis and better overall survival,whereas infiltrating growth pattern alone of primary carcinoma could predict the replacement liver metastasis and worse overall survival(P<0.05).On WES analysis,primary carcinoma with desmoplastic liver metastasis showed mutations in APC(4/5);TP53(3/5);KRAS,PIK3CA,and FAT4(2/5);BRCA-1,BRCA2,BRAF,and DNAH5(1/5),whereas primary carcinoma with replacement liver metastasis showed mutations in APC and TP53(3/5);KRAS,FAT4,DNH5,SMAD,ERBB2,ERBB3,LRP1,and SDK1(1/5).CONCLUSION The HGPs,TBS,and CDR of primary CRC as well as the presence of specific genetic mutations such as those in PIK3CA could be used to predict the HGPs of liver metastasis,response to therapy,and patients’prognosis.

Structural shift of gut microbiota during chemopreventive effects of epigallocatechin gallate on colorectal carcinogenesis in mice

AIM To investigate the effect of epigallocatechin gallate(EGCG) on structural changes of gut microbiota in colorectal carcinogenesis.METHODS An azoxymethane(AOM)/dextran sodium sulfate(DSS)-induced colitis mouse model was established. Fortytwo female FVB/N mice were randomly divided into the following three groups: group 1(10 mice, negative control) was treated with vehicle, group 2(16 mice, positive control) was treated with AOM plus vehicle, and group 3(16 mice, EG) was treated with AOM plus EGCG. For aberrant crypt foci(ACF) evaluation, the colons were rapidly took out after sacrifice, rinsed with saline, opened longitudinally, laid flat on a polystyrene board, and fixed with 10% buffered formaldehyde solution before being stained with 0.2% methylene blue in saline. For tumor evaluation, the colon was macroscopically inspected and photographed, then the total number of tumors was enumerated and tumor size measured. For histological examination, the fixed tissues were paraffin-embedded and sectioned at 5 mm thickness. Microbial genomic DNA was extracted from fecal and intestinal content samples using a commercial kit. The V4 hypervariable regions of 16 S r RNA were PCR-amplified with the barcoded fusion primers. Using the best hit classification option, the sequences from each sample were aligned to the RDP 16 S r RNA training set to classify the taxonomic abundance in QIIME. Statistical analyses were then performed.RESULTS Treatment of mice with 1% EGCG caused a significant decrease in the mean number of ACF per mouse, when compared with the model mice treated with AOM/DSS(5.38 ± 4.24 vs 13.13 ± 3.02, P < 0.01). Compared with the positive control group, 1% EGCG treatment dependently decreased tumor load per mouse by 85%(33.96 ± 6.10 vs 2.96 ± 2.86, respectively, P < 0.01). All revealed that EGCG could inhibit colon carcinogenesis by decreasing the number of precancerous lesions as well as solid tumors, with reduced tumor load and delayed histological progression of CRC. During the cancerization, the diversity of gut microbiota increased, potential carcinogenic bacteria such as Bacteroides were enriched, and the abundance of butyrate-producing bacteria(Clostridiaceae, Ruminococcus, etc.) decreased continuously. In contrast, the structure of gut microbiota was relatively stable during the intervention of EGCG on colon carcinogenesis. Enrichment of probiotics(Bifidobacterium, Lactobacillu, etc.) might be a potential mechanism for EGCG's effects on tumor suppression. Via bioinformatics analysis, principal coordinate analysis and cluster analysis of the tumor formation process, we found that the diversity of gut microbiota increased in the tumor model group while that in the EGCG interfered group(EG) remained relatively stable.CONCLUSION Gut microbiota imbalance might be a potential mechanism for the prevention of malignant transformation by EGCG, which is significant for diagnosis, treatment, prognosis evaluation, and prevention of colorectal cancer.

Use of blood-based biomarkers for early diagnosis and surveillance of colorectal cancer

Early screening for colorectal cancer(CRC) holds the key to combat and control the increasing global burden of CRC morbidity and mortality. However, the current available screening modalities are severely inadequate because of their high cost and cumbersome preparatory procedures that ultimately lead to a low participation rate. People simply do not like to have colonoscopies. It would be ideal, therefore, to develop an alternative modality based on blood biomarkers as the first line screening test. This will allow for the differentiation of the general population from high risk individuals. Colonoscopy would then become the secondary test, to further screen the high risk segment of the population. This will encourage participation and therefore help to reach the goal of early detection and thereby reduce the anticipated increasing global CRC incidence rate. A blood-based screening test is anappealing alternative as it is non-invasive and poses minimal risk to patients. It is easy to perform, can be repeated at shorter intervals, and therefore would likely lead to a much higher participation rate. This review surveys various blood-based test strategies currently under investigation, discusses the potency of what is available, and assesses how new technology may contribute to future test design.

Human colorectal cancer cells frequently express IgG and display unique Ig repertoire

BACKGROUND There is growing evidence proving that many human carcinomas, including colon cancer, can overexpress immunoglobulin(Ig); the non B cancer cell-derived Ig usually displayed unique V(D)J rearrangement pattern that are distinct from B cell-derived Ig. Especially, the cancer-derived Ig plays important roles in cancer initiation, progression, and metastasis. However, it still remains unclear if the colon cancer-derived Ig can display unique V(D)J pattern and sequencing, which can be used as novel target for colon cancer therapy.AIM To investigate the Ig repertoire features expressed in human colon cancer cells.METHODS Seven cancerous tissue samples of colon adenocarcinoma and corresponding noncancerous tissue samples were sorted by fluorescence-activated cell sorting using epithelial cell adhesion molecule as a marker for epithelial cells. Ig repertoire sequencing was used to analyze the expression profiles of all 5 classes of Ig heavy chains(IgH) and the Ig repertoire in colon cancer cells and corresponding normal epithelial cells.RESULTS We found that all 5 IgH classes can be expressed in both colon cancer cells and normal epithelial cells. Surprisingly, unlike the normal colonic epithelial cells that expressed 5 Ig classes, our results suggested that cancer cells most prominently express IgG. Next, we found that the usage of Ig in cancer cells caused the expression of some unique Ig repertoires compared to normal cells. Some VH segments, such as VH3-7, have been used in cancer cells, and VH3-74 was frequently present in normal epithelial cells. Moreover, compared to the normal cell-derived Ig, most cancer cell-derived Ig showed unique VHDJH patterns.Importantly, even if the same VHDJH pattern was seen in cancer cells and normal cells, cancer cell-derived IgH always displayed distinct hypermutation hot points.CONCLUSION We found that colon cancer cells could frequently express IgG and unique IgH repertoires, which may be involved in carcinogenesis of colon cancer. The unique IgH repertoire has the potential to be used as a novel target in immune therapy for colon cancer.

Gut mucosal microbiota profiles linked to colorectal cancer recurrence

BACKGROUND Emerging evidence links gut microbiota to various human diseases including colorectal cancer(CRC)initiation and development.However,gut microbiota profiles associated with CRC recurrence and patient prognosis are not completely understood yet,especially in a Chinese cohort.AIM To investigate the relationship between gut mucosal microbiota profiles and CRC recurrence and patient prognosis.METHODS We obtained the composition and structure of gut microbiota collected from 75 patients diagnosed with CRC and 26 healthy controls.The patients were followed up by regular examination to determine whether tumors recurred.Triplet-paired samples from on-tumor,adjacent-tumor and off-tumor sites of patients diagnosed with/without CRC recurrence were analyzed to assess spatial-specific patterns of gut mucosal microbiota by 16S ribosomal RNA sequencing.Next,we carried out bioinformatic analyses,Kaplan-Meier survival analyses and Cox regression analyses to determine the relationship between gut mucosal microbiota profiles and CRC recurrence and patient prognosis.RESULTS We observed spatial-specific patterns of gut mucosal microbiota profiles linked to CRC recurrence and patient prognosis.A total of 17 bacterial genera/families were identified as potential biomarkers for CRC recurrence and patient prognosis,including Anaerotruncus,Bacteroidales,Coriobacteriaceae,Dialister,Eubacterium,Fusobacterium,Filifactor,Gemella,Haemophilus,Mogibacteriazeae,Pyramidobacter,Parvimonas,Porphyromonadaceae,Slackia,Schwartzia,TG5 and Treponema.CONCLUSION Our work suggests that intestinal microbiota can serve as biomarkers to predict the risk of CRC recurrence and patient death.

Correlation between invasive microbiota in margin-surrounding mucosa and anastomotic healing in patients with colorectal cancer

BACKGROUND Impaired anastomotic healing is one of the major complications resulting from radical resection in colorectal cancer(CRC).Accumulating evidence suggests that intestinal microbiota is correlated with anastomotic healing.AIM To explore the microbiota structural shift in margin-surrounding mucosa and evaluate the predictive ability of selected bacterial taxa for impaired anastomotic healing.METHODS Margin-surrounding mucosa samples derived from 37 patients were collected to characterize the microbial community structure by 16 s r RNA gene sequencing.The patients were divided into two groups according to the healing status of anastomoses:well-healing group(n=30)and impaired-healing group(n=7).Statistic differences in bacteria taxa were compared by Wilcoxon test and chisquared test.The predictive ability of the selected bacterial taxa for the healing status of anastomoses was evaluated by the area under the receiver operator characteristic curve.RESULTS Community structure shifts were observed in the impaired-healing group andwell-healing group.Six bacterial species were found to be significantly correlated with anastomotic healing,and among these species,Alistipes shahii,Dialister pneumosintes,and Corynebacterium suicordis were considered as the predictive factors.Taking the known risk factor age into consideration,Alistipes shahii,Dialister pneumosintes,and Corynebacterium suicordis improved predictive ability for the healing status of anastomoses.CONCLUSION These data show that Alistipes shahii,Dialister pneumosintes,and Corynebacterium suicordis could be considered as supplementary factors in the prediction of anastomosis healing status in patients after CRC radical resection.

Differential expression of a novel colorectal cancer differentiation-related gene in colorectal cancer

AIM: To investigate SBA2 expression in CRC cell lines and surgical specimens of CRC and autologous healthy mucosa. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was used for relative quantification of SBA2 mRNA levels in 4 human CRC cell lines with different grades of differentiation and 30 clinical samples. Normalization of the results was achieved by simultaneous amplification of beta-actin as an internal control. RESULTS: In the exponential range of amplification, fairly good linearity demonstrated identical amplification efficiency for SBA2 and beta-actin (82%). Markedly lower levels of SBA2 mRNA were detectable in tumors, as compared with the coupled normal counterparts P【0.01). SBA2 expression was significantly (0.01】P 【 0.05) correlated with the grade of differentiation in CRC, with relatively higher levels in well-differentiated samples and lower in poorly-differentiated cases. Of the 9 cases with lymph nodes affected, 78% (7/9) had reduced SBA2 mRNA expression in contrast to 24% (5/21) in non-metastasis samples 0.01】P【0.05). CONCLUSION: SBA2 gene might be a promising novel biomarker of cell differentiation in colorectal cancer and its biological features need further studies.