American Society for Gastrointestinal Endoscopy-European Society of Gastrointestinal Endoscopy guideline on primary endoscopic bariatric and metabolic therapies for adults with obesity.

This joint ASGE-ESGE guideline provides an evidence-based summary and recommendations regarding the role of endoscopic bariatric and metabolic therapies (EBMTs) in the management of obesity. The document was developed using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) framework. It evaluates the efficacy and safety of EBMT devices and procedures that currently have CE mark or FDA-clearance/approval, or that had been approved within five years of document development. The guideline suggests the use of EBMTs plus lifestyle modification in patients with a BMI of ≥ 30 kg/m2, or with a BMI of 27.0-29.9 kg/m2 with at least 1 obesity-related comorbidity. Furthermore, it suggests the utilization of intragastric balloons and devices for endoscopic gastric remodeling (EGR) in conjunction with lifestyle modification for this patient population.

Microenvironment, systemic inflammatory response and tumor markers considering consensus molecular subtypes of colorectal cancer.

Introduction: Colorectal carcinomas (CRC) are one of the most frequent malignancies worldwide. Based on gene expression profile analysis, CRCs can be classified into four distinct subtypes also known as the consensus molecular subtypes (CMS), which predict biological behaviour. Besides CMS, several other aspects of tumor microenvironment (TME) and systemic inflammatory response (SIR) influence the outcome of CRC patients. TME and inflammation have important role in the immune (CMS1) and mesenchymal (CMS4) subtypes, however, the relationship between these and systemic inflammation has not been assessed yet. Our objective was to evaluate the connection between CMS, TME and SIR, and to analyze the correlation between these markers and routinely used tumor markers, such as CEA (Carcinoembryonic Antigen) and CA19-9 (Carbohydrate Antigen 19-9). Methods: FFPE (Formalin Fixed Paraffin Embedded) samples of 185 CRC patients were collected. TME was described using tumor-stroma ratio (TSR), Klintrup-Makinen (KM) grade, and Glasgow Microenvironment Score (GMS). CMS classification was performed on tissue microarray using MLH1, PMS2, MSH2 and MSH6, and pan-cytokeratin, CDX2, FRMD6, HTR2B and ZEB1 immunohistochemical stains. Pre-operative tumor marker levels and inflammatory markers [C-reactive protein - CRP, albumin, absolute neutrophil count (ANC), absolute lymphocyte count (ALC), absolute platelet count (APC)] and patient history were retrieved using MedSolution database. Results: Amongst TME-markers, TSR correlated most consistently with adverse clinicopathological features (p < 0.001) and overall survival (p < 0.001). Elevated CRP and modified Glasgow Prognostic Score (mGPS) were associated with worse outcome and aggressive phenotype, similarly to tumor markers CEA and CA19-9. Stroma-Tumor Marker score (STM score), a new combined score of CA19-9 and TSR delivered the second best prognostication after mGPS. Furthermore, CMS4 showed association with TSR and several laboratory markers (albumin and platelet derived factors), but not with other SIR descriptors. CMS did not show any association with CEA and CA19-9 tumor markers. Conclusion: More routinely available TME, SIR and tumor markers alone and in combination deliver reliable prognostic data for choosing the patients with higher risk for propagation. CMS4 is linked with high TSR and poor prognosis, but in overall, CMS-classification showed only limited effect on SIR- and tumor-markers.

American Society for Gastrointestinal Endoscopy-European Society of Gastrointestinal Endoscopy guideline on primary endoscopic bariatric and metabolic therapies for adults with obesity.

This joint ASGE-ESGE guideline provides an evidence-based summary and recommendations regarding the role of endoscopic bariatric and metabolic therapies (EBMTs) in the management of obesity. The document was developed using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) framework. It evaluates the efficacy and safety of EBMT devices and procedures that currently have CE mark or FDA-clearance/approval, or that had been approved within five years of document development. The guideline suggests the use of EBMTs plus lifestyle modification in patients with a BMI of ≥30 kg/m2, or with a BMI of 27.0-29.9 kg/m2 with at least 1 obesity-related comorbidity. Furthermore, it suggests the utilization of intragastric balloons and devices for endoscopic gastric remodeling (EGR) in conjunction with lifestyle modification for this patient population.

Curriculum for bariatric endoscopy and endoscopic treatment of the complications of bariatric surgery: European Society of Gastrointestinal Endoscopy (ESGE) Position Statement.

Obesity is a chronic, relapsing, degenerative, multifactorial disease that is associated with many co-morbidities. The global increasing burden of obesity has led to calls for an urgent need for additional treatment options. Given the rapid expansion of bariatric endoscopy and bariatric surgery across Europe, the European Society of Gastrointestinal Endoscopy (ESGE) has recognized the need to formalize and enhance training in bariatric endoscopy and the endoscopic treatment of bariatric surgical adverse events. This manuscript represents the outcome of a formal Delphi process resulting in an official Position Statement of the ESGE and provides a framework to develop and maintain skills in bariatric endoscopy and the endoscopic treatment of bariatric surgical adverse events. This curriculum is set out in terms of the prerequisites prior to training, minimum number of procedures, the steps for training and quality of training, and how competence should be defined and evidenced before independent practice. 1: ESGE recommends that every endoscopist should have achieved competence in upper gastrointestinal endoscopy before commencing training in bariatric endoscopy and the endoscopic treatment of bariatric surgical adverse events. 2: Trainees in bariatric endoscopy and the endoscopic treatment of the complications of bariatric surgery should have basic knowledge of the definition, classification, and social impact of obesity, its pathophysiology, and its related co-morbidities. The recognition and management of gastrointestinal diseases that are more common in patients with obesity, along with participation in multidisciplinary teams where obese patients are evaluated, are mandatory. 3 : ESGE recommends that competency in bariatric endoscopy and the endoscopic treatment of the complications of bariatric surgery can be learned by attending validated training courses on simulators initially, structured training courses, and then hands-on training in tertiary referral centers.

Cisteína oral N-Acetyl versus indometacina retal para prevenção de pancreatite pós-CPRE: um ensaio controlado randomizado multinacional multicêntrico / Oral n-acetyl cysteine versus rectal indomethacin for prevention of post ercp pancreatitis: a multicenter multinational randomized controlled trial

ABSTRACT Background This multicenter multinational RCT designed to compare the efficacy of suppository indomethacin and NAC for prevention of PEP. Methods: During a 6-month period, all of the ERCP cases in seven referral centers were randomly assigned to receive either 1200 mg oral NAC, indomethacin suppository 100 mg, 1200 mg oral NAC plus indomethacin suppository 100 mg or placebo 2 hours before ERCP. The primary outcomes were the rate and severity of any PEP. Results: A total of 432 patients included (41.4% male). They were originally citizens of 6 countries (60.87% Caucasian). They were randomly allocated to receive either NAC (group A, 84 cases), rectal indomethacin (group B, 138 cases), NAC + rectal indomethacin (group C, 115 cases) or placebo (group D, 95 cases). The rate of PEP in groups A, B and C in comparison with placebo were 10.7%, 17.4%, 7.8% vs 20% (P=0.08, 0.614 & 0.01 respectively). The NNT for NAC, indomethacin and NAC + indomethacin was 11, 38 and 8 respectively. Conclusion: Oral NAC is more effective than rectal indomethacin when compared to placebo for prevention of PEP and the combination of NAC and Indomethacin had the lowest incidence of PEP and may have synergistic effect in preventing of PEP (IRCT20201222049798N1; 29/12/2020).

RESUMO Contexto: Este estudo randomizado, controlado multicêntrico e multinacional foi projetado para comparar a eficácia da indometacina supositório e N-acetil cisteína (NAC) para prevenção de pancreatite pós colangiografia endoscópica. Métodos: Durante um período de 6 meses, todos os pacientes submetidos à CPRE em sete centros de referência foram aleatoriamente atribuídos para receber 1200 mg de NAC oral, supositório de indometacina 100 mg, 1200 mg de NAC oral mais supositório de indometacina 100 mg ou placebo 2 horas antes do procedimento. Os resultados primários foram a taxa e a gravidade de qualquer pancreatite pós procedimento (PPP). Resultados: Um total de 432 pacientes foram incluídos (41,4% do sexo masculino). Eram originalmente cidadãos de seis países (60,87% caucasianos). Foram alocados aleatoriamente para receber NAC (grupo A, 84 casos), indometacina retal (grupo B, 138 casos), NAC + indometacina retal (grupo C, 115 casos) ou placebo (grupo D, 95 casos). A taxa de PPP nos grupos A, B e C em comparação com o placebo foi de 10,7%, 17,4%, 7,8% vs 20% (P=0,08, 0,614 e 0,01, respectivamente). Conclusão A NAC oral é mais eficaz do que a indometacina retal quando comparado ao placebo para prevenção de PPP e a combinação de NAC e indometacina teve a menor incidência de PPP e pode ter efeito sinérgico na sua prevenção de PPP. (IRCT20201222049798N1; 29/12/2020).

ORAL N-ACETYL CYSTEINE VERSUS RECTAL INDOMETHACIN FOR PREVENTION OF POST ERCP PANCREATITIS: A MULTICENTER MULTINATIONAL RANDOMIZED CONTROLLED TRIAL.

BACKGROUND: This multicenter multinational RCT designed to compare the efficacy of suppository indomethacin and NAC for prevention of PEP. METHODS: During a 6-month period, all of the ERCP cases in seven referral centers were randomly assigned to receive either 1200 mg oral NAC, indomethacin suppository 100 mg, 1200 mg oral NAC plus indomethacin suppository 100 mg or placebo 2 hours before ERCP. The primary outcomes were the rate and severity of any PEP. RESULTS: A total of 432 patients included (41.4% male). They were originally citizens of 6 countries (60.87% Caucasian). They were randomly allocated to receive either NAC (group A, 84 cases), rectal indomethacin (group B, 138 cases), NAC + rectal indomethacin (group C, 115 cases) or placebo (group D, 95 cases). The rate of PEP in groups A, B and C in comparison with placebo were 10.7%, 17.4%, 7.8% vs 20% (P=0.08, 0.614 & 0.01 respectively). The NNT for NAC, indomethacin and NAC + indomethacin was 11, 38 and 8 respectively. CONCLUSION: Oral NAC is more effective than rectal indomethacin when compared to placebo for prevention of PEP and the combination of NAC and Indomethacin had the lowest incidence of PEP and may have synergistic effect in preventing of PEP (IRCT20201222049798N1; 29/12/2020).

Digital image analysis provides robust tissue microenvironment-based prognosticators in patients with stage I-IV colorectal cancer.

In patients with colorectal cancer (CRC), a promising marker is tumor-stroma ratio (TSR). Quantification issues highlight the importance of precise assessment that might be solved by artificial intelligence-based digital image analysis systems. Some alternatives have been offered so far, although these platforms are either proprietary developments or require additional programming skills. Our aim was to validate a user-friendly, commercially available software running in everyday computational environment to improve TSR assessment and also to compare the prognostic value of assessing TSR in 3 distinct regions of interests, like hotspot, invasive front, and whole tumor. Furthermore, we compared the prognostic power of TSR with the newly suggested carcinoma percentage (CP) and carcinoma-stroma percentage (CSP). Slides of 185 patients with stage I-IV CRC with clinical follow-up data were scanned and evaluated by a senior pathologist. A machine learning-based digital pathology software was trained to recognize tumoral and stromal compartments. The aforementioned parameters were evaluated in the hotspot, invasive front, and whole tumor area, both visually and by machine learning. Patients were classified based on TSR, CP, and CSP values. On multivariate analysis, TSR-hotspot was found to be an independent prognostic factor of overall survival (hazard ratio for TSR-hotspotsoftware: 2.005 [95% confidence interval (CI): 1.146-3.507], P = .011, for TSR-hotspotvisual: 1.781 [CI: 1.060-2.992], P = .029). Also, TSR was an independent predictor for distant metastasis and local relapse in most settings. Generally, software performance was comparable to visual evaluation and delivered reliable prognostication in more settings also with CP and CSP values. This study presents that software-assisted evaluation is a robust prognosticator. Our approach used a less sophisticated and thus easily accessible software without the aid of a convolutional neural network; however, it was still effective enough to deliver reliable prognostic information.

Traditional Serrated Adenoma of the Gallbladder, a Case Report.

While overwhelming majority of laparoscopic cholecystectomy specimens performed for gallstones or cholecystitis show rather typical findings, sometimes polypoid structures are also removed. These can be related to cholesterolosis or conventional adenomas, but occasionally extraordinary findings do emerge. In our case, a 67-year old lady with typical complaints of cholecystitis underwent routine laparoscopic cholecystectomy. Preoperative ultrasound revealed a polypoid mass with inflammation and without suspicion for malignancy. Microscopic examination showed partly conventional, low-grade dysplastic crypts forming a villous and rather complex structure. Ectopic crypt foci, slit-like serration pattern and serrated dysplasia with eosinophylic cytoplasm and centrally located nuclei were seen throughout the lesion, thus a traditional serrated adenoma (TSA) of the gallbladder was diagnosed. TSA represents the rarest subtype of serrated lesions in the colon and extracolonic manifestations are sporadically reported. Until now only a single case of a serrated adenoma was reported from the gallbladder. Here we describe the detailed clinical, pathological and molecular findings of our case and discuss these in the light of current literature data regarding this field.

Irreversible JNK blockade overcomes PD-L1-mediated resistance to chemotherapy in colorectal cancer.

Colorectal cancer (CRC) cells have low or absent tumor cell PD-L1 expression that we previously demonstrated can confer chemotherapy resistance. Here, we demonstrate that PD-L1 depletion enhances JNK activity resulting in increased BimThr116 phosphorylation and its sequestration by MCL-1 and BCL-2. Activated JNK signaling in PD-L1-depeted cells was due to reduced mRNA stability of the CYLD deubiquitinase. PD-L1 was found to compete with the ribonuclease EXOSC10 for binding to CYLD mRNA. Thus, loss of PD-L1 promoted binding and degradation of CYLD mRNA by EXOSC10 which enhanced JNK activity. An irreversible JNK inhibitor (JNK-IN-8) reduced BimThr116 phosphorylation and unsequestered Bim from MCL-1 and BCL-2 to promote apoptosis. In cells lacking PD-L1, treatment with JNK-IN-8, an MCL-1 antagonist (AZD5991), or their combination promoted apoptosis and reduced long-term clonogenic survival by anticancer drugs. Similar effects of the JNK inhibitor on cell viability were observed in CRC organoids with suppression of PD-L1. These data indicate that JNK inhibition may represent a promising strategy to overcome drug resistance in CRC cells with low or absent PD-L1 expression.

Randomized Phase II Trial of Polyphenon E versus Placebo in Patients at High Risk of Recurrent Colonic Neoplasia.

Polyphenon E (Poly E) is a green tea polyphenol preparation whose most active component is epigallocatechin gallate (EGCG). We studied the cancer preventive efficacy and safety of Poly E in subjects with rectal aberrant crypt foci (ACF), which represent putative precursors of colorectal cancers. Eligible subjects had prior colorectal advanced adenomas or cancers, and had ≥5 rectal ACF at a preregistration chromoendoscopy. Subjects (N = 39) were randomized to 6 months of oral Poly E (780 mg EGCG) daily or placebo. Baseline characteristics were similar by treatment arm (all P >0.41); 32 of 39 (82%) subjects completed 6 months of treatment. The primary endpoint was percent reduction in rectal ACF at chromoendoscopy comparing before and after treatment. Among 32 subjects (15 Poly E, 17 placebo), percent change in rectal ACF number (baseline vs. 6 months) did not differ significantly between study arms (3.7% difference of means; P = 0.28); total ACF burden was also similar (-2.3% difference of means; P = 0.83). Adenoma recurrence rates at 6 months were similar by arm (P > 0.35). Total drug received did not differ significantly by study arm; 31 (79%) subjects received ≥70% of prescribed Poly E. Poly E was well tolerated and adverse events (AE) did not differ significantly by arm. One subject on placebo had two grade 3 AEs; one subject had grade 2 hepatic transaminase elevations attributed to treatment. In conclusion, Poly E for 6 months did not significantly reduce rectal ACF number relative to placebo. Poly E was well tolerated and without significant toxicity at the dose studied. PREVENTION RELEVANCE: We report a chemoprevention trial of polyphenon E in subjects at high risk of colorectal cancer. The results show that polyphenon E was well tolerated, but did not significantly reduce the number of rectal aberrant crypt foci, a surrogate endpoint biomarker of colorectal cancer.

[Comparison of the traditional triple and a new bismuth-containing quadruple therapy in the first-line eradication of Helicobacter pylori]. / A Helicobacter pylori-fertozés elso vonalbeli megszüntetésére alkalmazott hagyományos hármas és egy új, bizmuttartalmú négyes kezelés összehasonlítása.

Introduction and aim: As the efficacy of the first-line traditional treatment used to eradicate Helicobacter pylori (H. p.) decreased below 75% in Hungary, a new protocol had to be created. Method: Supposing the success rate of the traditional therapy (14-day double dose of proton pump inhibitor [PPI], 1000 mg amoxicillin b.i.d., 500 mg clarithromycin b.i.d. [PAC]) to be 75% and the efficacy of the new protocol (10-day 120 mg bismuth dicitrate q.i.d., double dose PPI b.i.d., 500 mg tetracycline q.i.d. and 500 mg tinidazole b.i.d. [BQT]) to be 90%, we calculated 109 patients on each arm. Patients were recruited after upper gastrointestinal endoscopy from 5 endoscopic units in Vas county. The heterogeneity of groups, success rate and side effects of both therapies were evaluated by Fisher exact test; p<0.05 was considered significant. Results: 110 patients were included in the BQT and 109 patients in the PAC group. There was no heterogeneity between the two groups in age, gender and indication of eradication. H. p. eradication was successful in 103/110 (93.6%) in the BQT and 81/109 (74.3%) in the PAC group (p<0.001). The odds ratio in the BQT group for successful eradication was 5.05 (95% confidence interval: 2.02-14.42) as compared to the PAC group (p<0.001). The side effects of the two groups were similar, in the BQT group the frequency was 34.5%. Conclusion: 10 day-long BQT containing double dose PPI with 120 mg bismuth dicitrate q.i.d., 500 mg tetracycline q.i.d. and 500 mg tinidazole b.i.d. is recommended as the first-line treatment for the eradication of H. p. because of its high efficacy and tolerable side effects. Orv Hetil. 2019; 160(34): 1340-1345.

Gene promoter and exon DNA methylation changes in colon cancer development - mRNA expression and tumor mutation alterations.

BACKGROUND: DNA mutations occur randomly and sporadically in growth-related genes, mostly on cytosines. Demethylation of cytosines may lead to genetic instability through spontaneous deamination. Aims were whole genome methylation and targeted mutation analysis of colorectal cancer (CRC)-related genes and mRNA expression analysis of TP53 pathway genes. METHODS: Long interspersed nuclear element-1 (LINE-1) BS-PCR followed by pyrosequencing was performed for the estimation of global DNA metlyation levels along the colorectal normal-adenoma-carcinoma sequence. Methyl capture sequencing was done on 6 normal adjacent (NAT), 15 adenomatous (AD) and 9 CRC tissues. Overall quantitative methylation analysis, selection of top hyper/hypomethylated genes, methylation analysis on mutation regions and TP53 pathway gene promoters were performed. Mutations of 12 CRC-related genes (APC, BRAF, CTNNB1, EGFR, FBXW7, KRAS, NRAS, MSH6, PIK3CA, SMAD2, SMAD4, TP53) were evaluated. mRNA expression of TP53 pathway genes was also analyzed. RESULTS: According to the LINE-1 methylation results, overall hypomethylation was observed along the normal-adenoma-carcinoma sequence. Within top50 differential methylated regions (DMRs), in AD-N comparison TP73, NGFR, PDGFRA genes were hypermethylated, FMN1, SLC16A7 genes were hypomethylated. In CRC-N comparison DKK2, SDC2, SOX1 genes showed hypermethylation, while ERBB4, CREB5, CNTN1 genes were hypomethylated. In certain mutation hot spot regions significant DNA methylation alterations were detected. The TP53 gene body was addressed by hypermethylation in adenomas. APC, TP53 and KRAS mutations were found in 30, 15, 21% of adenomas, and in 29, 53, 29% of CRCs, respectively. mRNA expression changes were observed in several TP53 pathway genes showing promoter methylation alterations. CONCLUSIONS: DNA methylation with consecutive phenotypic effect can be observed in a high number of promoter and gene body regions through CRC development.

Multilevel omic data clustering reveals variable contribution of methylator phenotype to integrative cancer subtypes.

AIM: We aimed to assess to what extent CpG island methylator phenotype (CIMP) contributes to cancer subtypes obtained by multilevel omic data analysis. MATERIALS & METHODS: 16 The Cancer Genome Atlas datasets encompassing three data layers in 4688 tumor samples were analyzed. We identified cancer integrative subtypes (ISs) by the use of similarity network fusion and consensus clustering. CIMP high (CIMP-H) associated ISs were profiled by gene sets and transcriptional regulators enrichment analysis. RESULTS & CONCLUSION: In nine out of 16 cancer datasets CIMP-H clusters significantly overlaped with unique ISs. The contribution of CIMP-H on integrative molecular profiling is variable; therefore, only in a subset of cancer types does CIMP-H contribute to homogenous integrative subtype. CIMP-H associated ISs are heterogenous groups with regard to deregulated pathways and transcriptional regulators.

Colorectal adenoma and cancer detection based on altered methylation pattern of SFRP1, SFRP2, SDC2, and PRIMA1 in plasma samples.

Aberrant methylation is one of the most frequent epigenetic alterations that can contribute to tumor formation. Cell-free DNA can originate from tumor tissue; therefore, the evaluation of methylation markers in cell-free DNA can be a promising method for cancer screening. Our aim was to develop a panel of biomarkers with altered methylation along the colorectal adenoma-carcinoma sequence in both colonic tissue and plasma. Methylation of selected CpG sites in healthy colonic (n = 15), adenoma (n = 15), and colorectal cancer (n = 15) tissues was analyzed by pyrosequencing. MethyLight PCR was applied to study the DNA methylation of SFRP1, SFRP2, SDC2, and PRIMA1 gene promoters in 121 plasma and 32 biopsy samples. The effect of altered promoter methylation on protein expression was examined by immunohistochemistry. Significantly higher (P < 0.05) DNA methylation levels were detected in the promoter regions of all 4 markers, both in CRC and adenoma tissues compared with healthy controls. Methylation of SFRP1, SFRP2, SDC2, and PRIMA1 promoter sequences was observed in 85.1%, 72.3%, 89.4%, and 80.9% of plasma samples from patients with CRC and 89.2%, 83.8%, 81.1% and 70.3% from adenoma patients, respectively. When applied as a panel, CRC patients could be distinguished from controls with 91.5% sensitivity and 97.3% specificity [area under the curve (AUC) = 0.978], while adenoma samples could be differentiated with 89.2% sensitivity and 86.5% specificity (AUC = 0.937). Immunohistochemical analysis indicated decreasing protein levels of all 4 markers along the colorectal adenoma-carcinoma sequence. Our findings suggest that this methylation biomarker panel allows non-invasive detection of colorectal adenoma and cancer from plasma samples.

Construction of a multiplex mutation hot spot PCR panel: the first step towards colorectal cancer genotyping on the GS Junior platform.

Background: To support cancer therapy, development of low cost library preparation techniques for targeted next generation sequencing (NGS) is needed. In this study we designed and tested a PCR-based library preparation panel with limited target area for sequencing the top 12 somatic mutation hot spots in colorectal cancer on the GS Junior instrument. Materials and Methods: A multiplex PCR panel was designed to amplify regions of mutation hot spots in 12 selected genes (APC, BRAF, CTNNB1, EGFR, FBXW7, KRAS, NRAS, MSH6, PIK3CA, SMAD2, SMAD4, TP53). Amplicons were sequenced on a GS Junior instrument using ligated and barcoded adaptors. Eight samples were sequenced in a single run. Colonic DNA samples (8 normal mucosa; 33 adenomas; 17 adenocarcinomas) as well as HT-29 and Caco-2 cell lines with known mutation profiles were analyzed. Variants found by the panel on APC, BRAF, KRAS and NRAS genes were validated by conventional sequencing. Results: In total, 34 kinds of mutations were detected including two novel mutations (FBXW7 c.1740:C>G and SMAD4 c.413C>G) that have not been recorded in mutation databases, and one potential germline mutation (APC). The most frequently mutated genes were APC, TP53 and KRAS with 30%, 15% and 21% frequencies in adenomas and 29%, 53% and 29% frequencies in carcinomas, respectively. In cell lines, all the expected mutations were detected except for one located in a homopolymer region. According to re-sequencing results sensitivity and specificity was 100% and 92% respectively. Conclusions: Our NGS-based screening panel denotes a promising step towards low cost colorectal cancer genotyping on the GS Junior instrument. Despite the relatively low coverage, we discovered two novel mutations and obtained mutation frequencies comparable to literature data. Additionally, as an advantage, this panel requires less template DNA than sequence capture colon cancer panels currently available for the GS Junior instrument.

Circadian Rhythm of Methylated Septin 9, Cell-Free DNA Amount and Tumor Markers in Colorectal Cancer Patients.

To determine the level of cell-free DNA (cfDNA), Septin 9 (SEPT9) and tumor markers (CEA, AFP, CA19-9, TPA, CA72-4). Plasma samples were collected four times a day (06:00, 12:00, 18:00, 24:00) from 9 patients with CRC (5 stage I-II, 4 stage III-IV), from one with colorectal adenoma and from one healthy control. CfDNA was isolated, quantified and bisulfite-converted. CfDNA and methylated SEPT9 were determined by RT-PCR. Plasma levels of conventional tumor markers were also measured. The lowest cfDNA concentrations were observed at 24:00 and 18:00 in stage I-III patients. In stage IV samples low cfDNA level (mean 48.2 ng/ml) were observed at several time points (6:00, 12:00, 18:00). The highest cfDNA levels were measured at 6:00 and 12:00 in CRC I-III stages and at 24:00 in stage IV samples (78.65 ng/ml). Higher in-day differences were found in stage II (43-48%) than in stage I samples (22%). Interestingly, the highest SEPT9 methylation level was found at 24:00 in most CRC cases, in contrast to the cfDNA levels. At 24:00, all cancer and adenoma cases were positive for SEPT9 methylation. At other time points (6:00, 12:00, 18:00) only 77.7% of CRC samples showed SEPT9 positivity. Stage I samples were SEPT9 positive only at 24:00. CEA and CA19-9 levels displayed correlation with the amount of cfDNA in case of late stage cases. Daytime activity can influence SEPT9 positivity in cases with low concentration of cfDNA. Thus, it may improve screening sensitivity by collecting samples earlier in the morning.

Comprehensive DNA Methylation and Mutation Analyses Reveal a Methylation Signature in Colorectal Sessile Serrated Adenomas.

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

Aberrant DNA methylation of WNT pathway genes in the development and progression of CIMP-negative colorectal cancer.

The WNT signaling pathway has an essential role in colorectal carcinogenesis and progression, which involves a cascade of genetic and epigenetic changes. We aimed to analyze DNA methylation affecting the WNT pathway genes in colorectal carcinogenesis in promoter and gene body regions using whole methylome analysis in 9 colorectal cancer, 15 adenoma, and 6 normal tumor adjacent tissue (NAT) samples by methyl capture sequencing. Functional methylation was confirmed on 5-aza-2'-deoxycytidine-treated colorectal cancer cell line datasets. In parallel with the DNA methylation analysis, mutations of WNT pathway genes (APC, ß-catenin/CTNNB1) were analyzed by 454 sequencing on GS Junior platform. Most differentially methylated CpG sites were localized in gene body regions (95% of WNT pathway genes). In the promoter regions, 33 of the 160 analyzed WNT pathway genes were differentially methylated in colorectal cancer vs. normal, including hypermethylated AXIN2, CHP1, PRICKLE1, SFRP1, SFRP2, SOX17, and hypomethylated CACYBP, CTNNB1, MYC; 44 genes in adenoma vs. NAT; and 41 genes in colorectal cancer vs. adenoma comparisons. Hypermethylation of AXIN2, DKK1, VANGL1, and WNT5A gene promoters was higher, while those of SOX17, PRICKLE1, DAAM2, and MYC was lower in colon carcinoma compared to adenoma. Inverse correlation between expression and methylation was confirmed in 23 genes, including APC, CHP1, PRICKLE1, PSEN1, and SFRP1. Differential methylation affected both canonical and noncanonical WNT pathway genes in colorectal normal-adenoma-carcinoma sequence. Aberrant DNA methylation appears already in adenomas as an early event of colorectal carcinogenesis.

Exosomes in colorectal carcinoma formation: ALIX under the magnifying glass.

Exosomes are small membrane vesicles that have important roles in transporting a great variety of bioactive molecules between epithelial compartment and their microenvironment during tumor formation including colorectal adenoma-carcinoma sequence. We tested the mRNA expression of the top 25 exosome-related markers based on ExoCharta database in healthy (n=49), adenoma (n=49) and colorectal carcinoma (n=49) patients using Affymetrix HGU133 Plus2.0 microarrays. Most related genes showed significantly elevated expression including PGK1, PKM, ANXA5, ENO1, HSP90AB1 and MSN during adenoma-carcinoma sequence. Surprisingly, the expression of ALIX (ALG 2-interacting protein X), involved in multivesicular body (MVB) and exosome formation, was significantly reduced in normal vs adenoma (P=5.02 × 10(-13)) and in normal vs colorectal carcinoma comparisons (P=1.51 × 10(-10)). ALIX also showed significant reduction (P<0.05) at the in situ protein level in the epithelial compartment of adenoma (n=35) and colorectal carcinoma (n=37) patients compared with 27 healthy individuals. Furthermore, significantly reduced ALIX protein levels were accompanied by their gradual transition from diffuse cytoplasmic expression to granular signals, which fell into the 0.6-2 µm diameter size range of MVBs. These ALIX-positive particles were seen in the tumor nests, including tumor-stroma border, which suggest their exosome function. MVB-like structures were also detected in tumor microenvironment including α-smooth muscle actin-positive stromal cells, budding off cancer cells in the tumor front as well as in cancer cells entrapped within lymphoid vessels. In conclusion, we determined the top aberrantly expressed exosome-associated markers and revealed the transition of diffuse ALIX protein signals into a MVB-like pattern during adenoma-carcinoma sequence. These tumor-associated particles seen both in the carcinoma and the surrounding microenvironment can potentially mediate epithelial-stromal interactions involved in the regulation of tumor growth, metastatic invasion and therapy response.

Aging related methylation influences the gene expression of key control genes in colorectal cancer and adenoma.

AIM: To analyze colorectal carcinogenesis and age-related DNA methylation alterations of gene sequences associated with epigenetic clock CpG sites. METHODS: In silico DNA methylation analysis of 353 epigenetic clock CpG sites published by Steve Horvath was performed using methylation array data for a set of 123 colonic tissue samples [64 colorectal cancer (CRC), 42 adenoma, 17 normal; GEO accession number: GSE48684]. Among the differentially methylated age-related genes, secreted frizzled related protein 1 (SFRP1) promoter methylation was further investigated in colonic tissue from 8 healthy adults, 19 normal children, 20 adenoma and 8 CRC patients using bisulfite-specific PCR followed by methylation-specific high resolution melting (MS-HRM) analysis. mRNA expression of age-related "epigenetic clock" genes was studied using Affymetrix HGU133 Plus2.0 whole transcriptome data of 153 colonic biopsy samples (49 healthy adult, 49 adenoma, 49 CRC, 6 healthy children) (GEO accession numbers: GSE37364, GSE10714, GSE4183, GSE37267). Whole promoter methylation analysis of genes showing inverse DNA methylation-gene expression data was performed on 30 colonic samples using methyl capture sequencing. RESULTS: Fifty-seven age-related CpG sites including hypermethylated PPP1R16B, SFRP1, SYNE1 and hypomethylated MGP, PIPOX were differentially methylated between CRC and normal tissues (P < 0.05, Δß ≥ 10%). In the adenoma vs normal comparison, 70 CpG sites differed significantly, including hypermethylated DKK3, SDC2, SFRP1, SYNE1 and hypomethylated CEMIP, SPATA18 (P < 0.05, Δß ≥ 10%). In MS-HRM analysis, the SFRP1 promoter region was significantly hypermethylated in CRC (55.0% ± 8.4 %) and adenoma tissue samples (49.9% ± 18.1%) compared to normal adult (5.2% ± 2.7%) and young (2.2% ± 0.7%) colonic tissue (P < 0.0001). DNA methylation of SFRP1 promoter was slightly, but significantly increased in healthy adults compared to normal young samples (P < 0.02). This correlated with significantly increased SFRP1 mRNA levels in children compared to normal adult samples (P < 0.05). In CRC tissue the mRNA expression of 117 age-related genes were changed, while in adenoma samples 102 genes showed differential expression compared with normal colonic tissue (P < 0.05, logFC > 0.5). The change of expression for several genes including SYNE1, CLEC3B, LTBP3 and SFRP1, followed the same pattern in aging and carcinogenesis, though not for all genes (e.g., MGP). CONCLUSION: Several age-related DNA methylation alterations can be observed during CRC development and progression affecting the mRNA expression of certain CRC- and adenoma-related key control genes.