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Colitis-associated colorectal carcinoma (CAC) occurs in inflammatory bowel disease (IBD) because of the “chronic inflammation-dysplasia-cancer” carcinogenesis pathway characterized by p53 alterations in the early stages. Recently, gastric metaplasia (GM) has been described as the initial event of the serrated colorectal cancer (CRC) process, resulting from chronic stress on the colon mucosa. The aim of the study is to characterize CAC analyzing p53 alterations and microsatellite instability (MSI) to explore their relationship with GM using a series of CRC and the adjacent intestinal mucosa. Immunohistochemistry was performed to assess p53 alterations, MSI and MUC5AC expression as a surrogate for GM. The p53 mut-pattern was found in more than half of the CAC, most frequently stable (MSS) and MUC5AC negative. Only six tumors were unstable (MSI-H), being with p53 wt-pattern (p = 0.010) and MUC5AC positive (p = 0.005). MUC5AC staining was more frequently observed in intestinal mucosa, inflamed or with chronic changes, than in CAC, especially in those with p53 wt-pattern and MSS. Based on our results, we conclude that, as in the serrated pathway of CRC, in IBD GM occurs in inflamed mucosa, persists in those with chronic changes and disappears with the acquisition of p53 mutations. info:eu-repo/semantics/publishedVersion

Supplementary data Supplementary Methods Supplementary Table 1. Characteristics of Epicolon I patients (n=532) Supplementary Table 2. Characteristics of Clinical cohort patients (n=251) Supplementary Table 3. Primer sequences Supplementary Figure 1. Schematic representation of TFAP2E gene. The gene encoding transcription factor AP-2 epsilon (TFAP2E) is located on chromosome 1p34. In the schematic overview, exons are represented by blue boxes and introns with blue lines. The TFAP2E gene has two cytosine-phosphate-guanine (CpG) islands underscoring the potential of gene expression by means of CpG hypermethylation (green boxes). We used two quantitative methods, QMSP and pyrosequencing, covering both CpG islands in order to correlate methylation levels at these two locations. Supplementary Figure 2. Methylation analysis of TFAP2E CpG islands in CRC tissues. Calculation of cutoff value of TFAP2E Intron 3 methylation. Results comparing methylation of the TFAP2E CpG1 (Promoter/Exon 1) and CpG2 (Intron 3) in CRC tissues (N=50) by (A) Bisulfite pyrosequencing (% Methylation) (B-C) Bisulfite pyrosequencing results of TFAP2E Intron 3 methylation in normal colon mucosa from healthy subjects N-N (N=21); and CRC patients-matched tumor-and non-tumor samples N-C (N=41);(N=41). (D) Whitin-subject Receiver Operating Characteristics (ROC) analysis performed on measured TFAP2E % (percentage of methylation) values of patient-matched tumor- (N-N; N=21) and non-tumor (N-C; N=41) samples, respectively. The area under the ROC-curve was calculated as 0.73 (95% CI:0.62-0.82), indicating that the probability of observing a higher TFAP2E value in a tumor-sample of a patient compared to its related non-tumor sample is about 73%. Supplementary Figure 3. Correlation between TFAP2E intron 3 methylation and IHC expression. When methylation was analysed as a continuous variable using linear regression analysis, we observed a trend towards negative correlation with IHC protein expression; however, this correlation was significant only when combining lack of and low TFAP2E expression (IHC scores 0 & 1) versus strong TFAP2E staining (IHC scores 2 & 3) (p= 0.015). Linear regression analysis comparing TFAP2E methylation and expression in CRC tissues (N=314) using IHC results by binary scoring system: positive/negative (A) staining scores 0 & 1 vs 2 & 3(B) and the staining intensity scale 0 to 3 (C). Correlation and performance measures between TFAP2E methylation and expression in CRC tissues using IHC and methylation qualitative results (staining scores 0 & 1 vs 2 & 3; (D). Supplementary Figure 1. Schematic representation of TFAP2E gene. The gene encoding transcription factor AP-2 epsilon (TFAP2E) is located on chromosome 1p34. In the schematic overview, exons are represented by blue boxes and introns with blue lines. The TFAP2E gene has two cytosine-phosphate-guanine (CpG) islands underscoring the potential of gene expression by means of CpG hypermethylation (green boxes). We used two quantitative methods, QMSP and pyrosequencing, covering both CpG islands in order to correlate methylation levels at these two locations. Supplementary Figure 2. Methylation analysis of TFAP2E CpG islands in CRC tissues. Calculation of cutoff value of TFAP2E Intron 3 methylation. Results comparing methylation of the TFAP2E CpG1 (Promoter/Exon 1) and CpG2 (Intron 3) in CRC tissues (N=50) by (A) Bisulfite pyrosequencing (% Methylation) (B-C) Bisulfite pyrosequencing results of TFAP2E Intron 3 methylation in normal colon mucosa from healthy subjects N-N (N=21); and CRC patients-matched tumor-and non-tumor samples N-C (N=41);(N=41). (D) Whitin-subject Receiver Operating Characteristics (ROC) analysis performed on measured TFAP2E % (percentage of methylation) values of patient-matched tumor- (N-N; N=21) and non-tumor (N-C; N=41) samples, respectively. The area under the ROC-curve was calculated as 0.73 (95% CI:0.62-0.82), indicating that the probability of observing a higher TFAP2E value in a tumor-sample of a patient compared to its related non-tumor sample is about 73%. Supplementary Figure 3. Correlation between TFAP2E intron 3 methylation and IHC expression. When methylation was analysed as a continuous variable using linear regression analysis, we observed a trend towards negative correlation with IHC protein expression; however, this correlation was significant only when combining lack of and low TFAP2E expression (IHC scores 0 & 1) versus strong TFAP2E staining (IHC scores 2 & 3) (p= 0.015). Linear regression analysis comparing TFAP2E methylation and expression in CRC tissues (N=314) using IHC results by binary scoring system: positive/negative (A) staining scores 0 & 1 vs 2 & 3(B) and the staining intensity scale 0 to 3 (C). Correlation and performance measures between TFAP2E methylation and expression in CRC tissues using IHC and methylation qualitative results (staining scores 0 & 1 vs 2 & 3; (D). Supplementary Figure 4. Epicolon I. Overall Survival Stage IV Patients: TFAP2E IHC Expression. Disease Free Survival Stage II and III Patients: TFAP2E IHC Expression. (A) Overall survival of patients with stage IV disease, according to TFAP2E expression status comparing staining scores 0 & 1 (Low expression) vs staining scores 2 & 3 (High expression;). (B) Overall survival of patients that received or did not receive (C) 5-FU based chemotherapy according to TFAP2E expression status. (D) Disease Free Survival (DFS) of patients with stage II and III CRC, according to TFAP2E expression status comparing staining scores 0 & 1 (Low expression) vs staining scores 2 & 3 (High expression) (E) DFS of stages II+III CRC patients that received or did not receive (F)) adjuvant chemotherapy according to TFAP2E expression status.

Purpose: A recent study reported that 5-fluorouracil (5-FU)-based chemotherapy is less effective in treating patients with advanced colorectal cancer demonstrating hypermethylation of the TFAP2E gene. The aim of our study was to confirm and validate these findings in large, uniformly treated, well-characterized patient cohorts.Experimental Design: Two cohorts of 783 patients with colorectal cancer: 532 from a population-based, multicenter cohort (EPICOLON I) and 251 patients from a clinic-based trial were used to study the effectiveness of TFAP2E methylation and expression as a predictor of response of colorectal cancer patients to 5-FU–based chemotherapy. DNA methylation status of the TFAP2E gene in patients with colorectal cancer was assessed by quantitative bisulfite pyrosequencing analysis. IHC analysis of the TFAP2E protein expression was also performed.Results: Correlation between TFAP2E methylation status and IHC staining was performed in 607 colorectal cancer samples. Among 357 hypermethylated tumors, only 141 (39.6%) exhibited loss of protein expression. Survival was not affected by TFAP2E hypermethylation in stage IV patients [HR, 1.21; 95% confidence interval (CI), 0.79–1.87; log-rank P = 0.6]. In stage II–III cases, disease-free survival was not influenced by TFAP2E hypermethylation status in 5-FU–treated (HR, 0.91; 95% CI, 0.52–1.59; log-rank P = 0.9) as well as in nontreated patients (HR, 0.88; 95% CI, 0.5–1.54; log-rank P = 0.7).Conclusions: TFAP2E hypermethylation does not correlate with loss of its protein expression. Our large, systematic, and comprehensive study indicates that TFAP2E methylation and expression may not play a major role in predicting response to 5-FU–based chemotherapy in patients with colorectal cancer. Clin Cancer Res; 24(12); 2820–7. ©2018 AACR.

Supplementary Figure 4. Epicolon I. Overall Survival Stage IV Patients: TFAP2E IHC Expression. Disease Free Survival Stage II and III Patients: TFAP2E IHC Expression. (A) Overall survival of patients with stage IV disease, according to TFAP2E expression status comparing staining scores 0 & 1 (Low expression) vs staining scores 2 & 3 (High expression;). (B) Overall survival of patients that received or did not receive (C) 5-FU based chemotherapy according to TFAP2E expression status. (D) Disease Free Survival (DFS) of patients with stage II and III CRC, according to TFAP2E expression status comparing staining scores 0 & 1 (Low expression) vs staining scores 2 & 3 (High expression) (E) DFS of stages II+III CRC patients that received or did not receive (F)) adjuvant chemotherapy according to TFAP2E expression status.

Supplementary Figure 3. Correlation between TFAP2E intron 3 methylation and IHC expression. When methylation was analysed as a continuous variable using linear regression analysis, we observed a trend towards negative correlation with IHC protein expression; however, this correlation was significant only when combining lack of and low TFAP2E expression (IHC scores 0 & 1) versus strong TFAP2E staining (IHC scores 2 & 3) (p= 0.015). Linear regression analysis comparing TFAP2E methylation and expression in CRC tissues (N=314) using IHC results by binary scoring system: positive/negative (A) staining scores 0 & 1 vs 2 & 3(B) and the staining intensity scale 0 to 3 (C). Correlation and performance measures between TFAP2E methylation and expression in CRC tissues using IHC and methylation qualitative results (staining scores 0 & 1 vs 2 & 3; (D).

Purpose: The present study aimed to determine the prevalence of MUTYH mutations in patients with multiple colonic polyps and to explore the best strategy for diagnosing MUTYH-associated polyposis (MAP) in these patients.Experimental Design: This study included 405 patients with at least 10 colonic polyps each. All cases were genetically tested for the three most frequent MUTYH mutations. Whole-gene analysis was performed in heterozygous patients and in 216 patients lacking the three most frequent mutations. Polyps from 56 patients were analyzed for the KRAS-Gly12Cys and BRAF V600E somatic mutations.Results: Twenty-seven (6.7%) patients were diagnosed with MAP, of which 40.8% showed serrated polyps. The sensitivity of studying only the three common variants was 74.1%. Of 216 patients without any monoallelic mutation in common variants, whole-gene analysis revealed biallelic pathogenic mutation in only one. G396D mutation was associated with serrated lesions and older age at diagnosis. There was a strong association between germinal MUTYH mutation and KRAS Gly12Cys somatic mutation in polyps. BRAF V600E mutation was found in 74% of serrated polyps in MUTYH-negative patients and in none of the polyps of MAP patients.Conclusions: We observed a low frequency of MUTYH mutations among patients with multiple adenomatous and serrated polyps. The MAP phenotype frequently included patients with serrated polyps, especially when G396D mutation was involved. Our results show that somatic molecular markers of polyps can be useful in identifying MAP cases and support the need for the complete MUTYH gene analysis only in patients heterozygous for recurrent variants. Clin Cancer Res; 20(5); 1158–68. ©2014 AACR.

CCR Translation for This Article from Prevalence and Characteristics of MUTYH-Associated Polyposis in Patients with Multiple Adenomatous and Serrated Polyps

Supplementary Figure 1. Schematic representation of TFAP2E gene. The gene encoding transcription factor AP-2 epsilon (TFAP2E) is located on chromosome 1p34. In the schematic overview, exons are represented by blue boxes and introns with blue lines. The TFAP2E gene has two cytosine-phosphate-guanine (CpG) islands underscoring the potential of gene expression by means of CpG hypermethylation (green boxes). We used two quantitative methods, QMSP and pyrosequencing, covering both CpG islands in order to correlate methylation levels at these two locations.

Supplementary Figure 2. Methylation analysis of TFAP2E CpG islands in CRC tissues. Calculation of cutoff value of TFAP2E Intron 3 methylation. Results comparing methylation of the TFAP2E CpG1 (Promoter/Exon 1) and CpG2 (Intron 3) in CRC tissues (N=50) by (A) Bisulfite pyrosequencing (% Methylation) (B-C) Bisulfite pyrosequencing results of TFAP2E Intron 3 methylation in normal colon mucosa from healthy subjects N-N (N=21); and CRC patients-matched tumor-and non-tumor samples N-C (N=41);(N=41). (D) Whitin-subject Receiver Operating Characteristics (ROC) analysis performed on measured TFAP2E % (percentage of methylation) values of patient-matched tumor- (N-N; N=21) and non-tumor (N-C; N=41) samples, respectively. The area under the ROC-curve was calculated as 0.73 (95% CI:0.62-0.82), indicating that the probability of observing a higher TFAP2E value in a tumor-sample of a patient compared to its related non-tumor sample is about 73%.

The prediction of microsatellite instability (MSI) in colorectal cancer (CRC) using deep learning (DL) techniques directly from hematoxylin and eosin stained slides (H&E) has been shown feasible by independent works. Nonetheless, when available, relevant information from clinical, oncological and family history could be used to further inform DL predictions. The present work analyzes the effects from leveraging multimodal inputs and multitask supervision in a previously published DL system for the prediction of MSI in CRC (xDEEP-MSI). xDEEP-MSI was a multiple bias rejecting DL system based on adversarial networks trained and validated in 1788 patients from a total of 25 participating centers from EPICOLON and HGUA projects. In the present work, xDEEP-MSI is further enriched with weakly supervised learning in multiple molecular alterations (MSI status, K-RAS and BRAF mutations and Lynch Syndrome confirmed by germline mutations), adapted to multimodal inputs with variable degree of completeness (image, age, gender, localization of CRC, revised Bethesda criteria, Amsterdam II criteria and additional oncological history) and a self-supervised multiple instance learning that integrates multiple image-tiles, to obtain patient-level predictions. The AUC, including all three selected tissues (tumor epithelium, mucin and lymphocytic regions) and 5 magnifications, increases from 0.9 ± 0.03, to 0.94 ± 0.02. The sensibility and specificity reaches 92.5% 95%CI(79.6-98.4%) and 93.4% 95%CI(90.0-95.8%) respectively. To the best of our knowledge this is the first work that jointly uses multimodal inputs, multiple instance learning and multiple molecular supervision for the prediction of MSI in CRC from H&E, demonstrating their gains in performance. Prospective validation in an external independent dataset is still required.

To assess whether the main genetic differences observed in high-grade gliomas (HGG) will present different MR imaging and MR spectroscopy correlates that could be used to better characterize lesions in the clinical setting. Seventy-nine patients with histologically confirmed HGG were recruited. Immunohistochemistry analyses for isocitrate dehydrogenase gene 1 (IDH1), alpha thalassemia mental retardation X-linked gene (ATRX), Ki-67, and p53 protein expression were performed. Tumour radiological features were examined on MR images. Metabolic profile and infiltrative pattern were assessed with MR spectroscopy. MR features were analysed to identify imaging-molecular associations. The Kaplan-Meier method and the Cox regression model were used to identify survival prognostic factors. In total, 17.7% of the lesions were IDH1-mutated, 8.9% presented ATRX-mutated, 70.9% presented p53 unexpressed, and 22.8% had Ki-67 > 5%. IDH1 wild-type tumours had higher levels of mobile lipids (p = 0.001). The tumour-infiltrative pattern was higher in HGG with unexpressed p53 (p = 0.009). Mutated ATRX tumours presented higher levels of glutamate and glutamine (Glx) (p = 0.001). An association was observed between Glx tumour levels (p = 0.038) and Ki-67 expression (p = 0.008) with the infiltrative pattern. Survival analyses identified IDH1 status, age, and tumour choline levels as independent predictors of prognostic significance. Our results suggest that IDH1-wt tumours are more necrotic than IDH1-mut. And that the presence of an infiltrative pattern in HGG is associated with loss of p53 expression, Ki-67 index, and Glx levels. Finally, tumour choline levels could be used as a predictive factor in survival in addition to the IDH1 status to provide a more accurate prediction of survival in HGG patients. • IDH1-wt tumours present higher levels of mobile lipids than IDH1-mut. • Mutated ATRX tumours exhibit higher levels of glutamate and glutamine. • Loss of p53 expression, Ki-67 expression, and glutamate and glutamine levels may contribute to the presence of an infiltrative pattern in HGG.

BACKGROUND: Mismatch repair (MMR) deficiency is the hallmark of tumours from Lynch syndrome (LS), sporadic MLH1 hypermethylated and Lynch-like syndrome (LLS), but there is a lack of understanding of the variability in their mutational profiles based on clinical phenotypes. The aim of this study was to perform a molecular characterisation to identify novel features that can impact tumour behaviour and clinical management. METHODS: We tested 105 MMR-deficient colorectal cancer tumours (25 LS, 35 LLS and 45 sporadic) for global exome microsatellite instability, cancer mutational signatures, mutational spectrum and neoepitope load. RESULTS: Fifty-three percent of tumours showed high contribution of MMR-deficient mutational signatures, high level of global exome microsatellite instability, loss of MLH1/PMS2 protein expression and included sporadic tumours. Thirty-one percent of tumours showed weaker features of MMR deficiency, 62% lost MSH2/MSH6 expression and included 60% of LS and 44% of LLS tumours. Remarkably, 9% of all tumours lacked global exome microsatellite instability. Lastly, HLA-B07:02 could be triggering the neoantigen presentation in tumours that show the strongest contribution of MMR-deficient tumours. CONCLUSIONS: Next-generation sequencing approaches allow for a granular molecular characterisation of MMR-deficient tumours, which can be essential to properly diagnose and treat patients with these tumours in the setting of personalised medicine.

24 p.-10 fig.-3 tab., D-amino acid oxidase (DAAO) is an enzyme that catalyzes the oxidation of D-amino acids generating H2O2. The enzymatic chimera formed by DAAO bound to the choline-binding domain of N-acetylmuramoyl-L-alanine amidase (CLytA) induces cytotoxicity in several pancreatic and colorectal carcinoma and glioblastoma cell models. In the current work, we determined whether the effect of CLytA-DAAO immobilized in magnetic nanoparticles, gold nanoparticles, and alginate capsules offered some advantages as compared to the free CLytA-DAAO. Results indicate that the immobilization of CLytA-DAAO in magnetic nanoparticles increases the stability of the enzyme, extending its time of action. Besides, we compared the effect induced by CLytA-DAAO with the direct addition of hydrogen peroxide, demonstrating that the progressive generation of reactive oxygen species by CLytA-DAAO is more effective in inducing cytotoxicity than the direct addition of H2O2. Furthermore, a pilot study has been initiated in biopsies obtained from pancreatic and colorectal carcinoma and glioblastoma patients to evaluate the expression of the main genes involved in resistance to CLytA-DAAO cytotoxicity. Based on our findings, we propose that CLytA-DAAO immobilized in magnetic nanoparticles could be effective in a high percentage of patients and, therefore, be used as an anti-cancer therapy for pancreatic and colorectal carcinoma and glioblastoma., This research was funded by Foundation for the Promotion of Health and Biomedical Research of Valencia Region (FISABIO), grant number UGP-19-063, and co-supported by PRECIPITA crowdfunding platform from Spanish Foundation for Science and Technology (FECYT) and donations from Association of women affected by breast cancer in Elche and the region (AMACMEC) to M.S., Miguel Servet Program from Instituto de Salud Carlos III (CP19/00095) to C.d.J.R. and grants from the Spanish Ministry of Economy, Industry and Competitiveness, grant numbers BIO2013-47684-R and BIO2016-79323-R, and the RETICS-FEDER RICET, RD16/0027/0010, to J.M.S. The CIBER of Enfermedades Respiratorias (CIBERES) is an initiative of the Spanish Instituto de Salud Carlos III and Spanish National Research Council (CSIC Grant 201820I132).

23 p.-11 fig., The combination of the choline binding domain of the amidase N-acetylmuramoyl-L-alanine (CLytA)-D-amino acid oxidase (DAAO) (CLytA-DAAO) and D-Alanine induces cell death in several pancreatic and colorectal carcinoma and glioblastoma cell lines. In glioblastoma cell lines, CLytA-DAAO-induced cell death was inhibited by a pan-caspase inhibitor, suggesting a classical apoptotic cell death. Meanwhile, the cell death induced in pancreatic and colon carcinoma cell lines is some type of programmed necrosis. In this article, we studied the mechanisms that trigger CLytA-DAAO-induced cell death in pancreatic and colorectal carcinoma and glioblastoma cell lines and we acquire a further insight into the necrotic cell death induced in pancreatic and colorectal carcinoma cell lines. We have analyzed the intracellular calcium mobilization, mitochondrial membrane potential, PARP-1 participation and AIF translocation. Although the mitochondrial membrane depolarization plays a crucial role, our results suggest that CLytA-DAAO-induced cell death is context dependent. We have previously detected pancreatic and colorectal carcinoma cell lines (Hs766T and HT-29, respectively) that were resistant to CLytA-DAAO-induced cell death. In this study, we have examined the putative mechanism underlying the resistance in these cell lines, evaluating both detoxification mechanisms and the inflammatory and survival responses. Overall, our results provide a better understanding on the cell death mechanism induced by CLytA-DAAO, a promising therapy against cancer., This research was funded by Spanish Instituto de Salud Carlos III, grant number PI01202025, and donations from Association of women affected by breast cancer in Elche and the region (AMACMEC) to M.S., Miguel Servet Program from Instituto de Salud Carlos III (CP19/00095) to C.d.J.R. and grants from the Spanish Ministry of Economy, Industry and Competitiveness, grant numbers BIO2013-47684-R and BIO2016-79323-R, and the RETICS-FEDER RICET, RD16/0027/0010, to J.M.S. The CIBER of Enfermedades Respiratorias (CIBERES) is an initiative of the Spanish Instituto de Salud Carlos III and Spanish National Research Council (CSIC Grant 201820I132).

To assess whether the main genetic differences observed in high-grade gliomas (HGG) will present different MR imaging and MR spectroscopy correlates that could be used to better characterize lesions in the clinical setting.Seventy-nine patients with histologically confirmed HGG were recruited. Immunohistochemistry analyses for isocitrate dehydrogenase gene 1 (IDH1), alpha thalassemia mental retardation X-linked gene (ATRX), Ki-67, and p53 protein expression were performed. Tumour radiological features were examined on MR images. Metabolic profile and infiltrative pattern were assessed with MR spectroscopy. MR features were analysed to identify imaging-molecular associations. The Kaplan-Meier method and the Cox regression model were used to identify survival prognostic factors.In total, 17.7% of the lesions were IDH1-mutated, 8.9% presented ATRX-mutated, 70.9% presented p53 unexpressed, and 22.8% had Ki-67 5%. IDH1 wild-type tumours had higher levels of mobile lipids (p = 0.001). The tumour-infiltrative pattern was higher in HGG with unexpressed p53 (p = 0.009). Mutated ATRX tumours presented higher levels of glutamate and glutamine (Glx) (p = 0.001). An association was observed between Glx tumour levels (p = 0.038) and Ki-67 expression (p = 0.008) with the infiltrative pattern. Survival analyses identified IDH1 status, age, and tumour choline levels as independent predictors of prognostic significance.Our results suggest that IDH1-wt tumours are more necrotic than IDH1-mut. And that the presence of an infiltrative pattern in HGG is associated with loss of p53 expression, Ki-67 index, and Glx levels. Finally, tumour choline levels could be used as a predictive factor in survival in addition to the IDH1 status to provide a more accurate prediction of survival in HGG patients.• IDH1-wt tumours present higher levels of mobile lipids than IDH1-mut. • Mutated ATRX tumours exhibit higher levels of glutamate and glutamine. • Loss of p53 expression, Ki-67 expression, and glutamate and glutamine levels may contribute to the presence of an infiltrative pattern in HGG.

19 p.-8 fig.-1 tab., D-amino acid oxidase (DAAO) catalyzes the oxidation of D-amino acids generating hydrogen peroxide, a potential producer of reactive oxygen species. In this study, we used a CLytA-DAAO chimera, both free and bound to magnetic nanoparticles, against colon carcinoma, pancreatic adenocarcinoma, and glioblastoma cell lines. We found that the enzyme induces cell death in most of the cell lines tested and its efficiency increases significantly when it is immobilized in nanoparticles. We also tested this enzyme therapy in non-tumor cells, and we found that there is not cell death induction, or it is significantly lower than in tumor cells. The mechanism triggering cell death is apparently a classical apoptosis pathway in the glioblastoma cell lines, while in colon and pancreatic carcinoma cell lines, CLytA-DAAO-induced cell death is a necrosis. Our results constitute a proof of concept that an enzymatic therapy, based on magnetic nanoparticles-delivering CLytA-DAAO, could constitute a useful therapy against cancer and besides it could be used as an enhancer of other treatments such as epigenetic therapy, radiotherapy, and treatments based on DNA repair., This research was funded by Spanish Instituto de Salud Carlos III, grant number PI01202025, and donations from Association of women affected by breast cancer in Elche and the region (AMACMEC) to MS and grants from the Spanish Ministry of Economy, Industry and Competitiveness, grant numbers BIO2013-47684-R and BIO2016-79323-R, and the RETICS-FEDER RICET, RD16/0027/0010, to JMS. The CIBER of Enfermedades Respiratorias (CIBERES) is an initiative of the Spanish Instituto de Salud Carlos III and Spanish National Research Council (CSIC Grant 201820I132).

During surgical procedures for gliomas, tissue material obtained from cavitational ultrasonic surgical aspirators (CUSAs) is generally discarded but can actually exceed the amount and quality of certain tumour core resections (TCRs). Despite reports indicating the suitability of CUSA-derived material for diagnosis and research, its use is still marginal. We extended these conclusions to formalin-fixed, paraffin-embedded (FFPE) samples, the most common format for archival tumour tissue in anatomical pathology departments, by conducting for the first time RNA-seq analysis in CUSA aspirates. We compared the molecular diagnosis of somatic mutations used in the clinical routine and the gene expression profiles of fixed solid material from CUSA aspirates and TCRs from the same patients in selected gliomas encompassing grades II to IV. Despite the characteristic heterogeneity of gliomas, we found substantial similarities between the corresponding aspirates and TCRs that included transcriptional signatures associated with glioma subtypes. Based on these results, we confirmed that CUSA-fixed biomaterials from glioma surgeries are appropriate for downstream applications and biomarkers screening.

Gliomas are the most common brain tumors and include a variety of histologic types and grades of malignancy. They arise from glial cells and represent approximately 70% of the primary brain tumors. According to the criteria of the World Health Organization (WHO), the majority of gliomas can be classified into four grades of malignancy (I-IV). Virus infection, especially by DNA viruses and retroviruses, which may cause insertion of viral DNA sequences into the host genome, often triggers the host defense mechanisms. Particularly, the DNA methylation machinery can be activated to cause the methylation of foreign movable viral sequences and, therefore, silence viral gene expression. Several studies have shown the presence of Human Cytomegalovirus (HCMV) in glioblastoma, suggesting that the virus may participate in tumor pathogenesis. But this relationship is controversial because many other studies did not detect HCMV in these tumors. This study aims to detect the presence of HCMV in several samples of human glioma (94 formalin-fixed, paraffin-embedded samples and 28 snap-frozen samples) by different sensitive techniques. We have been unable to detect HCMV DNA and proteins in glioma samples. Therefore, arguments used so far to conclude that HCMV is an oncomodulator virus in gliomas must be, in our view, seriously reconsidered. This study was supported by Biomedical Research Foundations of the Alicante University Hospital (FCVI HGUA Código E-04); and the Elche University Hospital (FIBElx 08/2010).

Glioblastoma multiforme (GBM) is a poor prognosis type of tumour due to its resistance to chemo and radiotherapy. SOCS1 and SOCS3 have been associated with tumour progression and response to treatments in different kinds of cancers, including GBM. In this study, cell lines of IDH-wildtype GBM from primary cultures were obtained, and the role of SOCS1 and SOCS3 in the radiotherapy response was analysed. Fifty-two brain aspirates from GBM patients were processed, and six new cell lines of IDH-wildtype GBM were established. These new cell lines were characterized according to the WHO classification of CNS tumours. SOCS1 and SOCS3 expression levels were determined, at mRNA level by Q-PCR, at protein level by immunocytochemistry, and Western blot analysis. The results showed that SOCS1 and SOCS3 are overexpressed in GBM, as compared to a non-tumoral brain RNA pool. SOCS1 and SOCS3 expression were reduced by siRNA, and it was found that SOCS3 inhibition increases radioresistance in GBM cell lines, suggesting a key role of SOCS3 in radioresistant acquisition. In addition, radioresistant clonal populations obtained by selective pressure on these cell cultures also showed a significant decrease in SOCS3 expression, while SOCS1 remained unchanged. Furthermore, the induction of SOCS3 expression, under a heterologous promoter, in a radiotherapy resistant GBM cell line increased its radiosensitivity, supporting an important implication of SOCS3 in radiotherapy resistance acquisition. Finally, the treatment with TSA in the most radioresistant established cell line produced an increase in the effect of radiotherapy, that correlated with an increase in the expression of SOCS3. These effects of TSA disappeared if the increase in the expression of SOCS3 prevented with an siRNA against SOCS3. Thus, SOCS3 signal transduction pathway (JAK/STAT) could be useful to unmask new putative targets to improve radiotherapy response in GBM. This article has been funded by a grant from “Instituto de Salud Carlos III” (Grant PI012/02025), co-supported by FEDER funds, to M. Saceda; “Fundación ERESA” small project to M.Saceda, FISABIO grant (UGP-15-237) to V. M. Barberá, and a donation from AFECANCER to M.Saceda.

The utility of molecular markers for predicting the risk of metachronous advanced colorectal lesions (MACLs) remains poorly investigated. We examined the relationship between somatic hypermethylation in polyps at baseline and the risk of developing MACL. This retrospective cohort study included 281 consecutive patients with colonic polyps who were enrolled between 2007 and 2009 and followed-up until 2014. MACLs were defined as adenomas of &gt, 10 mm, high-grade dysplasia, or with a villous component, and serrated lesions of &gt, 10 mm or with dysplasia. In total, 595 polyps were removed at baseline colonoscopy and analyzed for pathological characteristics and CpG island methylator phenotype (CIMP) using the MS-MLPA (Methylation-Specific -- Multiplex Ligation-dependent Probe Amplification) technique. Forty-five patients (16.0%) showed at least one CIMP+ polyp. MACL risk was higher in patients with CIMP+ polyps (odds ratio (OR), 4.50, 95% CI, 1.78&ndash, 11.4, p = 0.002). Patients with CIMP+ polyps also exhibited shorter time to MACL development (33.8 months vs. 50.1 months, p &lt, 0.001), even with adjustment for polyp size and number (OR, 2.40, 95% CI, 1.33&ndash, 4.34). Adding CIMP analysis improved the sensitivity (57.0% to 70.9%), negative predictive value (71.1% to 77.3%), and overall accuracy (49.8% to 52.0%) for MACL risk estimation. These results highlight that CIMP may be a useful marker for endoscopic surveillance.

The study aims to identify the relevance of immunohistochemistry (IHC), copy number aberrations (CNA) and epigenetic disorders in BRCAness breast cancers (BCs). We studied 95 paraffin included BCs, of which 41 carried BRCA1/BRCA2 germline mutations and 54 were non hereditary (BRCAX/Sporadic). Samples were assessed for BRCA1ness and CNAs by Multiplex Ligation-dependent Probe Amplification (MLPA); promoter methylation (PM) was assessed by methylation-specific-MLPA and the expression of miR-4417, miR-423-3p, miR-590-5p and miR-187-3p by quantitative RT-PCR. IHC markers Ki67, ER, PR, HER2, CK5/6, EGFR and CK18 were detected with specific primary antibodies (DAKO, Denmark). BRCAness association with covariates was performed using multivariate binary logistic regression (stepwise backwards Wald option). BRCA1/2 mutational status (p = 0.027), large tumor size (p = 0.041) and advanced histological grade (p = 0.017) among clinic-pathological variables; ER (p < 0.001) among IHC markers; MYC (p < 0.001) among CNA; APC (p = 0.065), ATM (p = 0.014) and RASSF1 (p = 0.044) among PM; and miR-590-5p (p = 0.001), miR-4417 (p = 0.019) and miR-423 (p = 0.013) among microRNA expression, were the selected parameters significantly related with the BRCAness status. The logistic regression performed with all these parameters selected ER+ as linked with the lack of BRCAness (p = 0.001) and MYC CNA, APC PM and miR-590-5p expression with BRCAness (p = 0.014, 0.045 and 0.007, respectively). In conclusion, the parameters ER expression, APC PM, MYC CNA and miR-590-5p expression, allowed detection of most BRCAness BCs. The identification of BRCAness can help establish a personalized medicine addressed to predict the response to specific treatments.

Lynch syndrome (LS) is a common cause of hereditary colorectal cancer (CRC). Some CRC patients develop mismatch repair deficiency without germline pathogenic mutation, known as Lynch-like syndrome (LLS). We compared the risk of CRC in first-degree relatives (FDRs) in LLS and LS patients. LLS was diagnosed when tumors showed immunohistochemical loss of MSH2, MSH6, and PMS2, or loss of MLH1 with BRAF wild type, and/or no MLH1 methylation and absence of pathogenic mutation in these genes. CRC and other LS-related neoplasms were followed in patients diagnosed with LS and LLS and among their FDRs. Standardized incidence ratios (SIRs) were calculated for CRC and other neoplasms associated with LS among FDRs of LS and LLS patients. In total, 205 LS (1205 FDRs) and 131 LLS families (698 FDRs) had complete pedigrees. FDRs of patients with LLS had a high incidence of CRC (SIR, 2.08, 95% confidence interval (CI), 1.56&ndash, 2.71), which was significantly lower than that in FDRs of patients with LS (SIR, 4.25, 95% CI, 3.67&ndash, 4.90, p &lt, 0.001). The risk of developing other neoplasms associated with LS also increased among FDR of LLS patients (SIR, 2.04, 95% CI, 1.44&ndash, 2.80) but was lower than that among FDR of patients with LS (SIR, 5.01, 95% CI, 4.26&ndash, 5.84, 0.001). FDRs with LLS have an increased risk of developing CRC as well as LS-related neoplasms, although this risk is lower than that of families with LS. Thus, their management should take into account this increased risk.

BACKGROUND & AIMS: Lynch syndrome is characterized by DNA mismatch repair (MMR) deficiency. Some patients with suspected Lynch syndrome have DNA MMR deficiencies but no detectable mutations in genes that encode MMR proteins-this is called Lynch-like syndrome (LLS). There is no consensus on management of patients with LLS. We collected data from a large series of patients with LLS to identify clinical and pathology features. METHODS: We collected data from a nationwide-registry of patients with colorectal cancer (CRC) in Spain. We identified patients whose colorectal tumors had loss of MSH2, MSH6, PMS2, or MLH1 (based on immunohistochemistry), without the mutation encoding V600E in BRAF (detected by real-time PCR), and/or no methylation at MLH1 (determined by methylation-specific multiplex ligation-dependent probe amplification), and no pathogenic mutations in MMR genes, BRAF, or EPCAM (determined by DNA sequencing). These patients were considered to have LLS. We collected data on demographic, clinical, and pathology features and family history of neoplasms. The chi(2) test was used to analyze the association between qualitative variables, followed by the Fisher exact test and the Student t test or the Mann-Whitney test for quantitative variables. RESULTS: We identified 160 patients with LLS; their mean age at diagnosis of CRC was 55 years and 66 patients were female (41%). The Amsterdam I and II criteria for Lynch syndrome were fulfilled by 11% of cases and the revised Bethesda guideline criteria by 65% of cases. Of the patients with LLS, 24% were identified in universal screening. There were no proportional differences in sex, indication for colonoscopy, immunohistochemistry, pathology findings, or personal history of CRC or other Lynch syndrome-related tumors between patients who met the Amsterdam and/or Bethesda criteria for Lynch syndrome and patients identified in universal screening for Lynch syndrome, without a family history of CRC. CONCLUSIONS: Patients with LLS have homogeneous clinical, demographic, and pathology characteristics, regardless of family history of CRC.

Purpose: A recent study reported that 5-fluorouracil (5-FU)-based chemotherapy is less effective in treating patients with advanced colorectal cancer demonstrating hypermethylation of the TFAP2E gene. The aim of our study was to confirm and validate these findings in large, uniformly treated, well-characterized patient cohorts. Experimental Design: Two cohorts of 783 patients with colorectal cancer: 532 from a population-based, multicenter cohort (EPICOLON I) and 251 patients from a clinic-based trial were used to study the effectiveness of TFAP2E methylation and expression as a predictor of response of colorectal cancer patients to 5-FU–based chemotherapy. DNA methylation status of the TFAP2E gene in patients with colorectal cancer was assessed by quantitative bisulfite pyrosequencing analysis. IHC analysis of the TFAP2E protein expression was also performed. Results: Correlation between TFAP2E methylation status and IHC staining was performed in 607 colorectal cancer samples. Among 357 hypermethylated tumors, only 141 (39.6%) exhibited loss of protein expression. Survival was not affected by TFAP2E hypermethylation in stage IV patients [HR, 1.21; 95% confidence interval (CI), 0.79–1.87; log-rank P = 0.6]. In stage II–III cases, disease-free survival was not influenced by TFAP2E hypermethylation status in 5-FU–treated (HR, 0.91; 95% CI, 0.52–1.59; log-rank P = 0.9) as well as in nontreated patients (HR, 0.88; 95% CI, 0.5–1.54; log-rank P = 0.7). Conclusions: TFAP2E hypermethylation does not correlate with loss of its protein expression. Our large, systematic, and comprehensive study indicates that TFAP2E methylation and expression may not play a major role in predicting response to 5-FU–based chemotherapy in patients with colorectal cancer. Clin Cancer Res; 24(12); 2820–7. ©2018 AACR.

Objective The aim of this study was to validate a molecular classification of colorectal cancer (CRC) based on microsatellite instability (MSI), CpG island methylator phenotype (CIMP) status, BRAF, and KRAS and investigate each subtype's response to chemotherapy. Design This retrospective observational study included a population-based cohort of 878 CRC patients. We classified tumours into five different subtypes based on BRAF and KRAS mutation, CIMP status, and MSI. Patients with advanced stage II (T4N0M0) and stage III tumours received 5-fluoruracil (5-FU)-based chemotherapy or no adjuvant treatment based on clinical criteria. The main outcome was disease-free survival (DFS). Results Patients with the combination of microsatellite stable (MSS) tumours, BRAF mutation and CIMP positive exhibited the worst prognosis in univariate (log rank P < 0.0001) and multivariate analyses (hazard ratio 1.75, 95% CI 1.05-2.93, P = 0.03) after adjusting for age, sex, chemotherapy, and TNM stage. Treatment with 5-FU-based regimens improved prognosis in patients with the combination of MSS tumours, KRAS mutation and CIMP negative (log rank P = 0.003) as well as in patients with MSS tumours plus BRAF and KRAS wild-type and CIMP negative (log-rank P < 0.001). After adjusting for age, sex, and TNM stage in the multivariate analysis, only patients with the latter molecular combination had independently improved prognosis after adjuvant chemotherapy (hazard ratio 2.06, 95% CI 1.24-3.44, P = 0.005). Conclusion We confirmed the prognostic value of stratifying CRC according to molecular subtypes using MSI, CIMP status, and somatic KRAS and BRAF mutation. Patients with traditional chromosomally unstable tumours obtained the best benefit from adjuvant 5-FU-based chemotherapy. This work was supported by the Instituto de Salud Carlos III (PI08/0726, INT-09/208, PI11/2630, INT-12-078, INT13-196, PI14/01386), FISABIO-ISABIAL foundation (UGP-13-221, UGP-14-265), and the Asociación Española contra el Cáncer (Fundación Científica GCB13131592CAST). Eva Hernández-Illán received a grant from Instituto de Salud Carlos III (FI12/00233). Mar Giner-Calabuig received a grant from VALi+d. EXP ACIF/2016/002. Miren Alustiza received a predoctoral grant from ISABIAL (UGP-16-138). Other financial support was obtained from Asociación Española de Gastroenterología grants (Gonzalo Miño 2014, Tamarite 2015, Grupo de endoscopia 2016).

555 Background: The role of genetic profile of polyps to predict advanced metachronous lesions (AML) remains unknown. The aim is to study the relation between genetic profile of polyps and both risk of AMLs and time to develop them in surveillance. Methods: 308 patients with colonic polyps were consecutively enrolled between 2007 and 2009 for this cohort study, and followed up to 2014 (median 26 months, range 63). Variables as age, sex, smoking, weight, number of colonoscopies and number and characteristics of polyps were collected. 995 polyps were analyzed for somatic mutations on BRAF and KRAS genes using allelic discrimination by real-time PCR and direct DNA sequenciation, respectively. High level of methylation on CpG islands (CIMP-H) was also tested using MS-MLPA. AML was defined by a size higher than 9mm, high grade dysplasia or villous component. Risk of developing AML for individual genetic markers was studied using Chi-square tests and logistic regression. Log-rank test with Kaplan Meier survival curves and Cox-regression model were also performed. Multivariate analysis were adjusted by sex, age, familial colorectal cancer, smoking and features of AML in first colonoscopy. Results: 21% of polyps in first colonoscopy were CIMP-H. KRAS and BRAF mutations accounted for 25% and 17% of polyps, respectively. In univariate analysis, KRAS-mutated polyps were related to higher risk of AML in surveillance (52% KRAS-mutated polyps vs 31% non-mutated; p = 0.01). Similar results were obtained regarding CIMP-H (77% CIMP-H polyps vs 38% non-CIMP; p = 0.005). Logistic regression showed CIMP-H as the unique genetic marker of risk for AML (OR 11.41, 95% CI 2.04-63.70; p = 0.006). Regarding time to develop AML, shorter intervals were found related to CIMP-H (median of 31 vs 48 months in non-CIMP-H; p = 0.002) and KRAS-mutations (median of 36 vs 49 months in non-mutated; p = 0.029) in univariate analysis. Multivariate analysis highlighted CIMP-H as the unique independent marker associated to shorter time to develop AML (HR 4.01, 95% CI 1.36-10.46; p = 0.01). Conclusions: Presence of CIMP-H in polyps associates higher risk of subsequent AML and shorter interval to their development. Genetic profile of polyps emerges as useful tool for colonoscopy surveillance.

Background Colorectal cancers (CRCs) that have microsatellite instability (MSI) and mutL homolog 1 (MLH1) immunoloss are observed in 3 clinical scenarios: Lynch syndrome (LS), sporadic MSI CRC, and Lynch-like syndrome (LLS). v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) mutational analysis is used to differentiate LS from sporadic MSI CRC. The role of MLH1 promoter methylation status for the differential diagnosis of these clinical forms is not well established. The objectives of this study were: 1) to analyze MLH1 promoter methylation in MLH1-deficient CRCs by pyrosequencing, and 2) to assess its role in the differential diagnosis of MLH1-deficient CRCs. Methods In total, 165 CRCs were analyzed, including LS (n = 19), MSI BRAF-mutated CRC (n = 37), MSI BRAF wild-type CRC (n = 60), and a control group of CRCs without MSI (microsatellite stable [MSS] CRC; n = 49). MLH1 promoter methylation status was analyzed by pyrosequencing, and the ability of different strategies to identify LS was assessed. Results The average ± standard deviation methylation in LS (9% ± 7%) was significantly lower than that in MSI BRAF-mutated CRC (42% ± 17%; P < .001) and in MSI BRAF wild-type CRC (25% ± 19%; P = .002). Somatic MLH1 hypermethylation was detected in 3 patients (15.8%) with LS, in 34 patients (91.9%) with MSI BRAF-mutated CRC, and in 37 patients (61.7%) with MSI BRAF wild-type tumors. Patients with MSI BRAF wild-type, unmethylated tumors (ie, LLS) had a stronger family history of CRC than those who had tumors with MLH1 methylation (P < .05). The sensitivity for ruling out LS was 100% for BRAF analysis, 84.2% for MLH1 methylation analysis, and 84.2% for the combination of both analyses. Conclusion Somatic MLH1 promoter methylation occurs in up to 15% of LS CRCs. Somatic BRAF analysis is the most sensitive strategy for ruling out LS. Patients who have CRCs with loss of MLH1 protein expression and neither BRAF mutation nor MLH1 methylation resemble patients with LS.

Purpose: The present study aimed to determine the prevalence of MUTYH mutations in patients with multiple colonic polyps and to explore the best strategy for diagnosing MUTYH-associated polyposis (MAP) in these patients. Experimental Design: This study included 405 patients with at least 10 colonic polyps each. All cases were genetically tested for the three most frequent MUTYH mutations. Whole-gene analysis was performed in heterozygous patients and in 216 patients lacking the three most frequent mutations. Polyps from 56 patients were analyzed for the KRAS-Gly12Cys and BRAF V600E somatic mutations. Results: Twenty-seven (6.7%) patients were diagnosed with MAP, of which 40.8% showed serrated polyps. The sensitivity of studying only the three common variants was 74.1%. Of 216 patients without any monoallelic mutation in common variants, whole-gene analysis revealed biallelic pathogenic mutation in only one. G396D mutation was associated with serrated lesions and older age at diagnosis. There was a strong association between germinal MUTYH mutation and KRAS Gly12Cys somatic mutation in polyps. BRAF V600E mutation was found in 74% of serrated polyps in MUTYH-negative patients and in none of the polyps of MAP patients. Conclusions: We observed a low frequency of MUTYH mutations among patients with multiple adenomatous and serrated polyps. The MAP phenotype frequently included patients with serrated polyps, especially when G396D mutation was involved. Our results show that somatic molecular markers of polyps can be useful in identifying MAP cases and support the need for the complete MUTYH gene analysis only in patients heterozygous for recurrent variants. Clin Cancer Res; 20(5); 1158–68. ©2014 AACR.

Lynch syndrome (LS) is caused by germline mutations in one of the four mismatch repair (MMR) genes. Defects in this pathway lead to microsatellite instability (MSI) in DNA tumors, which constitutes the molecular hallmark of this disease. Selection of patients for genetic testing in LS is usually based on fulfillment of diagnostic clinical criteria (i.e. Amsterdam criteria or the revised Bethesda guidelines). However, following these criteria PMS2 mutations have probably been underestimated as their penetrances appear to be lower than those of the other MMR genes. The use of universal MMR study-based strategies, using MSI testing and immunohistochemical (IHC) staining, is being one proposed alternative. Besides, germline mutation detection in PMS2 is complicated by the presence of highly homologous pseudogenes. Nevertheless, specific amplification of PMS2 by long-range polymerase chain reaction (PCR) and the improvement of the analysis of large deletions/duplications by multiplex ligation-dependent probe amplification (MLPA) overcome this difficulty. By using both approaches, we analyzed 19 PMS2-suspected carriers who have been selected by clinical or universal strategies and found five large deletions and one frameshift mutation in PMS2 in six patients (31%). Owing to the high incidence of large deletions found in our cohort, we recommend MLPA analysis as the first-line method for searching germline mutations in PMS2.

Background & Aims We investigated whether patients with multiple serrated polyps, but not meeting the World Health Organization criteria for serrated polyposis syndrome, and their relatives have similar risks for colorectal cancer (CRC) as those diagnosed with serrated polyposis. Methods We collected data from patients with more than 10 colonic polyps, recruited in 2008–2009 from 24 hospitals in Spain for a study of causes of multiple colonic polyps. We analyzed data from 53 patients who met the criteria for serrated polyposis and 145 patients who did not meet these criteria, but who had more than 10 polyps throughout the colon, of which more than 50% were serrated. We calculated age- and sex-adjusted standardized incidence ratios (SIRs) for CRC in both groups, as well as in their first-degree relatives. Results The prevalence of CRC was similar between patients with confirmed serrated polyposis and multiple serrated polyps (odds ratio, 1.35; 95% confidence interval [CI], 0.64–2.82; P = .40). The SIR for CRC in patients with serrated polyposis (0.51; 95% CI, 0.01–2.82) did not differ significantly from the SIR for CRC in patients with multiple serrated polyps (0.74; 95% CI, 0.20–1.90; P = .70). The SIR for CRC also did not differ significantly between first-degree relatives of these groups (serrated polyposis: 3.28, 95% CI, 2.16–4.77; multiple serrated polyps: 2.79, 95% CI, 2.10–3.63; P = .50). Kaplan–Meier analysis showed no differences in the incidence of CRC between groups during the follow-up period (log-rank, 0.6). Conclusions The risk of CRC in patients with multiple serrated polyps who do not meet the criteria for serrated polyposis, and in their first-degree relatives, is similar to that of patients diagnosed with serrated polyposis.

DNA methylation (DNAm) has been linked to changes in chromatin structure, gene expression and disease. The DNAm level can be affected by genetic variation; although, how this differs by CpG dinucleotide density and genic location of the DNAm site is not well understood. Moreover, the effect of disease causing variants on the DNAm level in a tissue relevant to disease has yet to be fully elucidated. To this end, we investigated the phenotypic profiles, genetic effects and regional genomic heritability for 196080 DNAm sites in healthy colorectum tissue from 132 unrelated Colombian individuals. DNAm sites in regions of low-CpG density were more variable, on average more methylated and were more likely to be significantly heritable when compared with DNAm sites in regions of high-CpG density. DNAm sites located in intergenic regions had a higher mean DNAm level and were more likely to be heritable when compared with DNAm sites in the transcription start site (TSS) of a gene expressed in colon tissue. Within CpG-dense regions, the propensity of the DNAm level to be heritable was lower in the TSS of genes expressed in colon tissue than in the TSS of genes not expressed in colon tissue. In addition, regional genetic variation was associated with variation in local DNAm level no more frequently for DNAm sites within colorectal cancer risk regions than it was for DNAm sites outside such regions. Overall, DNAm sites located in different genomic contexts exhibited distinguishable profiles and may have a different biological function.

Background and study aims: Endoscopic surveillance in patients with multiple colorectal polyps aims to reduce colorectal cancer (CRC) incidence and mortality, as well as the need for colorectal surgery. The aim of this study was to determine the risk of developing CRC or the need for surgery during endoscopic surveillance in a cohort of patients with multiple (10-100) colorectal polyps. Patients and methods: This was a multicentrer, longitudinal, observational study in 15 CRC high risk clinics in Spain, carried out between January 2009 and December 2010. Patients who were included in the EPIPOLIP trial and had at least 1 year of follow-up were included in the study. The primary outcome of interest was the incidence of CRC at least 1 year following the initial colonoscopy. The secondary outcome was the need for colorectal surgery. Results: A total of 265 patients were followed for a median of 3.8 years. Patients underwent a median of 5 colonoscopies, and 17 patients (6.4 %) were diagnosed with CRC. A total of 32 patients (12.1 %) underwent surgery, including 15 (5.7 %) for prophylaxis without a diagnosis of CRC. The corresponding incidence density rates for CRC and colorectal surgery were 1.4 (95% confidence interval [CI] 0.7 to 2.1) and 2.7 (95 % CI 1.7 to 3.6) per 100 patient-years, respectively. Only the presence of symptoms at first colonoscopy was independently associated with CRC diagnosis (hazard ratio [HR] 7.7, 95% CI 1.1 to 59.3) and colorectal surgery (HR 4.6, 95% CI 1.02 to 20.6). Conclusions: Patients with more than 10 neoplastic polyps required frequent colonoscopies within a short follow-up period. More than 10% of patients required colorectal surgery within 4 years, more than half for incident CRC.

We describe an association between TGFBR1 polymorphism rs868 and mismatch repair proficient hereditary colorectal cancers. This polymorphism results in more binding of TGFBR1 to let-7b-5p miRNA, which would result in lower expression of TGFBR1 and thus higher colorectal cancer risk.The purpose of this study was to identify novel colorectal cancer (CRC)-causing alleles in unexplained familial CRC cases. In order to do so, coding regions in five candidate genes (MGMT, AXIN2, CTNNB1, TGFBR1 and TGFBR2) were sequenced in 11 unrelated microsatellite-stable hereditary non-polyposis CRC (MSS HNPCC) cases. Selected genetic variants were genotyped in a discovery set of 27 MSS HNPCC cases and 85 controls. One genetic variant, rs67687202, in TGFBR1 emerged as significant (P = 0.002), and it was genotyped in a replication set of 87 additional MSS HNPCC-like cases and 338 controls where it was also significantly associated with MSS HNPCC cases (P = 0.041). In the combined genotype data, rs67687202 was associated with a moderate increase in CRC risk (OR = 1.68; 95% CI = 1.13-2.50; P = 0.010). We tested a highly correlated SNP rs868 in 723 non-familial CRC cases compared with 629 controls, and it was not significantly associated with CRC risk (P = 0.370). rs868 is contained in a let-7 miRNA binding site in the 3'UTR of TGFBR1, which might provide a functional basis for the association in MSS HNPCC. In luciferase assays, the risk-associated allele for rs868 was associated with half the luciferase expression in the presence of miRNA let-7b-5p compared with protective allele, suggesting more binding of let-7b-5p and less TGFBR1 expression. Thus, rs868 potentially is a CRC risk-causing allele. Our results support the concept that rs868 is associated with lower TGFBR1 expression thereby increasing CRC risk.