Exome sequencing of early-onset patients supports genetic heterogeneity in colorectal cancer.

Colorectal cancer (CRC) is a complex disease that can be caused by a spectrum of genetic variants ranging from low to high penetrance changes, that interact with the environment to determine which individuals will develop the disease. In this study, we sequenced 20 early-onset CRC patients to discover novel genetic variants that could be linked to the prompt disease development. Eight genes, CHAD, CHD1L, ERCC6, IGTB7, PTPN13, SPATA20, TDG and TGS1, were selected and re-sequenced in a further 304 early onset CRC patients to search for rare, high-impact variants. Although we found a recurring truncating variant in the TDG gene shared by two independent patients, the results obtained did not help consolidate any of the candidates as promising CRC predisposing genes. However, we found that potential risk alleles in our extended list of candidate variants have a tendency to appear at higher numbers in younger cases. This supports the idea that CRC onset may be oligogenic in nature and may show molecular heterogeneity. Further, larger and robust studies are thus needed to unravel the genetics behind early-onset CRC development, coupled with novel functional analyses and omic approaches that may offer complementary insight.

Psychological impact of multigene cancer panel testing in patients with a clinical suspicion of hereditary cancer across Spain.

OBJECTIVE: Patients' psychological reactions to multigene cancer panel testing might differ compared with the single-gene testing reactions because of the complexity and uncertainty associated with the different possible results. Understanding patients' preferences and psychological impact of multigene panel testing is important to adapt the genetic counselling model. METHODS: One hundred eighty-seven unrelated patients with clinical suspicion of hereditary cancer undergoing a 25-gene panel test completed questionnaires after pretest genetic counselling and at 1 week, 3 months, and 12 months after results to elicit their preferences regarding results disclosure and to measure their cancer worry and testing-specific distress and uncertainty. RESULTS: A pathogenic variant was identified in 38 patients (34 high penetrance and 4 moderate penetrance variants), and 54 patients had at least one variant of uncertain significance. Overall, cancer panel testing was not associated with an increase in cancer worry after results disclosure (P value = .87). Twelve months after results, carriers of a moderate penetrance variant had higher distress and uncertainty scores compared with carriers of high penetrance variants. Cancer worry prior to genetic testing predicted genetic testing specific distress after results, especially at long term (P value <.001). Most of the patients reported the wish to know all genetic results. CONCLUSIONS: Our results suggest that patients can psychologically cope with cancer panel testing, but distress and uncertainty observed in carriers of moderate penetrance cancer variants in this cohort warrant further research.

Candidate predisposing germline copy number variants in early onset colorectal cancer patients

Purpose. A great proportion of the heritability of colorectal cancer (CRC) still remains unexplained, and rare variants, as well as copy number changes, have been proposed as potential candidates to explain the so-called ‘missing heritability’. We aimed to identify rare high-to-moderately penetrant copy number variants (CNVs) in patients suspected of having hereditary CRC due to an early onset. Methods/patients. We have selected for genome-wide copy number analysis, 27 MMR-proficient early onset CRC patients (<50 years) without identifiable germline mutations in Mendelian genes related to this phenotype. Rare CNVs were selected by removing all CNVs detected at MAF >1% in the in-house control CNV database (n = 629 healthy controls). Copy number assignment was checked by duplex real-time quantitative PCR or multiplex ligation probe amplification. Somatic mutation analysis in candidate genes included: loss of heterozygosity studies, point mutation screening, and methylation status of the promoter. Results. We have identified two rare germline deletions involving the AK3 and SLIT2 genes in two patients. The search for a second somatic mutational event in the corresponding CRC tumors showed loss of heterozygosity in AK3, and promoter hypermethylation in SLIT2. Both genes have been previously related to colorectal carcinogenesis. Conclusions. These findings suggest that AK3 and SLIT2 may be potential candidates involved in genetic susceptibility to CRC (AU)

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Methylation of WNT target genes AXIN2 and DKK1 as robust biomarkers for recurrence prediction in stage II colon cancer.

Stage II colon cancer (CC) still remains a clinical challenge with patient stratification for adjuvant therapy (AT) largely relying on clinical parameters. Prognostic biomarkers are urgently needed for better stratification. Previously, we have shown that WNT target genes AXIN2, DKK1, APCDD1, ASCL2 and LGR5 are silenced by DNA methylation and could serve as prognostic markers in stage II CC patients using methylation-specific PCR. Here, we have extended our discovery cohort AMC90-AJCC-II (N=65) and methylation was analyzed by quantitative pyrosequencing. Subsequently, we validated the results in an independent EPICOLON1 CC cohort (N=79). Methylation of WNT target genes is negatively correlated to mRNA expression. A combination of AXIN2 and DKK1 methylation significantly predicted recurrences in univariate (area under the curve (AUC)=0.83, confidence interval (CI): 0.72-0.94, P<0.0001) analysis in stage II microsatellite stable (MSS) CC patients. This two marker combination showed an AUC of 0.80 (CI: 0.68-0.91, P<0.0001) in the EPICOLON1 validation cohort. Multivariate analysis in the Academic Medical Center (AMC) cohort revealed that both WNT target gene methylation and consensus molecular subtype 4 (CMS4) are significantly associated with poor recurrence-free survival (hazard ratio (HR)methylation: 3.84, 95% CI: 1.14-12.43; HRCMS4: 3.73, 95% CI: 1.22-11.48). CMS4 subtype tumors with WNT target methylation showed worse prognosis. Combining WNT target gene methylation and CMS4 subtype lead to an AUC of 0.89 (0.791-0.982, P<0.0001) for recurrence prediction. Notably, we observed that methylation of DKK1 is high in BRAF mutant and CIMP (CpG island methylator phenotype)-positive cancers, whereas AXIN2 methylation appears to be associated with CMS4. Methylation of AXIN2 and DKK1 were found to be robust markers for recurrence prediction in stage II MSS CC patients. Further validation of these findings in a randomized and prospective manner could pave a way to identify poor prognosis patients of stage II CC for AT.

An international survey of polypectomy training and assessment.

Background and study aims Colonic polypectomy is acknowledged to be a technically challenging part of colonoscopy. Training in polypectomy is recognized to be often inconsistent. This study aimed to ascertain worldwide practice in polypectomy training. Patients and methods An electronic survey was distributed to endoscopic trainees and trainers in 19 countries asking about their experiences of receiving and delivering training. Participants were also asked about whether formal polypectomy training guidance existed in their country. Results Data were obtained from 610 colonoscopists. Of these responses, 348 (57.0 %) were from trainers and 262 (43.0 %) from trainees; 6.6 % of trainers assessed competency once per year or less often. Just over half (53.1 %) of trainees had ever had their polypectomy technique formally assessed by any trainer. Approximately half the trainees surveyed (51.1 %) stated that the principles of polypectomy had only ever been taught to them intermittently. Of those trainees with the most colonoscopy experience, who had performed over 500 procedures, 48.2 % had had training on removing large polyps of over 10 mm; 46.2 % (121 respondents) of trainees surveyed held no record of the polypectomies they had performed. Only four of the 19 countries surveyed had specific guidelines on polypectomy training. Conclusions A significant number of competent colonoscopists have never been taught how to perform polypectomy. Training guidelines worldwide generally give little direction as to how trainees should acquire polypectomy skills. The learning curve for polypectomy needs to be defined to provide reliable guidance on how to train colonoscopists in this skill.

Candidate predisposing germline copy number variants in early onset colorectal cancer patients.

PURPOSE: A great proportion of the heritability of colorectal cancer (CRC) still remains unexplained, and rare variants, as well as copy number changes, have been proposed as potential candidates to explain the so-called 'missing heritability'. We aimed to identify rare high-to-moderately penetrant copy number variants (CNVs) in patients suspected of having hereditary CRC due to an early onset. METHODS/PATIENTS: We have selected for genome-wide copy number analysis, 27 MMR-proficient early onset CRC patients (<50 years) without identifiable germline mutations in Mendelian genes related to this phenotype. Rare CNVs were selected by removing all CNVs detected at MAF >1% in the in-house control CNV database (n = 629 healthy controls). Copy number assignment was checked by duplex real-time quantitative PCR or multiplex ligation probe amplification. Somatic mutation analysis in candidate genes included: loss of heterozygosity studies, point mutation screening, and methylation status of the promoter. RESULTS: We have identified two rare germline deletions involving the AK3 and SLIT2 genes in two patients. The search for a second somatic mutational event in the corresponding CRC tumors showed loss of heterozygosity in AK3, and promoter hypermethylation in SLIT2. Both genes have been previously related to colorectal carcinogenesis. CONCLUSIONS: These findings suggest that AK3 and SLIT2 may be potential candidates involved in genetic susceptibility to CRC.

The histone deacetylase sirtuin 2 is a new player in the regulation of platelet function.

BACKGROUND: Histone deacetylases (HDACs) play a key role in signaling in many cell types. However, little is known about the participation of HDACs, particularly sirtuins (SIRTs), in platelet reactivity. OBJECTIVE: To investigate the role of HDACs in platelets, we examined the effects of SIRT inhibition on platelet function and protein acetylation in human platelets. METHODS: We used washed platelets obtained from healthy subjects. Cambinol (SIRT1 and SIRT2 inhibitor), AGK2 (specific SIRT2 inhibitor) and EX527 (specific SIRT1 inhibitor) were used as SIRT inhibitors. Platelets were stimulated with collagen, thrombin, or U46619, and platelet responses were determined according to optical aggregometry findings, dense granule release, and cytosolic calcium levels (Fura-2AM fluorescence). Protein acetylation and phosphorylation were assessed by immunoblotting. RESULTS: SIRT inhibition remarkably reduced platelet responses (aggregation, granule release, and cytosolic calcium level; P < 0.05). SIRT2 was present in platelets at the level of mRNA and protein, and its specific inhibition reduced platelet responses. The acetylated protein pattern observed in resting platelets changed during platelet aggregation. Inhibition of SIRT2 increased the acetylation of Akt kinase, which in turn blocked agonist-induced Akt phosphorylation and glycogen synthase kinase-3ß phosphorylation, which are markers of Akt activity. Finally, collagen-induced aggregation provoked Akt acetylation. CONCLUSIONS: Regulation of protein acetylation by SIRT2 plays a central role in platelet function. The effects of SIRT2 are mediated in part by the acetylation and inhibition of Akt. These results open a new avenue for research into the control of platelet function, and may help to identify new therapeutic targets.

Effect of oral anticoagulants on the outcome of faecal immunochemical test.

BACKGROUND: We aimed to evaluate whether oral anticoagulants (OACs) alter faecal immunochemical test (FIT) performance in average-risk colorectal cancer (CRC) screening. METHODS: Individuals aged 50-69 years were invited to receive one FIT sample (cutoff 75 ng ml(-1)) between November 2008 and June 2011. RESULTS: Faecal immunochemical test was positive in 9.3% (21 out of 224) of users of OAC and 6.2% (365 out of 5821) of non-users (P-trend=0.07). The positive predictive value (PPV) for advanced neoplasia (AN) in non-users was 50.4% vs 47.6% in users (odds ratio, 0.70; 95% CI, 0.3-1.8; P=0.5). The PPV for AN in OAC more antiplatelets (aspirin or clopidogrel) was 75% (odds ratio, 2; 95% CI, 0.4-10.8; P=0.4). CONCLUSIONS: Oral anticoagulant did not significantly modify the PPV for AN in this population-based colorectal screening program. The detection rate of advanced adenoma was higher in the combination OAC more antiplatelets.

High incidence of large deletions in the PMS2 gene in Spanish Lynch syndrome families.

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.

A genome-wide association study on copy-number variation identifies a 11q11 loss as a candidate susceptibility variant for colorectal cancer.

Colorectal cancer (CRC) is a complex disease, and therefore its development is determined by the combination of both environmental factors and genetic variants. Although genome-wide association studies (GWAS) of SNP variation have conveniently identified 20 genetic variants so far, a significant proportion of the observed heritability is yet to be explained. Common copy-number variants (CNVs) are one of the most important genomic sources of variability, and hence a potential source to explain part of this missing genetic fraction. Therefore, we have performed a GWAS on CNVs to explore the relationship between common structural variation and CRC development. Phase 1 of the GWAS consisted of 881 cases and 667 controls from a Spanish cohort. Copy-number status was validated by quantitative PCR for each of those common CNVs potentially associated with CRC in phase I. Subsequently, SNPs were chosen as proxies for the validated CNVs for phase II replication (1,342 Spanish cases and 1,874 Spanish controls). Four common CNVs were found to be associated with CRC and were further replicated in Phase II. Finally, we found that SNP rs1944682, tagging a 11q11 CNV, was nominally associated with CRC susceptibility (p value = 0.039; OR = 1.122). This locus has been previously related to extreme obesity phenotypes, which could suggest a relationship between body weight and CRC susceptibility.

Inhibition of VEGF expression through blockade of Hif1α and STAT3 signalling mediates the anti-angiogenic effect of melatonin in HepG2 liver cancer cells.

BACKGROUND: Hepatocellular carcinoma (HCC) growth relies on angiogenesis via vascular endothelial growth factor (VEGF) release. Hypoxia within tumour environment leads to intracellular stabilisation of hypoxia inducible factor 1 alpha (Hif1α) and signal transducer and activator of transcription (STAT3). Melatonin induces apoptosis in HCC, and shows anti-angiogenic features in several tumours. In this study, we used human HepG2 liver cancer cells as an in vitro model to investigate the anti-angiogenic effects of melatonin. METHODS: HepG2 cells were treated with melatonin under normoxic or CoCl2-induced hypoxia. Gene expression was analysed by RT-qPCR and western blot. Melatonin-induced anti-angiogenic activity was confirmed by in vivo human umbilical vein endothelial cells (HUVECs) tube formation assay. Secreted VEGF was measured by ELISA. Immunofluorescence was performed to analyse Hif1α cellular localisation. Physical interaction between Hif1α and its co-activators was analysed by immunoprecipitation and chromatin immunoprecipitation (ChIP). RESULTS: Melatonin at a pharmacological concentration (1 mM) decreases cellular and secreted VEGF levels, and prevents HUVECs tube formation under hypoxia, associated with a reduction in Hif1α protein expression, nuclear localisation, and transcriptional activity. While hypoxia increases phospho-STAT3, Hif1α, and CBP/p300 recruitment as a transcriptional complex within the VEGF promoter, melatonin 1 mM decreases their physical interaction. Melatonin and the selective STAT3 inhibitor Stattic show a synergic effect on Hif1α, STAT3, and VEGF expression. CONCLUSION: Melatonin exerts an anti-angiogenic activity in HepG2 cells by interfering with the transcriptional activation of VEGF, via Hif1α and STAT3. Our results provide evidence to consider this indole as a powerful anti-angiogenic agent for HCC treatment.

European guidelines for quality assurance in colorectal cancer screening and diagnosis: overview and introduction to the full supplement publication.

Population-based screening for early detection and treatment of colorectal cancer (CRC) and precursor lesions, using evidence-based methods, can be effective in populations with a significant burden of the disease provided the services are of high quality. Multidisciplinary, evidence-based guidelines for quality assurance in CRC screening and diagnosis have been developed by experts in a project co-financed by the European Union. The 450-page guidelines were published in book format by the European Commission in 2010.  They include 10 chapters and over 250 recommendations, individually graded according to the strength of the recommendation and the supporting evidence. Adoption of the recommendations can improve and maintain the quality and effectiveness of an entire screening process, including identification and invitation of the target population, diagnosis and management of the disease and appropriate surveillance in people with detected lesions. To make the principles, recommendations and standards in the guidelines known to a wider professional and scientific community and to facilitate their use in the scientific literature, the original content is presented in journal format in an open-access Supplement of Endoscopy. The editors have prepared the present overview to inform readers of the comprehensive scope and content of the guidelines.

Pharmacogenomics in colorectal cancer: a genome-wide association study to predict toxicity after 5-fluorouracil or FOLFOX administration.

The development of genotyping technologies has allowed for wider screening for inherited causes of variable outcomes following drug administration. We have performed a genome-wide association study (GWAS) on 221 colorectal cancer (CRC) patients that had been treated with 5-fluorouracil (5-FU), either alone or in combination with oxaliplatin (FOLFOX). A validation set of 791 patients was also studied. Seven SNPs (rs16857540, rs2465403, rs10876844, rs10784749, rs17626122, rs7325568 and rs4243761) showed evidence of association (pooled P-values 0.020, 9.426E-03, 0.010, 0.017, 0.042, 2.302E-04, 2.803E-03) with adverse drug reactions (ADRs). This is the first study to explore the genetic basis of inter-individual variation in toxicity responses to the administration of 5-FU or FOLFOX in CRC patients on a genome-wide scale.

European guidelines for quality assurance in colorectal cancer screening and diagnosis. First Edition--Colonoscopic surveillance following adenoma removal.

Multidisciplinary, evidence-based guidelines for quality assurance in colorectal cancer screening and diagnosis have been developed by experts in a project coordinated by the International Agency for Research on Cancer. The full guideline document covers the entire process of population-based screening. It consists of 10 chapters and over 250 recommendations, graded according to the strength of the recommendation and the supporting evidence. The 450-page guidelines and the extensive evidence base have been published by the European Commission. The chapter on colonoscopic surveillance following adenoma removal includes 24 graded recommendations. The content of the chapter is presented here to promote international discussion and collaboration by making the principles and standards recommended in the new EU Guidelines known to a wider professional and scientific community. Following these recommendations has the potential to enhance the control of colorectal cancer through improvement in the quality and effectiveness of surveillance and other elements in the screening process, including multi-disciplinary diagnosis and management of the disease.

Modification of loco-regional microenvironment in brain tumors by spinal cord stimulation. Implications for radio-chemotherapy.

The effectiveness of radiotherapy and chemotherapy in high grade gliomas (HGG) depends on tumor micro-environment. We summarize our experience of the influence of spinal cord stimulation (SCS) on this micro-environment. Patients with HGG (n = 26) were assessed pre- and post-SCS, using: (1) Doppler in middle cerebral arteries (MCA) and (2) in common carotid arteries (CCA); (3) tumor blood-flow using single photon emission computed tomography (SPECT); (4) tumor-pO(2) (mmHg) using polarographic probes (eight tumor areas from five patients); and (5) tumor glucose metabolism using (18)F-fluoro-2-deoxyglucose ((18)FDG) positron emission tomography ((18)FDG-PET). Pre-SCS: tumor blood-flow was lower (P < 0.001) than peri-tumor areas and healthy contra-lateral areas. Tumor-pO(2) was lower (P < 0.042) than healthy tissue. Tumor glucose metabolism was higher than peri-tumor areas (P = 0.017) and healthy contra-lateral areas (P = 0.048). Post-SCS: there were increases in: MCA blood-flow (P ≤ 0.002), CCA blood-flow (P ≤ 0.013), tumor blood-flow (P = 0.033), tumor glucose metabolism (P = 0.027) and tumor-pO(2) (P = 0.022). The percentage of hypoxic values decreased (P = 0.007). SCS can modify tumor micro-environment. The potential usefulness of SCS in improving the effectiveness of radio-chemotherapy in HGG needs to be evaluated.

A two-phase case-control study for colorectal cancer genetic susceptibility: candidate genes from chromosomal regions 9q22 and 3q22.

BACKGROUND: Colorectal cancer (CRC) is the second cause of cancer-related death in the Western world. Much of the CRC genetic risk remains unidentified and may be attributable to a large number of common, low-penetrance genetic variants. Genetic linkage studies in CRC families have reported additional association with regions 9q22-31, 3q21-24, 7q31, 11q, 14q and 22q. There are several plausible candidate genes for CRC susceptibility within the aforementioned linkage regions including PTCH1, XPA and TGFBR1 in 9q22-31, and EPHB1 and MRAS in 3q21-q24. METHODS: CRC cases and matched controls were from EPICOLON, a prospective, multicentre, nationwide Spanish initiative, composed of two independent phases. Phase 1 corresponded to 515 CRC cases and 515 controls, whereas phase 2 consisted of 901 CRC cases and 909 controls. Genotyping was performed for 172 single-nucleotide polymorphisms (SNPs) in 84 genes located within regions 9q22-31 and 3q21-q24. RESULTS: None of the 172 SNPs analysed in our study could be formally associated with CRC risk. However, rs1444601 (TOPBP1) and rs13088006 (CDV3) in region 3q22 showed interesting results and may have an effect on CRC risk. CONCLUSIONS: TOPBP1 and CDV3 genetic variants on region 3q22 may modulate CRC risk. Further validation and meta-analysis should be undertaken in larger CRC cohorts.

Clinically important molecular features of Peruvian colorectal tumours: high prevalence of DNA mismatch repair deficiency and low incidence of KRAS mutations.

BACKGROUND: The incidence of colorectal cancer (CRC) in Peru has been increasing, and no data have been published on the molecular features. We explored the most relevant genetic events involved in colorectal carcinogenesis, with clinical implications. METHODS: Using immunohistochemistry for mismatch-repair (MMR) proteins (MLH1, MSH2, MSH6, and PMS2) and microsatellite instability analysis, we evaluated the status of 90 non-selected CRC Peruvian patients followed in a nationwide reference hospital for cancer (INEN, Lima). Tumours with loss of hMLH1 were evaluated further for hMLH1 promoter hypermethylation and all cases were evaluated for the presence of KRAS and BRAF-V600E mutations. RESULTS: MMR deficiency was found in 35 (38.8%) patients. We identified an unexpected association between MMR deficiency and older age. Among the 14 cases with loss of MLH1, 10 samples exhibited hypermethylation. Of the 90 cases evaluated, 15 (16.7%) carried KRAS mutations; we found one previously unreported mutation (G13R). CONCLUSIONS: Peruvian CRC tumours exhibited the highest prevalence of MMR deficiency reported to date. The expected hereditary component was also high. The age of onset of these MMR deficient tumours was greater than that observed for non-MMR deficient cases, suggesting the ineffectiveness of the Bethesda criteria for Lynch syndrome screening in Peru. Prospective studies are warranted to define the molecular characteristics of CRC in this population.