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.

[Management of elective surgical activity in a tertiary hospital during the SARS-CoV-2 pandemic]. / Gestión de la actividad quirúrgica electiva de un hospital terciario durante la pandemia por SARS-CoV-2.

INTRODUCTION: During the SARS-CoV-2 pandemic, elective surgical activity was reduced to a minimum. As both the number of cases and the hospitalization needs for this pathology decreased, we thought it appropriate to progressively recover scheduled surgical activity. This work describes how, even with the current alarm state, we were able to practically normalize this activity in a few weeks. METHODS: Two weeks before the intervention, the patients included in the waiting lists were contacted by telephone. After checking their health status and expressing their desire to undergo surgery, they were provided with recommendations to decrease the risk of coronavirus infection. Likewise, an exclusive circuit was established to carry out, 48 hours before the intervention, the detection of SARS-CoV-2 by means of exudates nasopharyngeal PCR. The results were evaluated by each surgical service and the anesthesiology service. In addition, asymptomatic Surgical Area professionals could undergo weekly screening for the early detection of coronavirus according to the recommendations of Occupational Health. RESULTS: In the midst of a pandemic, scheduled surgical activity was reduced by 85%. From the week of April 13, the operating rooms available were recovered, which allowed practically all surgical activity to be recovered the week of May 25. CONCLUSIONS: The creation of circuits and procedures to streamline surgical activity, still in full force of the state of alarm, has allowed us, in a few weeks, to recover almost all of it.

Three-year outcome after transanal versus laparoscopic total mesorectal excision in locally advanced rectal cancer: a multicenter comparative analysis.

BACKGROUND: For patients with mid and distal rectal cancer, robust evidence on long-term outcome and causal treatment effects of transanal total mesorectal excision (TaTME) is lacking. This multicentre retrospective cohort study aimed to assess whether TaTME reduces locoregional recurrence rate compared to laparoscopic total mesorectal excision (LapTME). METHODS: Consecutive patients with rectal cancer within 12 cm from the anal verge and clinical stage II-III were selected from three institutional databases. Outcome after TaTME (Nov 2011 - Feb 2018) was compared to a historical cohort of patients treated with LapTME (Jan 2000 - Feb 2018) using the inverse probability of treatment weights method. The primary endpoint was three-year locoregional recurrence. RESULTS: A total of 710 patients were analysed, 344 in the TaTME group and 366 in the LapTME group. At 3 years, cumulative locoregional recurrence rates were 3.6% (95% CI, 1.1-6.1) in the TaTME group and 9.6% (95% CI, 6.5-12.7) in the LapTME group (HR = 0.4; 95% CI, 0.23-0.69; p = 0.001). Three-year cumulative disease-free survival rates were 74.3% (95% CI, 68.8-79.8) and 68.6% (95% CI, 63.7-73.5) (HR = 0.82; 95% CI, 0.65-1.02; p = 0.078) and three-year overall survival 87.2% (95% CI, 82.7-91.7) and 82.2% (95% CI, 78.0-86.2) (HR = 0.74; 95% CI, 0.53-1.03; p = 0.077), respectively. In patients who underwent sphincter preservation procedures, TaTME was associated with a significantly better disease-free survival (HR = 0.78; 95% CI, 0.62-0.98; p = 0.033). CONCLUSIONS: These findings suggest that TaTME may improve locoregional recurrence and disease-free survival rates among patients with mid and distal locally advanced rectal cancer.

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.

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.

EACR-MRS conference on Seed and Soil: In Vivo Models of Metastasis.

New experimental tools are urgently required to better understand the metastatic process. The importance of such tools is underscored by the fact that many anti-cancer therapies are generally ineffective against established metastases. This makes a major contribution to the fact that metastatic spread is responsible for over 90% of cancer patient deaths. It was therefore timely that the recent "Seed and Soil: In Vivo Models of Metastasis" conference held in Berlin, Germany (27-29 of November 2017) aimed to give an in-depth overview of the latest research models and tools for studying metastasis, and to showcase recent findings from world-leading metastasis researchers. This Meeting Report summarises the major themes of this ground-breaking conference.

Improving survival and local control in rectal cancer in Catalonia (Spain) in the context of centralisation: A full cycle audit assessment.

BACKGROUND: Rectal cancer surgery in Catalonia has been involved in a process of centralisation. We assessed the impact of this health policy strategy on quality of care and clinical results. METHODS: We compared patterns of care and clinical outcomes of all rectal cancer patients receiving radical surgery for the first time in public hospitals in two time periods, before (2005 and 2007) and after (2011-2012) centralisation, analysing indicators of care quality according to the regional clinical practice guidelines. Clinical outcomes at two years were also assessed. RESULTS: A total of 3780 patients were included. From 2005 to 2012, the proportion of patients treated surgically for the first time in centres whose annual surgical caseload was more than 11 increased from 84.0% to 90.4%. The rate of locoregional recurrence at two years fell from 4.5 to 3.06/100 person-years (p = 0.005). The crude mortality rate at three months, one and two years was reduced by 55%, 40% and 34% (p < 0.001). CONCLUSION: Improvements in quality of care might be associated with the centralisation of surgery and with the selective focus effect derived from the process of auditing. Our results support the continuation of clinical auditing and surveillance of authorised centres.

ZEB1 and TCF4 reciprocally modulate their transcriptional activities to regulate Wnt target gene expression.

The canonical Wnt pathway (TCF4/ß-catenin) has important roles during normal differentiation and in disease. Some Wnt functions depend on signaling gradients requiring the pathway to be tightly regulated. A key Wnt target is the transcription factor ZEB1 whose expression by cancer cells promotes tumor invasiveness by repressing the expression of epithelial specification markers and activating mesenchymal genes, including a number of Wnt targets such as LAMC2 and uPA. The ability of ZEB1 to activate/repress its target genes depends on its recruitment of corepressors (CtBP, BRG1) or coactivators (p300) although conditions under which ZEB1 binds these cofactors are not elucidated. Here, we show that TCF4 and ZEB1 reciprocally modulate each other's transcriptional activity: ZEB1 enhances TCF4/ß-catenin-mediated transcription and, in turn, Wnt signaling switches ZEB1 from a repressor into an activator. In colorectal cancer (CRC) cells with active Wnt signaling, ZEB1 enhances transcriptional activation of LAMC2 and uPA by TCF4/ß-catenin. However, in CRC cells with inactive Wnt, ZEB1 represses both genes. Reciprocal modulation of ZEB1 and TCF4 activities involves their binding to DNA and mutual interaction. Wnt signaling turns ZEB1 into an activator by replacing binding of CtBP/BRG1 in favor of p300. Using a mouse model of Wnt-induced intestinal tumorigenesis, we found that downregulation of ZEB1 reduces the expression of LAMC2 in vivo. These results identify a mechanism through which Wnt and ZEB1 transcriptional activities are modulated, offering new approaches in cancer therapy.

MiR-320e is a novel prognostic biomarker in colorectal cancer.

BACKGROUND: Advances in early detection and treatment have improved outcomes in patients with colorectal cancer (CRC). However, there remains a need for robust prognostic and predictive biomarkers. We conducted a systematic discovery and validation of microRNA (miRNA) biomarkers in two clinical trial cohorts of CRC patients. METHODS: We performed an initial 'discovery' phase using Affymetrix miRNA expression arrays to profile stage III CRC patients with and without tumour recurrence (n=50 per group) at 3-years of follow-up. All patients received adjuvant 5-fluorouracil (5-FU) plus oxaliplatin, that is, FOLFOX, treatment. During 'validation', we analysed miRNAs using qRT-PCR in an independent cohort of 237 stage II-IV CRC patients treated with 5-FU-based chemotherapy, as well as in normal colonic mucosa from 20 healthy subjects. Association with disease recurrence, disease-free survival (DFS) and overall survival (OS) was examined using Cox proportional hazard models. RESULTS: In the discovery cohort, miR-320e expression was significantly elevated in stage III colon cancers from patients with vs without recurrence (95% confidence interval (CI)=1.14-1.42; P<0.0001). These results were then independently validated in stage II and III tumours. Specifically, increased miR-320e expression was associated with poorer DFS (hazard ratio (HR)=1.65; 95% CI=1.27-2.13; P=0.0001) and OS (HR=1.78; 95% CI=1.31-2.41; P=0.0003) in stage III CRC patients. CONCLUSIONS: In two clinical trial cohorts, a systematic biomarker discovery and validation approach identified miR-320e to be a novel prognostic biomarker that is associated with adverse clinical outcome in stage III CRC patients treated with 5-FU-based adjuvant chemotherapy. These findings have important implications for the personalised management of CRC patients.

Ascorbic acid PEG-2L is superior for early morning colonoscopies in colorectal cancer screening programs: A prospective non-randomized controlled trial

BACKGROUND: The quality of colon cleansing and the tolerability of anterograde preparation are essential to the success of colorectal cancer screening. AIM: To compare the tolerability and efficacy of low-volume preparations vs the standard regimen in individuals scheduled for an early morning colonoscopy. Study: Participants in a population-based colorectal cancer screening program using the fecal immunochemical test who were scheduled for a colonoscopy from 09:00 a.m. to 10:20 a.m. were prospectively included and assigned to: (1) control group (PEG-ELS 4L): PEG 4L and electrolytes; (2) group AscPEG-2L: a combination of PEG and ascorbic acid 2L; and (3) group PiMg: sodium picosulfate and magnesium citrate 500 mL plus 2 L of clear fluids. Tolerability was evaluated with a questionnaire and the quality of bowel preparation with the Boston Bowel Preparation Scale. RESULTS: A total of 292 participants were included: 98 in the PEG-ELS 4L control group, 96 in the AscPEG-2L study group and 98 in the PiMg study group. Low-volume treatments were better tolerated than the standard solution (AscPEG-2L 94.8% and PiMg 93.9% vs PEG-ELS 4L 75.5%; p < 0.0001). The effectiveness of AscPEG-2L was superior to that of PEG-ELS 4L and PiMg (p = 0.011 and p = 0.032, respectively). Patient acceptance was higher for single-dose than for split-dose administration but efficacy was higher with the split dose than with other doses. CONCLUSIONS: In early morning colonoscopies, ascPEG-2L appears to be the best option, especially when administered in a split-dose

ANTECEDENTES: La calidad de la limpieza del colon y la tolerancia a la preparación anterógrada son claves para el éxito de un programa de cribado de cáncer colorrectal. OBJETIVO: Comparar la tolerancia y eficacia de las preparaciones de volumen reducido frente a la preparación estándar en pacientes programados para colonoscopia a primera hora de la mañana. Estudio: Individuos del programa de cribado poblacional con test de sangre oculta en heces programados para colonoscopia entre las 09:00 y 10:20 a.m fueron prospectivamente asignados a: 1) Grupo Control (PEG-ELS 4L): PEG con electrolitos 4 litros; 2) Grupo AscPEG-2L: PEG más ácido ascórbico 2 litros; y 3) Groupo PiMg: picosulfato sódico más citrato de magnesio 500 ml seguido de 2 litros de líquidos claros. Se evaluó la tolerancia mediante cuestionario y la calidad mediante la Boston Bowel Preparation Scale. RESULTADOS: Se incluyeron 292 sujetos: 98 en el grupo control PEG-ELS 4L, 96 en el grupo a estudio AscPEG-2L y 98 en el grupo a estudio PiMg. Las soluciones de volumen reducido fueron mejor toleradas que la solución estándar (AscPEG-2L 94.8% y PiMg 93.9% vs PEG-ELS 4L 75.5%; p < 0.0001). La calidad de la preparación fue superior en el grupo AscPEG-2L que en el grupo control PEG-ELS 4L y grupo PiMg (p = 0.011 and p = 0.032, respectivamente). Las dosis partidas fueron peor aceptadas por los sujetos pero resultaron en una mayor calidad de la preparación. CONCLUSIONES: AscPEG-2L es la mejor opción para las colonoscopias programadas a primera hora de la mañana, especialmente cuando se administra en dosis partida

Ascorbic acid PEG-2L is superior for early morning colonoscopies in colorectal cancer screening programs: a prospective non-randomized controlled trial.

BACKGROUND: The quality of colon cleansing and the tolerability of anterograde preparation are essential to the success of colorectal cancer screening. AIM: To compare the tolerability and efficacy of low-volume preparations vs the standard regimen in individuals scheduled for an early morning colonoscopy. STUDY: Participants in a population-based colorectal cancer screening program using the fecal immunochemical test who were scheduled for a colonoscopy from 09:00 a.m. to 10:20 a.m. were prospectively included and assigned to: (1) control group (PEG-ELS 4L): PEG 4L and electrolytes; (2) group AscPEG-2L: a combination of PEG and ascorbic acid 2L; and (3) group PiMg: sodium picosulfate and magnesium citrate 500 mL plus 2L of clear fluids. Tolerability was evaluated with a questionnaire and the quality of bowel preparation with the Boston Bowel Preparation Scale. RESULTS: A total of 292 participants were included: 98 in the PEG-ELS 4L control group, 96 in the AscPEG-2L study group and 98 in the PiMg study group. Low-volume treatments were better tolerated than the standard solution (AscPEG-2L 94.8% and PiMg 93.9% vs PEG-ELS 4L 75.5%; p < 0.0001). The effectiveness of AscPEG-2L was superior to that of PEG-ELS 4L and PiMg (p = 0.011 and p = 0.032, respectively). Patient acceptance was higher for single-dose than for split-dose administration but efficacy was higher with the split dose than with other doses. CONCLUSIONS: In early morning colonoscopies, ascPEG-2L appears to be the best option, especially when administered in a split-dose.

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.

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.