Sietze T. Van Turenhout

Use of faecal markers in screening for colorectal neoplasia: a European group on tumor markers position paper

Several randomized controlled trials have shown that population‐based screening using faecal occult blood testing (FOBT) can reduce mortality from colorectal neoplasia. Based on this evidence, a number of countries have introduced screening for colorectal cancer (CRC) and high‐risk adenoma and many others are considering its introduction. The aim of this article is to critically review the current status of faecal markers as population‐based screening tests for these neoplasia. Most of the available faecal tests involve the measurement of either occult blood or a panel of DNA markers. Occult blood may be measured using either the guaiac faecal occult blood test (gFOBT) or a faecal immunochemical test (iFOBT). Although iFOBT may require a greater initial investment, they have several advantages over gFOBT, including greater analytical sensitivity and specificity. Their use results in improved clinical performance and higher uptake rates. Importantly for population screening, some of the iFOBTs can be automated and provide an adjustable cutoff for faecal haemoglobin concentration. However, samples for iFOBT, may be less stable after collection than for gFOBT. For new centres undertaking FOBT for colorectal neoplasia, the European Group on Tumour Markers recommends use of a quantitative iFOBT with an adjustable cutoff point and high throughput analysis. All participants with positive FOBT results should be offered colonoscopy. The panel recommends further research into increasing the stability of iFOBT and the development of improved and affordable DNA and proteomic‐based tests, which reduce current false negative rates, simplify sample transport and enable automated analysis.

Double-balloon endoscopy as the primary method for small-bowel video capsule endoscope retrieval

BACKGROUND Capsule retention in the small bowel is a known complication of small-bowel video capsule endoscopy. Surgery is the most frequently used method of capsule retrieval. OBJECTIVE To determine the incidence and causes of capsule retention and to describe double-balloon endoscopy (DBE) as the primary technique used for capsule retrieval. DESIGN Retrospective analysis of all video capsule studies was performed at our center, and evaluation of the outcome of DBE was the first method used to retrieve entrapped video capsules. SETTING Tertiary referral center. PATIENTS A total of 904 patients who underwent small-bowel video capsule endoscopy. INTERVENTIONS Capsule retrieval by DBE. MAIN OUTCOME MEASUREMENTS The number of patients in whom capsule retention occurred and the number of patients in whom an entrapped capsule could be retrieved by using DBE. RESULTS Capsule retention occurred in 8 patients (incidence 0.88%; 95% CI, 0.41%-1.80%) and caused acute small-bowel obstruction in 6 patients. All retained capsules were successfully removed during DBE. Five patients underwent elective surgery to treat the underlying cause of capsule retention. One patient required emergency surgery because of multiple small-bowel perforations. LIMITATIONS Retrospective design. CONCLUSIONS In our series, the incidence of capsule retention was low. DBE is a reliable method for removing retained capsules and might prevent unnecessary surgery. If surgery is required, preoperative capsule retrieval allows preoperative diagnosis, adequate staging in case of malignancy, and optimal surgical planning.

Proximal Fluid Proteome Profiling of Mouse Colon Tumors Reveals Biomarkers for Early Diagnosis of Human Colorectal Cancer

Purpose: Early detection of colorectal cancer (CRC) and its precursor lesions is an effective approach to reduce CRC mortality rates. This study aimed to identify novel protein biomarkers for the early diagnosis of CRC. Experimental Design: Proximal fluids are a rich source of candidate biomarkers as they contain high concentrations of tissue-derived proteins. The FabplCre;Apc15lox/+ mouse model represents early-stage development of human sporadic CRC. Proximal fluids were collected from normal colon and colon tumors and subjected to in-depth proteome profiling by tandem mass spectrometry. Carcinoembryonic antigen (CEA) and CHI3L1 human serum protein levels were determined by ELISA. Results: Of the 2,172 proteins identified, quantitative comparison revealed 192 proteins that were significantly (P < 0.05) and abundantly (>5-fold) more excreted by tumors than by controls. Further selection for biomarkers with highest specificity and sensitivity yielded 52 candidates, including S100A9, MCM4, and four other proteins that have been proposed as candidate biomarkers for human CRC screening or surveillance, supporting the validity of our approach. For CHI3L1, we verified that protein levels were significantly increased in sera from patients with adenomas and advanced adenomas compared with control individuals, in contrast to the CRC biomarker CEA. Conclusion: These data show that proximal fluid proteome profiling with a mouse tumor model is a powerful approach to identify candidate biomarkers for early diagnosis of human cancer, exemplified by increased CHI3L1 protein levels in sera from patients with CRC precursor lesions. Clin Cancer Res; 18(9); 2613–24. ©2012 AACR.

Video capsule endoscopy in patients with nonresponsive celiac disease.

Goals and Background: Discriminating between patients with nonresponsive but otherwise uncomplicated celiac disease (CD) and patients with refractory celiac disease (RCD) and/or lymphoma is difficult, especially as many abnormalities encountered in complicated CD are not within reach of conventional gastroduodenoscopy. We aimed to describe video capsule endoscopy (VCE) findings in patients with CD and persisting or relapsing symptoms despite a gluten-free diet and to identify VCE findings associated with poor prognosis. Methods: We retrospectively analyzed 48 VCE studies performed in adult patients with CD because of persisting or relapsing symptoms despite adherence to a gluten-free diet. Patients with either uncomplicated CD or RCD type I were considered to have a good prognosis, whereas patients with either RCD type II or enteropathy-associated T-cell lymphoma were considered to have a poor prognosis. Multivariate analysis was performed to identify VCE findings independently associated with either good or poor prognosis. Results: Proximal focal erythema (odds ratio, 6.7; 95% confidence interval, 1.2-38.7; P=0.033) and absence of progression of the capsule to the distal intestine (odds ratio, 16.5; 95% confidence interval, 1.2-224.9; P=0.035) were independently associated with poor prognosis. Of the 28 patients with none of these 2 features, none died during follow-up, compared with 2 (13.3%) of the 15 patients with one of both features, and 4 (80.0%) of the 5 patients with both the features. Conclusions: VCE is a minimally invasive endoscopic modality that could be of use in identifying patients with nonresponsive CD who are at risk of poor prognosis.

Anticipating implementation of colorectal cancer screening in The Netherlands: a nation wide survey on endoscopic supply and demand

BackgroundColorectal cancer (CRC) screening requires sufficient endoscopic resources. The present study aims to determine the Dutch endoscopic production and manpower for 2009, evaluate trends since 2004, determine additional workload which would be caused by implementation of a CRC screening program, and inventory colonoscopy rates performed in other European countries.MethodsAll Dutch endoscopy units (N = 101) were surveyed for manpower and the numbers of endoscopy procedures performed in 2009. Based on calculations in the report issued by the Dutch Health Council, future additional workload caused by faecal immunochemical test (FIT) screening was estimated. The number of colonoscopies performed in Europe was evaluated by a literature search and an email-inquiry.ResultsCompared to 2004, there was a 24% increase in total endoscopies (N = 505,226 in 2009), and a 64% increase in colonoscopies (N = 191,339 in 2009) in The Netherlands. The number of endoscopists had increased by 4.6% (N = 583 in 2009). Five years after stepwise implementation of FIT-based CRC screening, endoscopic capacity needs to be increased an additional 15%. A lack of published data on the number of endoscopies performed in Europe was found. Based on our email-inquiry, the number of colonoscopies per 100,000 inhabitants ranged from 126 to 3,031 in 15 European countries.ConclusionsOver the last years, endoscopic procedures increased markedly in The Netherlands without a corresponding increase in manpower. A FIT-based CRC screening program requires an estimated additional 15% increase in endoscopic procedures. It is very likely that current colonoscopy density varies widely across European countries.

Faecal immunochemical test accuracy in patients referred for surveillance colonoscopy: a multi-centre cohort study

BackgroundGiven the increasing burden on colonoscopy capacity, it has been suggested that faecal immunochemical test (FIT) results could guide surveillance colonoscopy intervals. Against this background, we have evaluated the test accuracy of single and double FIT sampling to detect colorectal cancer (CRC) and/or advanced adenomas in an asymptomatic colonoscopy-controlled high-risk population.MethodsCohort study of asymptomatic high-risk patients (personal history of adenomas/CRC or family history of CRC), who provided one or two FITs before elective colonoscopy. Test accuracy of FIT for detection of CRC and advanced adenomas was determined (cut-off level 50 ng/ml).Results1,041 patients provided a FIT (516 personal history of adenomas, 172 personal history of CRC and 353 family history of CRC). Five CRCs (0.5%) and 101 advanced adenomas (9.7%) were detected by colonoscopy. Single FIT sampling resulted in a sensitivity, specificity, PPV and NPV for CRC of 80%, 89%, 3% and 99.9%, respectively, and for advanced adenoma of 28%, 91%, 24% and 92%, respectively. Double FIT sampling did not result in a significantly higher sensitivity for advanced neoplasia. Simulation of multiple screening rounds indicated that sensitivity of FIT for advanced adenoma could reach 81% after 5 screening rounds.ConclusionsIn once-only FIT sampling before surveillance colonoscopy, 70% of advanced neoplasia were missed. A simulation approach indicates that multiple screening rounds may be more promising in detecting advanced neoplasia and could potentially alleviate endoscopic burden.

Novel stool-based protein biomarkers for improved colorectal cancer screening : a case-control study

Screening aims to lower the burden of colorectal cancer (CRC) by either preventing cancer from developing or detecting it at a curable stage (1). Although colonoscopy remains the gold standard for detecting colorectal tumors, for reasons of compliance and cost, most population-wide screening programs use noninvasive stool-based tests for triage to colonoscopy (2). The guaiac-based fecal occult blood test has been proven to reduce mortality from CRC (35). The newer fecal immunochemical test (FIT), which uses an antibody against human hemoglobin, outperforms the guaiac-based fecal occult blood test and is now used widely (69). Yet, the sensitivity of FIT for detecting CRC is suboptimal (79%) and even poorer (31%) for finding advanced colonic adenomas, precursor lesions with an increased risk for progression (10, 11). Molecular screening tests have the potential to detect colorectal tumors better than FIT (12, 13). Indeed, multitarget stool DNA testing combined with FIT has been shown to have a higher sensitivity than FIT alone (14), but it is too costly to implement in population-wide screening programs. In contrast, like hemoglobin in FIT, protein biomarkers can be translated into simple and cost-effective antibody-based screening tests (15). Ideally, these biomarkers also could be quantified in the small stool sample volumes used in FIT-based screening programs. However, thus far, alternative protein biomarkers have failed to improve current hemoglobin-based CRC stool-screening tests (12). Technologic advancements in mass spectrometry now allow for in-depth proteomics for biomarker discovery in complex biological samples (16, 17). A classic approach is to first identify discriminating markers in tissue or cell-line material and then validate them in the final analyte, such as stool. However, constituents of the analyte ultimately used for screeningsuch as bacterial proteases and glycosidases in stoolmay affect test performance, possibly leading to validation failure (12, 18). Therefore, discovering biomarkers directly in the biological sample taken for screening, namely stool, may be a powerful alternative. In the present study, we set out to identify proteins in stool that outperform or complement fecal hemoglobin as a biomarker for early detection of CRC and advanced adenomas. Methods A brief overview of the methods is given here; full sample details and methods are provided in Supplements 1 and 2. For an overview of the workflow, see Figure 1 of Supplement 1. Supplement 1. Supplementary Appendix Supplement 2. Supplementary Tables Sample Series Written informed consent was obtained from all persons who provided stool samples. The study was conducted in compliance with institutional ethical regulations. See the Appendix Table for clinicopathologic characteristics. Appendix Table. Clinicopathologic Characteristics of Stool Sample Series 1 and 2 Sample Series 1 Twenty-two stool subsamples (12 from patients with CRC and 10 from persons without colorectal neoplasia) were collected from a colonoscopy-controlled referral population at VU University Medical Center in Amsterdam, the Netherlands, between 2003 and 2006. Sample Series 2 Whole stool samples from 293 persons with CRC (n= 81), with advanced adenomas (n= 40), with nonadvanced adenomas (n= 43), or without colorectal neoplasia (n= 129) were collected from a colonoscopy-controlled referral population at several centers in the Netherlands and Germany between 2005 and 2012. Sample Series 3 Fecal immunochemical test samples from 72 persons with CRC (n= 14), advanced adenoma (n= 16), or nonadvanced adenoma (n= 18) or who did not have colorectal neoplasia (n= 24) from a colonoscopy-controlled referral population at Kennemer Gasthuis Hospital in Haarlem, the Netherlands, between 2012 and 2014. Proteomics Analysis With Nanoscale Liquid Chromatography Coupled to Tandem Mass Spectrometry Proteins were extracted from stool as described previously (19), with a few adaptations. Equal amounts of protein were run through sodium dodecyl sulfate polyacrylamide gel electrophoresis (Invitrogen) and processed further by in-gel tryptic digestion (20). Peptides were separated by nanoscale liquid chromatography and detected on an LTQ-FT hybrid mass spectrometer (Thermo Fisher) (sample series 1) or a Q Exactive mass spectrometer (Thermo Fisher) (sample series 2). Data acquisition was not successful for 2 CRC samples in series 2. Spectra obtained from tandem mass spectrometry were searched against the UniProt human reference proteome FASTA file, release January 2014, by using MaxQuant 1.4.1.2 (21). Protein abundance was quantified by label-free spectral counting (22). Antibody-Based Assays Fecal immunochemical test fluids from sample series 3 were analyzed by using antibody-based assays for myeloperoxidase (MPO), alpha-2-macroglobulin (A2M), retinol binding protein 4 (RBP4), and adiponectin and analyzed with Discovery Workbench 4.0 software (Meso Scale Diagnostics). Statistical Analysis Statistical analyses were performed in the R computing environment, version 3.1.1 (The R Foundation), including the packages rpart, pROC, gplots, and ggplot2 (2326). Spectral counts were subjected to global normalization (27). Hierarchical clustering was performed on log2 (normalized expression values plus 1) by using the Euclidean distance for sample clustering, the Spearman distance for protein clustering, and complete linkage in both. Heat maps show the Z scores for individual proteins. Univariate differential abundance analysis was performed by using the beta-binomial test (27). Proteins consistently more abundant in CRC than control samples in both series 1 and 2 (BenjaminiHochbergcorrected P value; Q 0.05) constituted input for selecting specific biomarker panels. Biomarker panels were defined by using 2 statistical methods on sample series 2, namely logistic regression (exhaustive search) and classification and regression tree (CART) analysis. Receiver-operating characteristic (ROC) analysis was used to evaluate the performance of protein panels in discriminating between samples with advanced adenoma or CRC and those without colorectal neoplasia. Areas under the curve (AUCs) from ROC curves were compared and evaluated for statistical difference by using the bootstrap method from the pROC package. To assess the statistical significance of the difference in sensitivity between any marker panel and hemoglobin at 95% specificity, the McNemar test was used. Spearman rank correlation, MannWhitney, or KruskalWallis tests were used to assess the relation between protein abundancy and tumor characteristics, such as size, location, stage (CRC), histology (advanced adenoma), and grade of dysplasia (advanced adenoma). Role of the Funding Source The funding sources had no role in the design, conduct, or reporting of the study or in the decision to submit the manuscript for publication. Results Proteomics Analysis of Human Stool Samples A total of 468 human proteins were identified in sample series 1 (Table 1 of Supplement 2 and Figure 2 of Supplement 1). Spectral counts for the alpha and beta chains of hemoglobin, known to be present in equal amounts (28), showed a strong correlation (= 0.95, P < 0.001) (Figure 3A of Supplement 1). Likewise, the S100 calcium binding protein A8 and A9 (S100A8 and S100A9) calprotectin subunits were strongly correlated (= 0.91, P < 0.001) (Figure 3B of Supplement 1). Unsupervised cluster analysis revealed that the protein profile of most CRC stool samples differed from that of control samples (Appendix Figure, A). Because these results confirm the feasibility of quantifying CRC-specific human proteins in stool samples, the analysis was extended to the second, larger series of samples. Appendix Figure. Unsupervised hierarchical cluster analysis of human protein levels in stool samples. Shown are clusters and heat maps for sample series 1 (A) and 2 (B). The bluered color scale of the heat maps depicts protein levels as measured in spectral counts (blue: low; red: high). Green- and red-coded samples in the legend bar represent control and CRC stool samples, respectively. CRC = colorectal cancer. Subsequent analysis of 291 stool samples (Appendix Table) revealed a total of 733 human proteins (Table 2 of Supplement 2 and Figure 2B of Supplement 1) and reidentified 78% of the proteins (367 of 468) from sample series 1. Also in this second sample series, protein levels of hemoglobin alpha and beta, as well as the calprotectin subunits, were highly correlated (= 0.94, P < 0.001, and = 0.8, P < 0.001, respectively) (Figure 3C and 3D of Supplement 1). Again, the CRC stool samples had a protein profile different from that of the control samples (Appendix Figure, B). In sample series 1 and 2, a total of 834 human proteins were detected, 367 (44%) of which were common to both series (Table 3 of Supplement 2 and Figure 2C of Supplement 1). Proteins Discriminating CRC From Control Samples Signals that arise during tumor development are most suitable as biomarkers. Therefore, most cancer screening tests are based on positive signals, so we focused on proteins that were more abundant in the CRC than the control samples. Differential abundance analysis (CRC vs. control samples; fold change >0, P 0.05) yielded 93 and 213 proteins in sample series 1 and 2, respectively, with 55 proteins common to both (Table 3 of Supplement 2). This list of 55 proteins decreased to 29 after correction for multiple testing (that is, Q 0.05) (Table). These 29 proteins included hemoglobin subunits alpha 1, beta, and delta (HBA1, HBB, and HBD). Because population-based screening requires high sensitivity combined with high specificity, specificity was fixed at 95% to evaluate the corresponding sensitivities. At a specificity of 95%, 6 proteinscomplement C3 (C3), A2M, haptoglobin (HP), complement C5 (C5), fibronectin 1 (FN1), and ceruloplasmin (CP)had a statistically significantly higher sensitivity than HBA1 (Figure

S1123 Double Versus Single Sampling of Fecal Immunochemical Tests for Colorectal Cancer Screening; Added Value or Added Costs?

Background Fecal immunochemical tests (FITs) are state of the art in colorectal cancer(CRC) screening. Sensitivity of a single FIT for advanced neoplasia is around 50%. Theoretically, as blood loss from colon tumors can be intermittent, sensitivity of FITs could improve by double sampling. This study aims to compare the sensitivity of single FIT sampling and double FIT sampling at different cut-off values, for the detection of advanced neoplasia. Methods All subjects (≥18 years) scheduled for elective colonoscopy in three participating centers in the Amsterdam area were asked to perform FITs (OC sensor®) on two consecutive days. FIT results were compared with colonoscopy and histology as gold standard. Test performance of single FIT was compared to the sensitivity of double FIT sampling. Double FIT sampling was considered positive if one of both FITs was higher than the cut-off value. Test performances were evaluated at cut-off values ranging from 50-150ng/ml (incremental steps of 25ng/ml). Results Of 1105 subjects who performed two FITs and underwent total colonoscopy, 140 (9,4%) had advanced neoplasia (AN), of which 34 were CRC and 106 were advanced adenomas (AA). Of the CRC cases, 70% were Dukes stage A or B (stage unknown in 2). Positivity rates for single FIT ranged from 11-17%, and for double FIT from 14-23%. At the same cut-off value for positivity, sensitivity of double FIT sampling was higher than sensitivity of single FIT sampling. For any particular specificity (e.g. 90%), the sensitivity of double FIT was slightly higher than that of single FIT at a lower cut-off value (see table), but these differences were not statistically significant. Conclusions Two fold sampling of FIT does increase sensitivity for advanced neoplasia. However, at a given specificity, sensitivity of double sampling is comparable to single sampling at a lower cutoff value. Sensitivity and specificity of single and double FIT testing for the detection of advanced neoplasia

930 Comparing Three Different Strategies of Double Sampling by Fecal Immunochemical Tests for Detection of Advanced Colorectal Neoplasm's

Background Fecal Immunochemical Tests (FITs) are widely used for Colorectal Cancer (CRC) screening. Some sampling schemes use one-day FIT testing, whereas others use twoday FIT testing. Data on sensitivity and specificity of two-day FIT testing are lacking. This study aims to compare the difference in sensitivity and specificity of three different strategies of two-day FIT sampling, at different cut-off values. Methods In three hospitals in the Amsterdam area, all subjects ≥18 years who were referred for elective colonoscopy were asked to perform a FIT (OC sensor®) on two consecutive days. Colonoscopy and histology were considered as gold standard. Two-day FIT sampling was defined positive in three different ways: 1) when at least one of two FITs was above the cut-off value (FIT+), 2) when both FITs were above the cut-off value(FIT++), and 3) when the geometric mean of two FITs was above the cut-off value (FITmean). Test performance of all three strategies was evaluated at cut-off values of 50, 75 and 100ng/ml. Results In 1105 subjects with complete colonoscopy, 140 (9,4%) AN were found (34 CRCs and 106 advanced adenomas). Of the CRC cases, 70% were Dukes stage A or B (stage unknown in 2). Positivity rates for FIT+ ranged from 16-23%, for FIT++ from 10-13% and for FITmean from 13-17% (see table). At any cut-off level, FIT++ resulted in the lowest and FIT+ in the highest sensitivity. FIT++ showed a higher specificity than both other strategies (see table). Conclusions The most sensitive strategy in two-day FIT sampling is defining the test positive when at least one of both FITs passes the cut off (FIT+), whereas the highest specificity is obtained when both FITs need to be positive for calling the tests positive (FIT++). At all cut-off values, FITmean offers substantially better specificity than FIT+, while sensitivity is substantially higher than FIT++. Sensitivity and specificity of three methods to use two-day FIT sampling for the detection of advanced neoplasia

Su1876 Proximal Fluid Proteome Profiling of Mouse Colon Tumors Reveals Biomarkers for Early Diagnosis of Human Colorectal Cancer

Purpose: Early detection of colorectal cancer (CRC) and its precursor lesions is an effective approach to reduce CRC mortality rates. This study aimed to identify novel protein biomarkers for the early diagnosis of CRC. Experimental Design: Proximal fluids are a rich source of candidate biomarkers as they contain high concentrations of tissue-derived proteins. The FabplCre;Apc 15lox/þ mouse model represents early-stage development of human sporadicCRC. Proximalfluids werecollected fromnormalcolon andcolontumors and subjected to in-depth proteome profiling by tandem mass spectrometry. Carcinoembryonic antigen (CEA) and CHI3L1 human serum protein levels were determined by ELISA. Results: Of the 2,172 proteins identified, quantitative comparison revealed 192 proteins that were significantly (P 5-fold) more excreted by tumors than by controls. Further selection for biomarkers with highest specificity and sensitivity yielded 52 candidates, including S100A9, MCM4,andfourotherproteinsthathavebeenproposedascandidatebiomarkersforhumanCRCscreening or surveillance, supporting the validity of our approach. For CHI3L1, we verified that protein levels were significantlyincreasedinserafrompatientswithadenomasandadvancedadenomascomparedwithcontrol individuals, in contrast to the CRC biomarker CEA. Conclusion: These data show that proximal fluid proteome profiling with a mouse tumor model is a powerful approach to identify candidate biomarkers for early diagnosis of human cancer, exemplified by increased CHI3L1 protein levels in sera from patients with CRC precursor lesions. Clin Cancer Res; 18(9); 2613-24. � 2012 AACR.