Frank A. Oort

Methylation of TFPI2 in Stool DNA: A Potential Novel Biomarker for the Detection of Colorectal Cancer

We have used a gene expression array-based strategy to identify the methylation of tissue factor pathway inhibitor 2 (TFPI2), a potential tumor suppressor gene, as a frequent event in human colorectal cancers (CRC). TFPI2 belongs to the recently described group of embryonic cell Polycomb group (PcG)-marked genes that may be predisposed to aberrant DNA methylation in early stages of colorectal carcinogenesis. Aberrant methylation of TFPI2 was detected in almost all CRC adenomas (97%, n = 56) and stages I to IV CRCs (99%, n = 115). We further explored the potential of TFPI2 as a biomarker for the early detection of CRC using stool DNA-based assays in patients with nonmetastatic CRC and average-risk noncancer controls who were candidates for screening. TFPI2 methylation was detected in stool DNA from stage I to III CRC patients with a sensitivity of 76% to 89% and a specificity of 79% to 93%. Detection of TFPI2 methylation in stool DNA may act as a useful adjunct to the noninvasive strategies for screening of CRCs in the future.

N-Myc Downstream-Regulated Gene 4 (NDRG4): A Candidate Tumor Suppressor Gene and Potential Biomarker for Colorectal Cancer

BACKGROUND Identification of hypermethylated tumor suppressor genes in body fluids is an appealing strategy for the noninvasive detection of colorectal cancer. Here we examined the role of N-Myc downstream-regulated gene 4 (NDRG4) as a novel tumor suppressor and biomarker in colorectal cancer. METHODS NDRG4 promoter methylation was analyzed in human colorectal cancer cell lines, colorectal tissue, and noncancerous colon mucosa by using methylation-specific polymerase chain reaction (PCR) and bisulfite sequencing. NDRG4 mRNA and protein expression were studied using real-time-PCR and immunohistochemistry, respectively. Tumor suppressor functions of NDRG4 were examined by colony formation, cell proliferation, and migration and invasion assays in colorectal cancer cell lines that were stably transfected with an NDRG4 expression construct. Quantitative methylation-specific PCR was used to examine the utility of NDRG4 promoter methylation as a biomarker in fecal DNA from 75 colorectal cancer patients and 75 control subjects. All P values are two-sided. RESULTS The prevalence of NDRG4 promoter methylation in two independent series of colorectal cancers was 86% (71/83) and 70% (128/184) compared with 4% (2/48) in noncancerous colon mucosa (P < .001). NDRG4 mRNA and protein expression were decreased in colorectal cancer tissue compared with noncancerous colon mucosa. NDRG4 overexpression in colorectal cancer cell lines suppressed colony formation (P = .014), cell proliferation (P < .001), and invasion (P < .001). NDRG4 promoter methylation analysis in fecal DNA from a training set of colorectal cancer patients and control subjects yielded a sensitivity of 61% (95% confidence interval [CI] = 43% to 79%) and a specificity of 93% (95% CI = 90% to 97%). An independent test set of colorectal cancer patients and control subjects yielded a sensitivity of 53% (95% CI = 39% to 67%) and a specificity of 100% (95% CI = 86% to 100%). CONCLUSIONS NDRG4 is a candidate tumor suppressor gene in colorectal cancer whose expression is frequently inactivated by promoter methylation. NDRG4 promoter methylation is a potential biomarker for the noninvasive detection of colorectal cancer in stool samples.

DNA methylation of phosphatase and actin regulator 3 detects colorectal cancer in stool and complements FIT

Using a bioinformatics-based strategy, we set out to identify hypermethylated genes that could serve as biomarkers for early detection of colorectal cancer (CRC) in stool. In addition, the complementary value to a Fecal Immunochemical Test (FIT) was evaluated. Candidate genes were selected by applying cluster alignment and computational analysis of promoter regions to microarray-expression data of colorectal adenomas and carcinomas. DNA methylation was measured by quantitative methylation-specific PCR on 34 normal colon mucosa, 71 advanced adenoma, and 64 CRC tissues. The performance as biomarker was tested in whole stool samples from in total 193 subjects, including 19 with advanced adenoma and 66 with CRC. For a large proportion of these series, methylation data for GATA4 and OSMR were available for comparison. The complementary value to FIT was measured in stool subsamples from 92 subjects including 44 with advanced adenoma or CRC. Phosphatase and Actin Regulator 3 (PHACTR3) was identified as a novel hypermethylated gene showing more than 70-fold increased DNA methylation levels in advanced neoplasia compared with normal colon mucosa. In a stool training set, PHACTR3 methylation showed a sensitivity of 55% (95% CI: 33–75) for CRC and a specificity of 95% (95% CI: 87–98). In a stool validation set, sensitivity reached 66% (95% CI: 50–79) for CRC and 32% (95% CI: 14–57) for advanced adenomas at a specificity of 100% (95% CI: 86–100). Adding PHACTR3 methylation to FIT increased sensitivity for CRC up to 15%. PHACTR3 is a new hypermethylated gene in CRC with a good performance in stool DNA testing and has complementary value to FIT. Cancer Prev Res; 5(3); 464–72. ©2011 AACR.

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.

Similar fecal immunochemical test results in screening and referral colorectal cancer

AIM To improve the interpretation of fecal immunochemical test (FIT) results in colorectal cancer (CRC) cases from screening and referral cohorts. METHODS In this comparative observational study, two prospective cohorts of CRC cases were compared. The first cohort was obtained from 10 322 average risk subjects invited for CRC screening with FIT, of which, only subjects with a positive FIT were referred for colonoscopy. The second cohort was obtained from 3637 subjects scheduled for elective colonoscopy with a positive FIT result. The same FIT and positivity threshold (OC sensor; ≥ 50 ng/mL) was used in both cohorts. Colonoscopy was performed in all referral subjects and in FIT positive screening subjects. All CRC cases were selected from both cohorts. Outcome measurements were mean FIT results and FIT scores per tissue tumor stage (T stage). RESULTS One hundred and eighteen patients with CRC were included in the present study: 28 cases obtained from the screening cohort (64% male; mean age 65 years, SD 6.5) and 90 cases obtained from the referral cohort (58% male; mean age 69 years, SD 9.8). The mean FIT results found were higher in the referral cohort (829 ± 302 ng/mL vs 613 ± 368 ng/mL, P = 0.02). Tissue tumor stage (T stage) distribution was different between both populations [screening population: 13 (46%) T1, eight (29%) T2, six (21%) T3, one (4%) T4 carcinoma; referral population: 12 (13%) T1, 22 (24%) T2, 52 (58%) T3, four (4%) T4 carcinoma], and higher T stage was significantly associated with higher FIT results (P < 0.001). Per tumor stage, no significant difference in mean FIT results was observed (screening vs referral: T1 498 ± 382 ng/mL vs 725 ± 374 ng/mL, P = 0.22; T2 787 ± 303 ng/mL vs 794 ± 341 ng/mL, P = 0.79; T3 563 ± 368 ng/mL vs 870 ± 258 ng/mL, P = 0.13; T4 not available). After correction for T stage in logistic regression analysis, no significant differences in mean FIT results were observed between both types of cohorts (P = 0.10). CONCLUSION Differences in T stage distribution largely explain differences in FIT results between screening and referral cohorts. Therefore, FIT results should be reported according to T stage.

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

FIT Performance in Early-Stage Colorectal Cancer—Response

See the original Letter to the Editor, [p. 1562][1] We would like to thank Dr. Boncz and colleagues for sharing their experience with their proprietary fecal immunochemical test (FIT) in colorectal cancer (CRC) screening in Hungary. Considering the Hungarian data, 2 questions arise. At first,

S1125 Differences in Fit Results Between Screening and Referred Colorectal Cancer Patients are Explained by Differences in Tissue Tumor Stage

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