Novel Stool-Based Protein Biomarkers for Improved Colorectal Cancer Screening: A Case-Control Study.

BACKGROUND: The fecal immunochemical test (FIT) for detecting hemoglobin is used widely for noninvasive colorectal cancer (CRC) screening, but its sensitivity leaves room for improvement. OBJECTIVE: To identify novel protein biomarkers in stool that outperform or complement hemoglobin in detecting CRC and advanced adenomas. DESIGN: Case-control study. SETTING: Colonoscopy-controlled referral population from several centers. PARTICIPANTS: 315 stool samples from one series of 12 patients with CRC and 10 persons without colorectal neoplasia (control samples) and a second series of 81 patients with CRC, 40 with advanced adenomas, and 43 with nonadvanced adenomas, as well as 129 persons without colorectal neoplasia (control samples); 72 FIT samples from a third independent series of 14 patients with CRC, 16 with advanced adenomas, and 18 with nonadvanced adenomas, as well as 24 persons without colorectal neoplasia (control samples). MEASUREMENTS: Stool samples were analyzed by mass spectrometry. Classification and regression tree (CART) analysis and logistic regression analyses were performed to identify protein combinations that differentiated CRC or advanced adenoma from control samples. Antibody-based assays for 4 selected proteins were done on FIT samples. RESULTS: In total, 834 human proteins were identified, 29 of which were statistically significantly enriched in CRC versus control stool samples in both series. Combinations of 4 proteins reached sensitivities of 80% and 45% for detecting CRC and advanced adenomas, respectively, at 95% specificity, which was higher than that of hemoglobin alone (P < 0.001 and P = 0.003, respectively). Selected proteins could be measured in small sample volumes used in FIT-based screening programs and discriminated between CRC and control samples (P < 0.001). LIMITATION: Lack of availability of antibodies prohibited validation of the top protein combinations in FIT samples. CONCLUSION: Mass spectrometry of stool samples identified novel candidate protein biomarkers for CRC screening. Several protein combinations outperformed hemoglobin in discriminating CRC or advanced adenoma from control samples. Proof of concept that such proteins can be detected with antibody-based assays in small sample volumes indicates the potential of these biomarkers to be applied in population screening. PRIMARY FUNDING SOURCE: Center for Translational Molecular Medicine, International Translational Cancer Research Dream Team, Stand Up to Cancer (American Association for Cancer Research and the Dutch Cancer Society), Dutch Digestive Foundation, and VU University Medical Center.

Decoy receptor 1 (DCR1) promoter hypermethylation and response to irinotecan in metastatic colorectal cancer.

Diversity in colorectal cancer biology is associated with variable responses to standard chemotherapy. We aimed to identify and validate DNA hypermethylated genes as predictive biomarkers for irinotecan treatment of metastatic CRC patients. Candidate genes were selected from 389 genes involved in DNA Damage Repair by correlation analyses between gene methylation status and drug response in 32 cell lines. A large series of samples (n=818) from two phase III clinical trials was used to evaluate these candidate genes by correlating methylation status to progression-free survival after treatment with first-line single-agent fluorouracil (Capecitabine or 5-fluorouracil) or combination chemotherapy (Capecitabine or 5-fluorouracil plus irinotecan (CAPIRI/FOLFIRI)). In the discovery (n=185) and initial validation set (n=166), patients with methylated Decoy Receptor 1 (DCR1) did not benefit from CAPIRI over Capecitabine treatment (discovery set: HR=1.2 (95%CI 0.7-1.9, p=0.6), validation set: HR=0.9 (95%CI 0.6-1.4, p=0.5)), whereas patients with unmethylated DCR1 did (discovery set: HR=0.4 (95%CI 0.3-0.6, p=0.00001), validation set: HR=0.5 (95%CI 0.3-0.7, p=0.0008)). These results could not be replicated in the external data set (n=467), where a similar effect size was found in patients with methylated and unmethylated DCR1 for FOLFIRI over 5FU treatment (methylated DCR1: HR=0.7 (95%CI 0.5-0.9, p=0.01), unmethylated DCR1: HR=0.8 (95%CI 0.6-1.2, p=0.4)). In conclusion, DCR1 promoter hypermethylation status is a potential predictive biomarker for response to treatment with irinotecan, when combined with capecitabine. This finding could not be replicated in an external validation set, in which irinotecan was combined with 5FU. These results underline the challenge and importance of extensive clinical evaluation of candidate biomarkers in multiple trials.

Spectrin repeat containing nuclear envelope 1 and forkhead box protein E1 are promising markers for the detection of colorectal cancer in blood.

Identifying biomarkers in body fluids may improve the noninvasive detection of colorectal cancer. Previously, we identified N-Myc downstream-regulated gene 4 (NDRG4) and GATA binding protein 5 (GATA5) methylation as promising biomarkers for colorectal cancer in stool DNA. Here, we examined the utility of NDRG4, GATA5, and two additional markers [Forkhead box protein E1 (FOXE1) and spectrin repeat containing nuclear envelope 1 (SYNE1)] promoter methylation as biomarkers in plasma DNA. Quantitative methylation-specific PCR was performed on plasma DNA from 220 patients with colorectal cancer and 684 noncancer controls, divided in a training set and a test set. Receiver operating characteristic analysis was performed to measure the area under the curve of GATA5, NDRG4, SYNE1, and FOXE1 methylation. Functional assays were performed in SYNE1 and FOXE1 stably transfected cell lines. The sensitivity of NDRG4, GATA5, FOXE1, and SYNE1 methylation in all stages of colorectal cancer (154 cases, 444 controls) was 27% [95% confidence interval (CI), 20%-34%), 18% (95% CI, 12%-24%), 46% (95% CI, 38%-54%), and 47% (95% CI, 39%-55%), with a specificity of 95% (95% CI, 93%-97%), 99% (95% CI, 98%-100%), 93% (95% CI, 91%-95%), and 96% (95% CI, 94%-98%), respectively. Combining SYNE1 and FOXE1, increased the sensitivity to 56% (95% CI, 48%-64%), while the specificity decreased to 90% (95% CI, 87%-93%) in the training set and to 58% sensitivity (95% CI, 46%-70%) and 91% specificity (95% CI, 80%-100%) in a test set (66 cases, 240 controls). SYNE1 overexpression showed no major differences in cell proliferation, migration, and invasion compared with controls. Overexpression of FOXE1 significantly decreased the number of colonies in SW480 and HCT116 cell lines. Overall, our data suggest that SYNE1 and FOXE1 are promising markers for colorectal cancer detection.

FOXE1 and SYNE1 genes hypermethylation panel as promising biomarker in colitis-associated colorectal neoplasia.

BACKGROUND: Colitis-associated colorectal cancer affects individuals with inflammatory bowel disease (IBD) more often and earlier than cancer in the general population. Colonoscopy provides the surveillance gold standard. Changes to the surveillance intervals depending on endoscopic activity have been made, given data demonstrating that this is an important predictor of future dysplasia or cancer, but adjuvant, noninvasive clinical tools are still warranted to improve surveillance outcomes and to assist in management and interpretation of dysplasia. Methylation markers may be able to do this. METHODS: SYNE1, FOXE1, NDRG4, and PHACTR3 genes were screened using methylation-specific PCR that permit the methylation status of the genes to be determined directly on biopsies. Ninety-three patients with long-standing IBD undergoing a cancer surveillance program, and 30 healthy controls were studied. These included colorectal adenocarcinomas on a background of IBD of various stages (n = 25), IBD-associated dysplastic lesions (n = 29), adenomas arising on a background of ulcerative colitis (n = 8), samples from patients with no evidence of dysplasia or cancer but long-standing IBD (n = 31), and symptomatic patients found to have normal colonoscopy (controls) (n = 30). RESULTS: Gene promotor hypermethylation of SYNE1 and FOXE1 genes varied significantly between the groups and was increasingly likely with increased disease severity. Neither occurred in controls, whereas promotor hypermethylation was detected in biopsies of 60% of patients with colitis-associated colorectal cancer for FOXE1 and 80% for SYNE1. Promotor hypermethylation of either gene was highly significantly different between the groups overall. CONCLUSIONS: FOXE1 and SYNE1 hypermethylation markers demonstrated significantly increased expression in neoplastic tissue. Promoter methylation analysis of these genes might be a useful marker of neoplasia in long-standing IBD.

Analytical sensitivity and stability of DNA methylation testing in stool samples for colorectal cancer detection.

BACKGROUND: Stool-based molecular tests hold large potential for improving colorectal cancer screening. Here, we investigated the analytical sensitivity of a DNA methylation assay on partial stool samples, and estimated the DNA degradation in stool over time. In addition, we explored the detection of DNA methylation in fecal immunochemical test (FIT) fluid. MATERIALS AND METHODS: Partial stool samples of colonoscopy-negative individuals were homogenized with stool homogenization buffer, spiked with different numbers of HCT116 colon cancer cells and kept at room temperature for 0, 24, 48, 72 and 144 h before DNA isolation. Analytical sensitivity was determined by the lowest number of cells that yielded positive test results by DNA methylation or mutation analysis. DNA methylation in FIT fluid was measured in 11 CRC patients and 20 control subjects. RESULTS: The analytical sensitivity for detecting DNA methylation was 3000 cells per gram stool, compared to 60000 cells per gram stool for detection of DNA mutations in the same stool samples. No degradation up to 72 h was noted when a conservation buffer was used. DNA methylation was detected in 4/11 CRC FIT samples and in none of the 20 control FIT samples. CONCLUSIONS: Methylation based stool DNA testing showed a high analytical sensitivity for tumor DNA in partial stool samples, which was hardly influenced by DNA degradation over time, provided an adequate buffer was used. The feasibility of detecting DNA methylation in FIT fluid demonstrates the opportunity to combine testing for occult blood with DNA methylation in the same collection device.

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.

Prostate cancer detected by methylated gene markers in histopathologically cancer-negative tissues from men with subsequent positive biopsies.

The goal of this retrospective, multicenter study was to evaluate the ability of a newly developed refinement of a quantitative methylation-specific PCR assay to detect prostate cancer in histopathologically negative biopsy samples collected from men who were later positively diagnosed during a follow-up biopsy procedure. Biomarkers tested in the assay included the much-studied glutathione-S-transferase P1 gene and others reported to be frequently methylated in prostate cancer. Core biopsy tissue from subjects with serial negative biopsies served as a negative control to assess assay specificity. As a positive control, biopsy core tissue from patients histopathologically diagnosed with prostate cancer was used to gauge true marker sensitivity in known cancer-containing specimens. Testing was completed in 971 archived paraffin-embedded tissue blocks from 264 men screened for prostate cancer. More samples were initially tested, but due to the advanced age of the paraffinized tissue, DNA quality for quantitative methylation-specific PCR analysis was insufficient in 34% of the available blocks. The glutathione-S-transferase P1 gene has been confirmed as a powerful indicator of the presence of prostate cancer cells. A sensitivity of 52% was observed in the "potentially false-negative first biopsies," with a corresponding specificity of 85% and the sensitivity in biopsy tissue cores containing histopathologically confirmed prostate cancer was 95%. An even higher sensitivity can be reached with RAR-2beta (84%) and APC (72%), with respective specificities of 48% and 50%. Gene methylation was detected in initial, negative biopsy tissue in men who were later diagnosed with prostate cancer. Testing for methylation in histopathologically negative biopsies could improve the early detection of prostate cancer.

Promoter DNA methylation of oncostatin m receptor-beta as a novel diagnostic and therapeutic marker in colon cancer.

In addition to genetic changes, the occurrence of epigenetic alterations is associated with accumulation of both genetic and epigenetic events that promote the development and progression of human cancer. Previously, we reported a set of candidate genes that comprise part of the emerging "cancer methylome". In the present study, we first tested 23 candidate genes for promoter methylation in a small number of primary colon tumor tissues and controls. Based on these results, we then examined the methylation frequency of Oncostatin M receptor-beta (OSMR) in a larger number of tissue and stool DNA samples collected from colon cancer patients and controls. We found that OSMR was frequently methylated in primary colon cancer tissues (80%, 80/100), but not in normal tissues (4%, 4/100). Methylation of OSMR was also detected in stool DNA from colorectal cancer patients (38%, 26/69) (cut-off in TaqMan-MSP, 4). Detection of other methylated markers in stool DNA improved sensitivity with little effect on specificity. Promoter methylation mediated silencing of OSMR in cell lines, and CRC cells with low OSMR expression were resistant to growth inhibition by Oncostatin M. Our data provide a biologic rationale for silencing of OSMR in colon cancer progression and highlight a new therapeutic target in this disease. Moreover, detection and quantification of OSMR promoter methylation in fecal DNA is a highly specific diagnostic biomarker for CRC.

GATA4 and GATA5 are potential tumor suppressors and biomarkers in colorectal cancer.

PURPOSE: The transcription factors GATA4 and GATA5 are involved in gastrointestinal development and are inactivated by promoter hypermethylation in colorectal cancer. Here, we evaluated GATA4/5 promoter methylation as potential biomarkers for noninvasive colorectal cancer detection, and investigated the role of GATA4/5 in colorectal cancer. EXPERIMENTAL DESIGN: Promoter methylation of GATA4/5 was analyzed in colorectal tissue and fecal DNA from colorectal cancer patients and healthy controls using methylation-specific PCR. The potential function of GATA4/5 as tumor suppressors was studied by inducing GATA4/5 overexpression in human colorectal cancer cell lines. RESULTS: GATA4/5 methylation was observed in 70% (63/90) and 79% (61/77) of colorectal carcinomas, respectively, and was independent of clinicopathologic features. Methylation frequencies in normal colon tissues from noncancerous controls were 6% (5 of 88, GATA4; P < 0.001) and 13% (13 of 100, GATA5; P < 0.001). GATA4/5 overexpression suppressed colony formation (P < 0.005), proliferation (P < 0.001), migration (P < 0.05), invasion (P < 0.05), and anchorage-independent growth (P < 0.0001) of colorectal cancer cells. Examination of GATA4 methylation in fecal DNA from two independent series of colorectal cancer patients and controls yielded a sensitivity of 71% [95% confidence interval (95% CI), 55-88%] and specificity of 84% (95% CI, 74-95%) for colorectal cancer detection in the training set, and a sensitivity of 51% (95% CI, 37-65%) and specificity of 93% (95% CI, 84-100%) in the validation set. CONCLUSIONS: Methylation of GATA4/5 is a common and specific event in colorectal carcinomas, and GATA4/5 exhibit tumor suppressive effects in colorectal cancer cells in vitro. GATA4 methylation in fecal DNA may be of interest for colorectal cancer detection.

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.

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.

Promoter methylation primarily occurs in tumor cells of patients with non-small cell lung cancer.

BACKGROUND: The distribution of promoter methylation throughout the lungs of patients with non-small cell lung cancer (NSCLC) is unknown. In this explorative study, we assessed the methylation status of the promoter region of 11 genes in brush samples of 3 well-defined endobronchial locations in patients with NSCLC and in brushes of former and current smokers without NSCLC. MATERIALS AND METHODS: The methylation status of RASSF1A, GATA4, GATA5, SFRP1, RARbeta2, DAPK, MGMT, p16, p14, CHFR and APC2 was determined in all samples using real-time methylation-specific PCR. RESULTS: Ten patients with NSCLC and 18 non-NSCLC controls were included. Eight patients had one or more methylated genes in their tumor brush. Promoter methylation of genes in proximal or contralateral locations was much less frequent than in tumor brushes, and almost exclusively occurred in normal tissue if the same gene was also methylated in the tumor brush. CONCLUSION: Promoter methylation almost exclusively occurred in tumor cells of patients with NSCLC.

Virologic therapy response significantly correlates with the number of active drugs as evaluated using a LiPA HIV-1 resistance scoring system.

BACKGROUND: Resistance testing is increasingly accepted as a tool in guiding the selection of human immunodeficiency virus type 1 (HIV-1) antiretroviral therapy in HIV-1 infected individuals who fail their current regimen. OBJECTIVES: To descriptively compare the correlation between virologic treatment response and results using three genotypic HIV-1 drug resistance interpretation systems: the VERSANT HIV-1 Resistance Assay (LiPA) system and two sequence-based interpretation systems. STUDY DESIGN: Specimens from 213 HIV-1-infected subjects, either starting (n=104) or switching to (n=109) a regimen of three or four antiretroviral drugs, were collected retrospectively at baseline and after 3 months of uninterrupted therapy. The correlation between viral load change and the number of predicted active drugs in the treatment regimen was assessed. An interpretation algorithm was recently developed to process VERSANT HIV-1 Resistance Assay (LiPA) data. The number of active drugs predicted using this algorithm was rank correlated with the viral load change over a 3-month treatment period. For comparison, a similar calculation was made using two sequence-based algorithms (REGA version 5.5 and VGI GuideLines Rules 4.0), both applied on the same sequences. RESULTS: Statistically significant (p<0.05) correlation coefficients for each of the three HIV-1 drug resistance interpretation systems were observed in the treatment-experienced subjects on a 3-drug regimen (-0.39, -0.38, and -0.42, respectively) as well as on a 4-drug regimen (-0.33, -0.31, and -0.37, respectively). However, no significant correlation was observed in treatment-naive subjects, probably due to the very low frequency of drug resistance in these subjects. CONCLUSION: All three genotypic drug resistance interpretation systems (LiPA version 1, REGA version 5.5, and VGI GuideLines Rules 4.0) were statistically significantly correlated with virologic therapy response as measured by viral load testing.