Detection of colorectal disease by stool defensin assay: an exploratory study.

BACKGROUND & AIMS: Alpha-defensins 1-3 (human neutrophil peptides [HNP]1-3), reported to be elevated in tumor tissue and serum of patients with colorectal cancer (CRC), have not been studied in stool. We evaluated the neoplasm specificity of HPN1-3 and their discriminant value as stool markers for CRC. METHODS: Protein and mRNA expression of HPN1-3 were assayed in CRC cell lines, microdissected CRC and normal epithelium, and white blood cells. HNP1-3 proteins in stools were quantified in blinded fashion from 30 normal subjects, 20 patients with CRC, 10 with a large colorectal adenoma, 10 with upper gastrointestinal cancer, and 10 with IBD. Stool lactoferrin was also quantified. RESULTS: HPN1-3 proteins were not detected in CRC cell lines but were high (>4000 ng/mL) in white blood cells. mRNA levels of HPN1-3 were comparably low in CRC cell lines, microdissected CRC, and normal colon epithelium, but they were >1000-fold and >30,000-fold higher in white blood cells and neutrophils, respectively. Mean stool HPN1-3 levels were 17 ng/mL with normals, 125 ng/mL with CRC, 62 ng/mL with adenoma, 63 ng/mL with upper gastrointestinal cancer, and 231 ng/mL with IBD (P < .01 for each patient group vs normals). HPN1-3 levels in IBD were higher than in CRC (P = .04). At 90% specificity, sensitivity of stool defensins was 35% for CRC, 40% for adenoma, 40% for upper gastrointestinal cancers, and 80% for IBD. Stool defensins and lactoferrin levels correlated (R2 = 0.70, P < .001). CONCLUSIONS: Alpha-defensins 1-3 levels are nonspecifically elevated in stools from patients with colorectal neoplasia and likely originate from white blood cells. Alpha-defensins 1-3 in stool might serve as markers of inflammatory bowel conditions.

A sensitive method to quantify human long DNA in stool: relevance to colorectal cancer screening.

Human long DNA in stool may reflect nonapoptotic exfoliation and has been used as a colorectal cancer (CRC) marker. Targeting human-specific Alu repeats represents a logical but untested approach. A real-time Alu PCR assay was developed for quantifying long human DNA in stool and evaluated in this study. The accuracy and reproducibility of this assay and the stability of long DNA during room temperature fecal storage were assessed using selected patient stools and stools added to human DNA. Thereafter, long DNA levels were determined in blinded fashion from 18 CRC patients and 20 colonoscopically normal controls. Reproducibility of real-time Alu PCR for quantifying fecal long DNA was high (r2 = 0.99; P < 0.01). Long DNA levels in nonbuffered stools stored at room temperature fell a median of 75% by 1 day and 81% by 3 days. An EDTA buffer preserved DNA integrity during such storage. Human long DNA was quantifiable in all stools but was significantly higher in stools from CRC patients than from normal controls (P < 0.05). At a specificity of 100%, the sensitivity of long DNA for CRC was 44%. Results indicate that real-time Alu PCR is a simple method to sensitively quantify long human DNA in stool. This study shows that not all CRCs are associated with increased fecal levels of long DNA. Long DNA degrades with fecal storage, and measures to stabilize this analyte must be considered for optimal use of this marker.

Frequent methylation of eyes absent 4 gene in Barrett's esophagus and esophageal adenocarcinoma.

Most esophageal adenocarcinomas arise within Barrett's esophagus but the cause of this increasingly prevalent condition remains unknown. Early detection improves survival and discriminant screening markers for Barrett's esophagus and cancer are needed. This study was designed to explore the natural history of eyes absent 4 (EYA4) gene methylation in the neoplastic progression of Barrett's esophagus and to evaluate methylated EYA4 as a candidate marker. Aberrant promoter methylation of EYA4 was studied by methylation-specific PCR using bisulfite-treated DNA from esophageal adenocarcinomas, Barrett's esophagus, and normal epithelia, and then confirmed by sequencing. Eight cancer cell lines were treated with the demethylation agent 5-aza-2'-deoxycytidine, and EYA4 mRNA expression with and without treatment was quantified by real-time reverse-transcription PCR. EYA4 hypermethylation was detected in 83% (33 of 40) of esophageal adenocarcinomas and 77% (27 of 35) of Barrett's tissues, but only in 3% (2 of 58) of normal esophageal and gastric mucosa samples (P < 0.001). The unmethylated cancer cell lines had much higher EYA4 mRNA expression than the methylated cancer cell lines. Demethylation caused by 5-aza-2'-deoxycytidine increased the mRNA expression level by a median of 3.2-fold in methylated cells, but its effect on unmethylated cells was negligible. Results indicate that aberrant promoter methylation of EYA4 is very common during tumorigenesis in Barrett's esophagus, occurs in early metaplasia, seems to be an important mechanism of down-regulating EYA4 expression, and represents an intriguing candidate marker for Barrett's metaplasia and esophageal cancer.

Aberrant methylation of the eyes absent 4 gene in ulcerative colitis-associated dysplasia.

BACKGROUND & AIMS: This study explored the eyes absent 4 (EYA4) gene promoter methylation in noncolitic colorectal tissues and assessed its discrimination for neoplasia in chronic ulcerative colitis (CUC). METHODS: The methylation status of noncolitic specimens was confirmed by direct bisulfite sequencing. Methylation-specific polymerase chain reaction (MSP) primers were designed to evaluate colorectal tissues, including 50 noncolitic patients comprising 24 normal epithelia, 14 polyps, and 12 cancers. The assay was tested on tissues from 67 CUC patients including 31 surveillance neoplasia-positive patients and nonneoplastic controls including 22 CUC surveillance-negative and 14 CUC short-disease duration. Remote colonic tissue was included from each of 27 of the 31 CUC neoplasia cases. The expression of EYA4 was quantified in cell lines by use of reverse-transcription polymerase chain reaction. RESULTS: Within noncolitic tissues, bisulfite sequencing showed EYA4 promoter hypermethylation in 80% (8 of 10) of colorectal cancers but in none (0 of 9) of the normal tissues. MSP was positive in 81% (21 of 26) of cancers and polyps and in only 4% (1 of 14) of normal mucosa. In CUC, MSP was positive in 81% (25 of 31) of neoplastic cases but in none (0 of 36) of the nonneoplastic controls. RNA expression was decreased in methylated compared with unmethylated cell lines (P < .001). Treatment with 5-Aza-2'-deoxycytidine (DAC)/Trichostatin (TSA) increased the overall messenger RNA expression (P = .005). CONCLUSIONS: The EYA4 gene promoter is hypermethylated commonly in sporadic and colitic neoplasia and may be associated with gene silencing. EYA4 methylation represents a candidate marker for CUC surveillance.

Aberrant methylation of secreted frizzled-related protein genes in esophageal adenocarcinoma and Barrett's esophagus.

Hypermethylation of secreted frizzled-related proteins (SFRP) genes frequently occurs with several cancers but has not been studied in esophageal adenocarcinoma or its precursor-Barrett's esophagus. To explore the role of SFRP methylation in the neoplastic progression of Barrett's esophagus and to evaluate methylated SFRP genes as biomarkers for Barrett's esophagus and cancer, methylation of SFRP genes was determined in esophageal adenocarcinomas, Barrett's esophagus and normal epithelia using methylation-specific PCR. Protein expression of SFRP genes was then assessed in these tissues by immunohistochemistry. The mRNA expression of SFRP genes was quantified by real-time reverse-transcription PCR in esophageal adenocarcinoma cell lines with and without demethylation by 5-aza-2'deoxycytidine and inhibition of deacetylation by trichostatin A treatment. Hypermethylation of SFRP1, 2, 4 and 5 was detected in 93%, 83%, 73% and 85% of 40 cancers; 81%, 89%, 78% and 73% of 37 Barrett's epithelia; 25%, 64%, 32% and 21% of 28 adjacent normal epithelia from Barrett's patients; and 10%, 67%, 0% and 13% of 30 normal esophagogastric epithelia from healthy individuals, respectively (p < 0.001 for SFRP1, 4 and 5; p < 0.05 for SFRP2). Protein expression of SFRP1, 2 and 4 was downregulated in 87%, 67% and 90% of cancers, and expression correlated inversely with grade and stage of cancers and with grade of dysplasia. Expression of SFRP2 and SFRP4 proteins was lower in cancers with corresponding gene methylation (p < 0.05). Demethylation treatment effectively re-expressed SFRP mRNA in cancer cell lines. Thus, hypermethylation of SFRP genes is a common early event in the evolution of esophageal adenocarcinoma, and methylation of SFRP1, 4 and 5 might serve as biomarkers for Barrett's neoplasia. Aberrant promoter methylation appears to functionally silence SFRP gene expression in esophageal adenocarcinoma.