p33ING1b methylation in fecal DNA as a molecular screening tool for colorectal cancer and precancerous lesions

The present study aimed to investigate the feasibility of detecting p33 inhibitor of growth 1b (p33ING1b) gene methylation in fecal DNA as a screening method for colorectal carcinoma (CRC) and precancerous lesions. The methylation of p33ING1b was analyzed in fecal samples from 61 patients with CRCs, 27 patients with precancerous lesions (advanced adenoma) and 20 normal individuals by nested methylation-specific polymerase chain reaction (nMSP) and fecal occult blood test. Methylated p33ING1b was detected in 73.77% of CRC patients and 62.96% of adenoma patients. By contrast, only 5% of normal individuals had methylated p33ING1b. These results indicated 73.77% sensitivity for detecting CRC, 62.96% sensitivity for detecting precancerous lesions and 95% specificity of the assay for detecting CRCs and precancerous lesions. The detection of p33ING1b methylation status by incubation of DNA contained in agarose beads for bisulfite modification, followed by nMSP, is a promising non-invasive screening method for CRCs and precancerous lesions.


Introduction
Colorectal carcinoma (CRC) is one of the leading causes of cancer-associated mortality worldwide. Five-year survival is 90% if the disease is diagnosed while still localized (i.e., confined to the wall of the bowel), but only 68% for regional disease (i.e., disease with lymph node involvement) and just 10% if distant metastases are present (1). Detection of early-stage cancer and precancerous lesions is a key measure in reducing the mortality rate. Fecal occult blood tests (FOBT) have reduced the mortality and morbidity of CRC. However, these have a significant false result rate. Alternative screening tests, including flexible sigmoidoscopy and colonoscopy, have been investigated and used as alternatives, but none have been shown to be as cost effective as FOBT in reducing mortality rates. DNA mutation detection is a potentially beneficial test, but has yet to demonstrate superior sensitivity and specificity in a clinical setting (2). Optimized screening methods and markers must be established with high sensitivity and specificity for early-stage cancers and precancerous lesions.
Additionally, it has been demonstrated that DNA hypermethylation can be detected in DNA from the feces of CRC patients, suggesting that fecal DNA methylation analysis is a promising approach to non-invasive screening for early colorectal lesions (12)(13)(14)(15). Although these reports have confirmed the potential for early detection of colon cancer-derived aberrantly methylated DNA in feces, the majority of these investigations analyzed only a small number of samples. Current detection methods and their sensitivity for known markers are not optimal (16,17). Thus, the development of improved screening methods and identification of new cancer markers is highly desirable.
The p33 inhibitor of growth 1b (p33 ING1b ) gene has been identified as a novel growth inhibitor and tumor suppressor gene, with reduced expression in ovarian, esophageal, gastric, brain and breast cancers. However, mutation of the p33 ING1b gene is rare (18)(19)(20)(21)(22). The p33 ING1b gene promoter region is rich in CpG dinucleotides (23), suggesting that other mechanisms, including DNA methylation, may contribute to the reduced expression of this gene. In the present study, the methylation of p33 ING1b in feces from patients with CRCs and precancerous lesions was analyzed and compared with normal controls. In addition, the sensitivity and specificity of this test were compared with those of the immunochemical FOBT (IFOBT), and the feasibility of detecting hypermethylation in feces as a non-invasive screening tool for CRCs and precancerous lesions was evaluated.

Materials and methods
Patients and specimens. In total, 61 CRC patients, 27 advanced adenoma (AA) patients and 20 patients with endoscopically normal colons undergoing colonoscopy for routine clinical examination at the First Affiliated Hospital of Guangxi Medical University (Nanning, China) were enrolled in this study. Fecal specimens were collected by patients, sent to the laboratory within 2 h after defecation and stored at -80˚C until analyzed. In addition, 37 cancer tissue samples resected by surgery were obtained from the 61 CRC patients. This study was approved by the ethical committee of Guangxi Medical University (Nanning, China). Written informed consent was obtained from the families of the patients.
IFOBT. All stool samples were examined using a single IFOBT with MagStream HemSp (Fujirebio, Tokyo, Japan), an immunochemical test for human hemoglobin. The FOBT was performed at the laboratory of the First Affiliated Hospital of Guangxi Medical University.
Fecal DNA isolation. Fecal DNA was isolated by the QIAamp DNA Stool mini kit (Qiagen, Hilden, Germany). The extracted DNA was quantitated by ultraviolet detection and stored at -20˚C.
CRC tissue DNA isolation. Tissue uvDNA isolation was performed by the conventional method of phenol/chloroform treatment followed by proteinase K digestion. The extracted DNA was stored at -20˚C.
Bisulfite modification. The bisulfite modification procedure was performed according to Olek et al (24) and Zhang et al (25) with minor modifications. In brief, 500 ng genomic DNA was denatured by incubation in 0.3 M NaOH for 15 min at 37˚C, then mixed with 2 volumes 2% low melting point agarose. Agarose/DNA mixtures were pipetted into chilled mineral oil to form agarose beads. Each bead was placed in an individual tube to which 200 µl aliquots of 5 M bisulfate solution were added [2.5 M sodium metabisulfite (Sigma, St. Louis, MO, USA) and 100 mM hydroquinone (Sigma); pH 5.0]. The reaction mixture was then incubated in the dark for 16 h at 50˚C. Treatment was completed by equilibration against 1 ml Tris-EDTA buffer followed by desulfonation in 500 µl NaOH (0.2 M). Finally, the beads were washed with 1 ml H 2 O and then used directly in polymerase chain reaction (PCR).
Nested methylation-specific PCR (nMSP). Blood DNA treated in vitro with CpG methyltransferase (M.SssI) (Fermentas, Hanover, MD, USA) was used as a positive control for methylated alleles, and placental DNA was used as a negative control for the nMSP assay. The bisulfite-modified DNA underwent nMSP in a blind manner using primer pairs designed to specifically amplify the methylated or unmethylated alleles of p33 ING1b . The sequences of PCR primers specific for methylated and unmethylated alleles of p33 ING1b and the sizes of the expected PCR products are summarized in Table I (22). Briefly, 3 µl bisulfite-modified DNA was added in a final volume of 25 µl PCR mixture containing 2.5 µl 10X PCR buffer, 2 µl deoxynucleotide triphosphates (2.5 mmol/l), 0.5 µl each nested primer (10 µmol/l) and 1 unit Long Taq polymerase (Tiangen Biotech (Beijing) Co., Ltd., Beijing, China). The PCR amplification protocol for stage 1 was as follows: 94˚C for 4 min, followed by 35 cycles at 94˚C for 30 sec, specific annealing temperature 53˚C for 30 sec and 72˚C for 30 sec, and finally 72˚C for 5 min. For stage 2 amplifications [PCR with the methylation-specific primers (Sangon Biotech (Shanghai) Co. Ltd., Shanghai, China)] 1 µl first-step PCR products was added in a final volume of 25 µl PCR mixture: 2.5 µl 10X PCR buffer, 2 µl deoxynucleotide triphosphates (2.5 mmol/l), 0.5 µl each MSP primer (10 µmol/l) and 1 unit Taq polymerase (Takara, Dalian, China). The PCR  (Table IV).

A B
The positive detection rates of FOBT in 61 CRCs, 27 AA cases and 20 control patients were 49.18, 33.33 and 10%, respectively (Table V). The positive rate of FOBT in cases of CRC was significantly higher than in the control group (P<0.01). However, there was no statistically significant difference between FOBT results in cases of AA and the control group (P>0.05).
The sensitivity, specificity, crude accuracy and Youden's index of the test for p33 ING1b methylation in fecal DNA were compared with those of the FOBT (Table VI) (26). Youden's index was used to measure the effectiveness of the diagnostic marker and is as follows: Youden's index (J) = Se(c) + Sp(c) -1 (where Se is senstivity and Sp is specificity). The p33 ING1b methylation test had a sensitivity of 73.77% for CRCs and 62.96% for detecting AAs, and a specificity, crude accuracy and Youden's index of 95%, 84.26% and 0.75, respectively. The FOBT had a sensitivity of 49.18% for CRCs and 33.33% for AAs, and a specificity, crude accuracy and Youden's index of 90%, 52.78% and 0.44, respectively (Table VI). The sensitivities of the p33 ING1b methylation test for CRCs and AAs were higher than those of FOBT (P<0.01 and P<0.05, respectively). There was no statistically significant difference between the specificities of the two methods (P>0.05). The crude accuracy of testing for p33 ING1b methylation in fecal DNA was higher than that of FOBT (P<0.01).

Discussion
Since misregulation of gene expression by aberrant DNA methylation is a well-characterized event in tumor biology and has been extensively documented for CRC (3)(4)(5)(6)(7)(8)(9)(10), identification of aberrantly methylated genes is a promising strategy for research, diagnostics and therapeutics (27). However, specific disadvantages associated with fecal DNA testing render it unsuitable for use in general population screening for CRC
In the present study, the feasibility of detecting methylated fecal DNA by nMSP as a screening tool for CRCs and precancerous lesions was explored. Although the routine method of DNA bisulfite modification with MSP is the most common method for detecting DNA methylation in previous studies (14,29,30), this method has not been optimized: The process is complicated, has low sensitivity (detects 1 methylated allele in up to 1,000 unmethylated alleles) due to sample damage and loss during bisfulfite modification, and is expensive when a commercial kit is used. Grunau et al (31) demonstrated that for the majority of routine PCR, 50 ng human template DNA yields sufficient PCR product. The authors found that ~90% of the template DNA is lost during treatment with bisulfite. In the present study, the concentration of fecal DNA extracted by the commercial kit was 67.35±13.17 ng/l, while some samples did not satisfy the requirements for bisulfite treatment and MSP. Under these circumstances, this method may result in an abnormally low detection rate of fecal DNA methylation. As demonstrated by Olek et al (24), bisulfite modification by incubation of DNA contained in agarose beads, followed by nMSP to detect DNA promoter methylation, is easy to perform and high levels of sensitivity can be reached. Treatment of DNA contained in agarose beads minimizes DNA degredation and prevents incomplete conversion of the DNA. The authors also succeeded in amplifying fragments of up to 3 kb, which cannot be achieved using conventional methods. The improved MSP procedure incorporates a nested, two-stage PCR approach, which is more sensitive than the conventional method (detects 1 methylated allele in up to 50,000 unmethylated alleles) (32). In the current study, as little as 10 pg DNA was amplified by treatment within agarose beads followed by nMSP, and even by general Taq polymerase can succeeded in nMSP. This level of sensitivity allows determination of methylation patterns in small fecal samples, advantageous for a high-sensitivity screening method for CRCs. In addition, this study demonstrated that DNA extracted from feces is of sufficient quality and quantity for the detection of DNA methylation by this method. This is also a less expensive test compared with commercial kits for bisulfite modification and PCR.
Next, the methylation status of p33 ING1b (14,30) have demonstrated that aberrant gene methylation may occur in the early stages of CRCs and even in precancerous lesions, and that this can be detected in feces, which has potential value in the noninvasive and early diagnosis of colorectal neoplasms. However, the low sensitivity and specificity of the assays suggest the need to develop more powerful techniques. The present results demonstrate that methylation of the p33 ING1b gene in feces can be considered as a biomarker for CRCs and precancerous lesions, however, these findings must be confirmed or improved upon in studies with larger sample sizes.
FOBT is the most widely used method to screen for CRCs. Although FOBT is valuable as a noninvasive screening method that reduces the risk of CRC-associated mortality (33,34), it has limited sensitivity that leads to numerous CRCs remaining undetected (35)(36)(37). This limitation shows that FOBT is not the best method for screening CRCs. By contrast, using aberrant gene methylation as a molecular marker offers a potentially powerful approach to population-based screening for CRCs and precancerous lesions (28). In the present study, the efficacies of the fecal p33 ING1b methylation test and FOBT for the identification of CRCs and precancerous lesions were compared. FOBT had sensitivities of 49.18, 33.33 and 10% for CRCs, precancerous lesions and the normal control group, respectively, which were lower than those for the detection of p33 ING1b methylation in feces (P<0.05 for AA and P<0.01 for CRCs). However, the two methods had high specificity for CRCs and precancerous lesions (≥90%) and the sensitivity of FOBT for CRCs was greater than that for the control group (P<0.01). No statistically significant difference in FOBT sensitivity for precancerous lesions and the normal control group was identified (P>0.05). These results suggest that FOBT may   be a useful method for screening CRCs but not for precancerous lesions, and that the detection of p33 ING1b methylation in feces may be more effective than FOBT in screening for CRCs and precancerous lesions. It is clear from this study that testing for p33 ING1b methylation in fecal DNA exhibited a higher sensitivity than FOBT without reduced specificity.
In conclusion, the results of the present study demonstrate the value of bisulfite modification of DNA contained in agarose beads, followed by nMSP, to detect fecal DNA promoter methylation, as a noninvasive and simple test, and suggest p33 ING1b methylation as a potential biomarker in screening for CRCs and precancerous lesions.