1. Abstract 1.1. Aims The identification of tumor-specific DNA methylation patterns in epithelial colorectal cells in feces has emerged as a novel and noninvasive screening method for colorectal cancer (CRC) and precancerous lesions. Our study investigates a new panel of DNA methylation biomarkers with high sensitivity and specificity for the early screening of CRC and advanced adenomas in a Chinese population. 1.2. Methods Target DNA biomarkers were isolated and subjected to real-time flurescence quantification analysis in fecal samples from 64 CRC patients, 72 advanced adenomas(AA) patients, 33 hyperplastic polyps(HP) patients and 59 healthy controls. Receiver operating characteristic (ROC) curve was performed to compare the diagnosis value of the DNA methylation biomarker panel with traditional fecal occult blood testing(FOBT). 1.3. Results The classification model built with methylation of SDC2, PPP2R5C and ADHFE1 showed a sensitivity of 90.63% for CRC, 59.72% for AA and 21.21% for HP, with the specificity of 91.53%. Fecal DNA methylation test is superior to FOBT for the diagnosis of CRC and AA (AUC: 0.85 vs. 0.71, P< 0.001), especially for AA (AUC: 0.82 vs. 0.64, P< 0.001). No noticeable correlation was found between the sensitivity of DNA methylation testing panel and the clinical characteristics. This panel was more accurate than FOBT in detecting CRC and AA for patients >50 years (47.15% vs 73.17%, P< 0.05). 1.4. Conclusion Our findings validated the methylation testing of SDC2, PPP2R5C and ADHFE1 in feces as a promising alternative for screening colorectal cancer and advanced adenomas.
Keywords: Colorectal cancer; DNA methylation marker; SDC2; PPP2R5C; ADHFE1
2. Introduction Colorectal cancer(CRC) is the third most common cancer and has ranked second in terms of mortality worldwide in 2020 [1]. The five-year survival rate exceeds 90% for patients with lesions still confined to the intestinal wall, but is only 68% for patients with local lymph node involvement and less than 10% if distant metastases occur [2]. Reducing the exposure to risk factors, such as sedentary lifestyle, obesity, smoking and alcohol consumption and unhealthy diatary habits, attributes to decrease the incidence and mortality rates of CRC, but most effectively, we need to improve our screening methods to distinguish those potential patients at early stage, and by polypectomy, to prolong their lifespan [3-5]. Currently, there are mainly two categories of CRC screening for the average-risk population: structual and stool-based examinations. Structual examinations mainly include colonoscopy, double-contrast barium enema (DCBE) and flexible sigmoidoscopy (FSIG); stool tests mainly include occult blood or exfoliated DNA testing [4]. Among them, colonoscopy is the most accurate, it allows visualization of full colon and rectum and can remove the lesions in a single session, which enable it of both diagnosis and treatment value. But colonoscopy also has limititions. It requires several days of unpleasant bowel preparation, and due to its invasiveness and the risk of bowel perfortion, patients are less likely to choose colonoscopy as their screeening option [6]. DCBE can also detects and evaluates entire bowel, and it provides a second chance for patients with contraindications or who have failed colonoscopies. But the extensive bowel preparation, lower sensitivity for adenomas and lack of access to polypectomy or biopsy limit the application within general public.
3. Materials and Methods 3.1. Study Design This research was approved by the ethics committee of Qingdao Central Hospital and affiliated Hospital of Qingdao University. A total of 228 participants were recuited from August 1, 2020 to March 1, 2021 in this study, and written consent was obtained. Participants would be excluded when they were found (1) the previous history of CRC or any other cancer;or (2) ungerwent bowel operation, chemotherapy or radiotherapy. Based on the pathology results, paticipants were gathered into four groups: Control group(n=59), HP (size﹤10mm) group(n=33), AA(size﹥10mm) group (n=72), and CRC group(n=64). Clinical features including age, sex, tumor location, TNM stage, differentiation degree, histology and pathology results were recorded. 3.2. Stool collection, DNA extraction, bisulfite treatment and qRT-PCR Stool samples (about 5g) were collected in 50-Ml tubes with 15mL preservative buffer at least 1 day prior to bowel preparation for colonoscopy, then all samples were stored at -80o C. All stool samples in preservative buffer were thawed and homogenized for 1 min with a shaking device. Each tube was centrifuged at 12,000 g for 15 minutes. The TIANamp stool DNA kit (TIANGEN BIOTECH Co., Ltd.) was used to extract human genomic DNA. Bisulfite conversion was performed to discriminate those methylated DNA with a DNA bisulfite conversion kit (TIANGEN BIOTECH Co., Ltd.). NanoDrop 2000(Thermo Scientific, MA, USA) was used to measure the DNA concentration. After purification and conversion, DNA was kept at -20o C. SLAN-96S(SHANGHAI HONGSHI BIOTECH Co.,LTD) was used to perform qPT-PCR, and three replication were conducted for each sample [15]. 3.3. Fecal occult blood testing(FOBT) FOBT (BeckmanCoulter, Fullerton, CA) were performed to all stool samples immediately once obtained. After 1 day, add a drop of peroxide catalyst to the opposite side of the test card. A blue reaction within one minute is considered positive.
4. Statistical Analysis Twenty-six sites of these three genes were detected. The methylation result was defined as the methylation index(MI). Based on the different number of methylated sites, the result was defined as positive when MI﹥5 and as negative when MI﹤5. The logistic regression was built to differentiate risk factors. Chi-aquare test and Fisher exact test were used as appropriate to evaluate the diagnostic values of stool DNA methylation testing and FOBT. Receiver operation curve(ROC), 95% confidence intervals(CI), sensitivity and specificity were implemented to evaluate the diagnostic accuracy. Statistical analysis was performed using Graphpad Prism 8 and IBM SPSS statistics software version 26.0. P values﹤0.05 were considered to be statistically significant.
5. Results 5.1. Clinical Characteristics of Subjects Stool samples were obtained from 228 participants who underwent routine colonoscopies at the Qingdao Central Hospital. The participants (aged 61.45±10.63y, 57.5% male) include 59 healthy controls (aged 52.83±12.38y, 44.10% male), 33 HP patients (aged 56.91±10.21y, 57.60% male), 72 AA patients(aged 61.00±8.61y, 54.20% male) and 64 CRC patients(aged 65.09±10.58y, 62.50% male). The mean age of colonoscopy-negative participants was younger than CRC patients, and the percentage of male was higher than female in the CRC group. Of 228 subjects enrolled, 2, 11, 20 and 25 were at stage I, II, III and IV of CRC, respectively. Meanwhile, 59.72% of advanced adenomas and 78.1% of malignant neoplasms were found in the left colon. The clinical information were presented in Table 1. 5.2. Clinical performance of DNA methlylation and FOBT in colorectal cancer screening As shown in Table 2, stool-based DNA methylation testing is more sensitive than FOBT in detecting CRC and precancerous lesions(74.26% vs. 47.06%, P﹤0.05). To be specific, the sensitivity of DNA methylation was 90.63% for CRC, 59.72% for AA, 21.21% for HP, respectively. For FOBT, the sensitivity was 70.31% for CRC, 26.39% for AA, 6.06% for HP. The specificity of DNA methylation and FOBT was up to 91.53% and 89.83%, hyperplastic polyps; AA, advanced adenomas; CRC, colorectal cancer. Left colon was defined as the rectum, sigmoid, and descending colon; Right colon was defined as the transverse colon, ascending colon and cecum. respectively. ROC was constructed to evaluate the diagnostic value. As shown in Figure 1, DNA methylation showed better performance than FOBT in CRC and advanced adenomas (AUC:0.85 [95% CI: 0.80-0.91] vs. 0.71 [95% CI: 0.64-0.78], P﹤0.05; Figure 1D). The AUC of the panel reached 0.91 [95% CI: 0.86-0.97]) in CRC detection (Figure 1A), and 0.82 [95% CI: 0.74-0.89]) in AA detection (Figure 1B), respectively. The diagnostic value in detecting polyps was almost equivalant between DNA methylation and FOBT (AUC:0.59 [95% CI: 0.46-0.72] vs. 0.55 [95% CI: 0.42- 0.68], P﹥0.05; Figure 1C).
6. Discussion Cancer prevention through screening requires detection of precancerous lesions. Traditionally, adenomas are regarded as the most necessary step to CRC. About more than half of people will develop colorectal adenomas during their lifetime, but merely 6% will undergo malignant transformation [17]. It is the adenomas that are most likely to progress that should be screened for. According to previous studies, those ≥ 1 cm in size or with highly atypical hyperplasia or villous structures are called advanced adenomas; these adenomas have the highest risk of malignant transformation and are often considered as the most relevant part of screening [17,18]. Early screening programs have been proved to be efficient in decreasing the incidence and mortality of colorectal cancer in many countries [19]. In China, FOBT is widely adopted in early detection of CRC, but is restricted due to its low sensitivity and dietary [20]. Previous studies have found fecal DNA methylation testing showed high accuracy of detecting CRC and advanced adenomas [21-23]. Cologuard, which combines an essay for hypermethylated biomarkers(BMP3, NDRG4) and mutant KRAS with FIT test, has been approved by FDA for CRC screening [24]. Multitarget stool DNA test was recmmended by American Cancer Society(ACS) as a new CRC screening option in the updated guideline [25]. Since tumor phenotypes, molecular patterns and genotypes are highly heterogeneous, a single or universal molecular marker for cancer or precancerous lesions is of limited significance and therefore a set of biomarkers are needed. In this study, we evaluated the diagnostic performances of a new panel of DNA methylation biomarkers, and it showed an overall sensitivity of 90.63% with AUC of 0.91 in detecting CRC not affected by sex, age, location, or tumor stage (P > 0.05), with a specificity of 91.53%. This panel also functioned well in detecting AA (sensitivity 59.72%, AUC=0.82), but had low performance in detecting HP (sensitivity 21.21%, AUC=0.59). We choose three methylation biomarkers (SDC2, PPP2R5C and ADHFE1). Hypermethylated SDC2 was found in all stages of CRC and the stool-based SDC2 methylation test had identical high sensitivity as Cologuard for screening CRC [9]. In a qPCR based mSDC2 assay reported in 2017, the sensitivity of detecting CRC and >1 cm adenomas was 81.1% and 58.2%, respectively, with a specificity of 93.3% [26], which is in line with our findings, implying that SDC2 was a candidate methylation biomarker for CRC screening. Protein phosphatase 2(PP2A) is a serine/threonine phosphatase, which serves as a tumor suppressor by involving in numerous negative signaling pathways of cell growth and division. Inhibition of PP2A activity has been reported to promote malignant transformation [27]. PPP2R5C interacts with PPP2R1A or other PP2A subunits through mutations at the protein–protein binding interface, which may interfere with assembly of the complex and thereby abrogate its cancer suppressive properties [28]. The downregulation and hypermethylation of ADHFE1 in colorectal cancer tissues are associated with poor differentiation and advanced TNM staging in CRC patients [29].
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Xuezhen Ma. A New Panel of DNA Methylation Biomarkers for Screening Colorectal Cancer and Precancerous Lesions in a Chinese Population. Annals of Clinical and Medical Case Reports 2021