Volume 131, Issue 5, Pages 1418-1430 (November 2006) Aristaless-like Homeobox-4 Gene Methylation Is a Potential Marker for Colorectal Adenocarcinomas  Matthias P.A. Ebert, Fabian Model, Suzanne Mooney, Kari Hale, Joe Lograsso, Lori Tonnes–Priddy, Juliane Hoffmann, Antal Csepregi, Christoph Röcken, Bela Molnar, Hans–Ulrich Schulz, Peter Malfertheiner, Catherine Lofton–Day  Gastroenterology  Volume 131, Issue 5, Pages 1418-1430 (November 2006) DOI: 10.1053/j.gastro.2006.08.034 Copyright © 2006 AGA Institute Terms and Conditions

Figure 1 Results of the APPCR analysis of pooled DNA from normal and adenoma tissues. A hypermethylated gene (ALX4) was detected in the adenoma DNA (box, arrow). R, RsaI; H, RsaI/HpaII; M, RsaI/MspI. Gastroenterology 2006 131, 1418-1430DOI: (10.1053/j.gastro.2006.08.034) Copyright © 2006 AGA Institute Terms and Conditions

Figure 2 Degree of methylation of the ALX4 gene as assessed by MethyLight assay as outlined in Materials and Methods. (A) ALX4 gene methylation in normal colon mucosa and matched colon cancer. (B) ALX4 gene methylation in matched cases from 11 patients with primary colorectal cancers and liver metastasis. (C) ALX4 gene methylation in primary colorectal cancers and metastasis. (D) ALX4 methylation in colorectal cancer cell lines. Gastroenterology 2006 131, 1418-1430DOI: (10.1053/j.gastro.2006.08.034) Copyright © 2006 AGA Institute Terms and Conditions

Figure 3 Bisulfite sequencing. Four cases of cancers with a high degree of methylation and their matched normal nonmalignant colon mucosa tissues were selected (inset). ALX4 gene methylation was confirmed by sequencing of bisulfite-treated genomic DNA of samples 1–4. N, normal mucosa; T, tumor. Numbers 1–4 correspond to cases 1–4 in the MethyLight assay (inset); MethyLight assay indicates the CpG sites that were covered by both the MethyLight assay and sequencing of the respective DNA fragments. Gastroenterology 2006 131, 1418-1430DOI: (10.1053/j.gastro.2006.08.034) Copyright © 2006 AGA Institute Terms and Conditions

Figure 4 ALX4 gene expression in colon cancer cell lines. Four colon cancer cell lines were analyzed for ALX4 gene expression by reverse-transcription PCR. In these 4 cell lines, ALX4 messenger RNA levels were below the level of detection; however, after treatment with the methylation inhibitor 5-aza-2′-deoxycytidine, Lovo cells readily exhibited ALX4 messenger RNA. β2M, β2-microglobulin; A, incubation with 5′-azadeocxycytidine; D, incubation with DMSO; −, no DNA; M, DNA ladder. Gastroenterology 2006 131, 1418-1430DOI: (10.1053/j.gastro.2006.08.034) Copyright © 2006 AGA Institute Terms and Conditions

Figure 5 Gene methylation in colorectal cancer. The methylation status of 6 genes in 47 colorectal cancers (TPEF/HPP1, APC, TIMP3, DAPK, Caveolin-2, p16) has been reported by our group recently.18 Now ALX4 gene methylation was assessed by MethyLight analysis in the same set of patients. While ALX4 and TPEF/HPP1 gene methylation occurred in the majority of colorectal cancers, the other 6 genes were less frequently methylated. Each box indicates a sample from a patient with colorectal cancer (n = 47). ■, methylated sample, PMR ≥4%; □, unmethylated sample, PMR <4%. Gastroenterology 2006 131, 1418-1430DOI: (10.1053/j.gastro.2006.08.034) Copyright © 2006 AGA Institute Terms and Conditions

Figure 6 ALX4 gene methylation in gastrointestinal and other neoplasias. Methylation of the ALX4 gene was analyzed using MethyLight analysis. Each box indicates a sample from a patient with the respective preneoplastic or neoplastic condition. ■, methylated sample, PMR ≥4%; □, unmethylated sample, PMR <4%; N, normal; T, tumor; D, dysplasia; L, liver; M, metastasis; IEN, intraepithelial neoplasia; SCC, squamous cell cancer; HCC, hepatocellular cancer; CCC, adenocarcinomas of the biliary tree including gallbladder cancer and cholangiocellular carcinomas. Gastroenterology 2006 131, 1418-1430DOI: (10.1053/j.gastro.2006.08.034) Copyright © 2006 AGA Institute Terms and Conditions

Figure 7 Serum ALX4 methylation levels in patients with colorectal cancer and adenomas. (A) The serum levels in 30 patients with colorectal cancer were significantly increased compared with noncancer controls (P < .0001). Mean ± SEM. (B) Receiver operating characteristic curve. The area under the curve generated from serum ALX4 gene methylation was 0.839 (95% confidence interval, 0.721–0.921) for all 30 patients with colorectal cancer. (C) Overall, there was no significant difference in the mean ALX4 methylation levels of patients with hyperplastic or adenomatous polyps compared with patients without polyps (normal). Mean ± SEM. Gastroenterology 2006 131, 1418-1430DOI: (10.1053/j.gastro.2006.08.034) Copyright © 2006 AGA Institute Terms and Conditions