Small bowel adenocarcinoma (SB-AC) is a very rare tumor entity. Epigenetic alterations, including hypermethylation of DNA mismatch repair genes and tumor suppressor genes, seem to be important for carcinogenesis in tumors of the gastrointestinal tract, but have not yet been investigated in SB-AC. In the current study, the prevalence of hypermethylation in a panel of genes involved in gastrointestinal carcinogenesis (hMLH1, HPP1, p14 ARF , p16 INK4A , APC) was determined in a series of SB-AC. Paraffin-embedded tumor samples from 56 patients with SB-AC who underwent surgical resection between January 1985 and December 2003 were investigated for hypermethylation by means of methylation-specific real-time PCR, and compared with our findings in a previously investigated series of 50 gastric adenocarcinomas. In comparison with adenocarcinomas of the stomach, SB-AC revealed a significantly higher rate of hypermethylation of HPP1 (86% versus 54%, p 5 0.0003), p16 INK4A (32% versus 10%, p 5 0.0006), and a significantly lower rate of hypermethylation of APC (48% versus 84%, p 5 0.0001). Hypermethylation of hMLH1 and p14 ARF was present in 23% and 9% of SB-AC, respectively. Locally advanced tumor categories (pT3/4) showed a higher rate of hypermethylation of HPP1 (90%) than did early tumor categories (pT1/2 categories, 40%; p 5 0.0036). This was also reflected by the correlation between the HPP1 hypermethylation and high UICC stage (p 5 0.02). No correlation was found between hypermethylation and other clinicopathologic parameters such as age, tumor grade and nodal status. Our findings suggest that hypermethylation of hMLH1, HPP1, p16 INK4A and APC is frequent in primary adenocarcinomas of the small bowel. The differences in the hypermethylation spectrum of small bowel and stomach cancer indicate significant epigenetic differences between these tumors. ' 2006 Wiley-Liss, Inc.Key words: small bowel carcinoma; hypermethylation; methylationspecific real-time PCR; gastric cancer Primary adenocarcinomas of the small bowel (SB-AC) are rare. 1-3 Surgeons and physicians are challenged by this tumor because of its infrequency, unspecific symptoms and typically delayed diagnosis. 1,3-5 Prognostic factors are the residual tumor (R category), tumor stage according to the UICC, lymph node metastasis, lymphatic vessel invasion and vascular invasion. [4][5][6] Mucosal contact time with bile acid solutions seems to be one important factor in the development of adenocarcinoma of the small bowel, 7 implicating bile as a potential carcinogen. Other predisposing conditions are small bowel adenomas, familial adenomatous polyposis, celiac disease, cystic fibrosis, peptic ulcer disease 8 and Crohn's disease. 9 Cases occurring in association with ulcerative colitis and backwash ileitis have also been described. 10 An important epigenetic mechanism for silencing tumor suppressor genes during carcinogenesis is the hypermethylation of CpG islands. 11 In certain cancer types, such as colorectal and endometrial cancers, loss of function of the DNA mis...