A. Steven Fleisher

Distinct methylation patterns of two APC gene promoters in normal and cancerous gastric epithelia

The adenomatous polyposis coli (APC) tumor suppressor gene is mutationally inactivated in both familial and sporadic forms of colorectal cancers. In addition, hypermethylation of CpG islands in the upstream portion of APC, a potential alternative mechanism of tumor suppressor gene inactivation, has been described in colorectal cancer. Because a subset of both gastric and colorectal cancers display the CpG island methylator phenotype, we hypothesized that epigenetic inactivation of APC was likely to occur in at least some gastric cancers. APC exhibits two forms of transcripts from exons 1A and 1B in the stomach. Therefore, we investigated CpG island methylation in the sequences upstream of exons 1A and 1B, i.e., promoters 1A and 1B, respectively. We evaluated DNAs from 10 gastric cancer cell lines, 40 primary gastric cancers, and 40 matching non-cancerous gastric mucosae. Methylated alleles of promoter 1A were present in 10 (100%) of 10 gastric cancer cell lines, 33 (82.5%) of 40 primary gastric cancers, and 39 (97.5%) of 40 non-cancerous gastric mucosae. In contrast, promoter 1B was unmethylated in all of these same samples. APC transcripts from exon 1A were not expressed in nine of the 10 methylated gastric cancer cell lines, whereas APC transcripts were expressed from exon 1B. Thus, expression from a given promoter correlated well with its methylation status. We conclude that in contrast to the colon, methylation of promoter 1A is a normal event in the stomach; moreover, promoter 1B is protected from methylation in the stomach and thus probably does not participate in this form of epigenetic APC inactivation.

Hypermethylation of the hMLH1 gene promoter is associated with microsatellite instability in early human gastric neoplasia

A significant portion of gastric cancers exhibit defective DNA mismatch repair, manifested as microsatellite instability (MSI). High-frequency MSI (MSI-H) is associated with hypermethylation of the human mut-L homologue 1 (hMLH1) mismatch repair gene promoter and diminished hMLH1 expression in advanced gastric cancers. However, the relationship between MSI and hMLH1 hypermethylation has not been studied in early gastric neoplasms. We therefore investigated hMLH1 hypermethylation, hMLH1 expression and MSI in a group of early gastric cancers and gastric adenomas. Sixty-four early gastric neoplasms were evaluated, comprising 28 adenomas, 18 mucosal carcinomas, and 18 carcinomas with superficial submucosal invasion but clear margins. MSI was evaluated using multiplex fluorescent PCR to amplify loci D2S123, D5S346, D17S250, BAT 25 and BAT 26. Methylation-specific PCR was performed to determine the methylation status of hMLH1. In two hypermethylated MSI-H cancers, hMLH1 protein expression was also evaluated by immunohistochemistry. Six of sixty-four early gastric lesions were MSI-H, comprising 1 adenoma, 4 mucosal carcinomas, and 1 carcinoma with superficial submucosal invasion. Two lesions (one adenoma and one mucosal carcinoma) demonstrated low-frequency MSI (MSI-L). The remaining 56 neoplasms were MSI-stable (MSI-S). Six of six MSI-H, one of two MSI-L, and none of thirty MSI-S lesions showed hMLH1 hypermethylation (P<0.001). Diminished hMLH1 protein expression was demonstrated by immunohistochemistry in two of two MSI-H hypermethylated lesions. hMLH1 promoter hypermethylation is significantly associated with MSI and diminished hMLH1 expression in early gastric neoplasms. MSI and hypermethylation-associated inactivation of hMLH1 are more prevalent in early gastric cancers than in gastric adenomas. Thus, hypermethylation-associated inactivation of the hMLH1 gene can occur early in gastric carcinogenesis.

Low prevalence of the APC I1307K sequence in Jewish and non-Jewish patients with inflammatory bowel disease.

A germline sequence alteration at codon 1307 of the APC gene (I1307K) has been reported in 6 – 7% of the Ashkenazi Jewish population in the United States. This alteration is believed to predispose the APC gene to a secondary mutation at the same locus, resulting in an increased risk of colorectal carcinoma. There is an increased risk of colorectal carcinoma in patients with inflammatory bowel disease (IBD), a relatively large proportion of whom are Ashkenazi Jews. We therefore sought to determine whether the I1307K sequence variant occurred in the germline DNA of IBD patients. To our surprise, we found this sequence in only two of 267 patients with IBD (0.7%), occurring in only 1.5% of Jewish IBD patients. The I1307K sequence variant was not found in 67 patients with esophageal cancer, 53 patients with gastric carcinoma (13 MSI-H and 44 MSI-negative), or ten patients with sporadic MSI-H colon cancer. These findings suggest that the I1307K sequence is relatively rare in the germline of Jewish as well as non-Jewish IBD patients. It does not appear to contribute to the increased colorectal cancer risk present in these patients.

Colon Polyps in Beckwith-Wiedemann Syndrome: Role of Imprinted Genes

Dear Sir: We read with interest recent articles emphasizing a possible role of epigenetic gene silencing by promoter hypermethylation in gastrointestinal cancers.1–3 In addition, we and others have observed that hypermethylation of the hMLH1 gene promoter is associated with loss of expression of hMLH1 protein and mismatch repair deficiency in sporadic colorectal,4 endometrial,5 and gastric cancer.6 DNA methylation is thought to be one of the mechanisms responsible for genetic imprinting, a phenomenon in which expression of a specific gene product is derived exclusively from one of the two parental alleles. This process may contribute to cancer by causing loss of function of growth-inhibitory genes, as occurs with the paternally imprinted genes H19 and p57kip2, located on chromosome 11p15, in Wilms’ tumors.7 In addition, genetic imprinting may contribute to cancer by causing gain of function of growth-promoting genes, as occurs when the maternally imprinted gene insulin-like growth factor 2 (IGF-2), also located on 11p15, is abnormally expressed in Wilms’ tumor due to loss of imprinting (LOI).8 A hereditary syndrome regarded as a paradigm of an imprinting disorder is the Beckwith– Wiedemann syndrome (BWS).9 Multiple developmental abnormalities, mental retardation, and childhood tumors, particularly Wilms’ tumor, characterize BWS. We have observed colonic polyposis in a patient with BWS, a condition not previously associated with colonic polyps or cancer. This patient had congenital mild mental retardation, hemihypertrophy, and a Wilms’ tumor resected at age 2. She also had thyroid cancer resected at age 21 and underwent a radical mastectomy for breast cancer at age 40. Abdominal computerized tomography suggested partial small bowel malrotation. At age 47, the patient underwent colonoscopy because of microcytic anemia with Hemoccult-positive stools. Six polyps were removed from the transverse and descending colon, several of which were tubular adenomas and one of which was a serrated adenoma. Upper gastrointestinal endoscopy showed no abnormalities. The genetic basis for BWS is complex and involves multiple different abnormalities of 11p15. Reported abnormalities include trisomy of 11p15 of paternal origin, uniparental disomies, chromosomal translocations, and LOI of genes on 11p15. Multiple imprinted genes on 11p15 that may play a role in cancer, including H19, IGF-2, and the cell cycle inhibitor p57kip2, have been linked to BWS.9 Recent observations on the role of abnormal gene imprinting in cancer suggest that patients with BWS might be at increased risk of colon cancer. The progression from normal mucosa to adenomatous polyps and then to colon cancer is thought to occur because of multiple molecular genetic events affecting critical ‘‘caretaker’’ and ‘‘gatekeeper’’ genes. In addition to mutations and chromosomal alterations, recent studies suggest that potentially reversible epigenetic hypermethylation and LOI may also contribute to colorectal cancer pathogenesis. Recently, LOI for IGF-2 was identified in a high proportion of sporadic colorectal cancers, particularly those with defective mismatch repair.10 Unlike most genetic alterations in sporadic colorectal cancer found only in tumors, LOI for IGF-2 was found in normal colon mucosa and peripheral blood, indicating that LOI may identify patients at risk of developing colon cancer.10 The finding of colonic polyps in our patient with BWS provides additional support for the contention that LOI of genes on 11p15 predisposes to the development of colon cancer. Further studies are warranted to determine whether patients with BWS should have more intensive colorectal cancer screening than the general population.