C. Nobre Leitão

Effect of folate supplementation on DNA methylation of rectal mucosa in patients with colonic adenomas: correlation with nutrient intake

We have evaluated the effect of folate supplementation (5 mg/day) on global deoxyribonucleic acid (DNA) methylation status of the rectal mucosa of 20 patients with resected colonic adenomas in a prospective, controlled, cross-over study. Baseline values of DNA methylation were inversely correlated with caloric (P = 0.03) and fat intake (P = 0.05) and patients harbouring multiple polyps consumed significantly more calories (P = 0.0006), fat (P = 0.009) and carbohydrates (P = 0.009) as compared to patients having one single lesion. Folate supplementation resulted in a significant decrease of DNA hypomethylation in 7/20 patients (P = 0.05) which returned to previous values after placebo treatment. This effect was significantly correlated with number of polyps, with all the responders presenting one single lesion, whereas 8/13 of the non-responders had multiple ones (chi2 = 7.17, P = 0.007). In conclusion, folate supplementation may decrease degree of DNA hypomethylation, but only in patients with one single polyp. In those with multiple lesions, other nutritional factors such as caloric and fat intake, may be more determinant.

DNA hypomethylation and proliferative activity are increased in the rectal mucosa of patients with long-standing ulcerative colitis

DNA methylation and DNA cytometric parameters were evaluated in the rectal mucosa from patients with extensive and long‐standing ulcerative colitis.

Chromosomal analysis of Barrett's cells: demonstration of instability and detection of the metaplastic lineage involved.

Barretts esophagus is lined by columnar and goblets cells with gastric and intestinal characteristics. Despite the association between goblet elements and malignancy, it was not demonstrated that other columnar cells lineages are not related to neoplasia. Chromosomal abnormalities were described in metaplasia adjacent to Barretts neoplasia, but it is unknown which metaplastic lineages are involved. This work assessed the frequency and the type of chromosomal abnormalities in Barretts esophagus without neoplasia and performed the identification of the metaplastic cells carrying chromosomal gains. Barretts esophagus biopsies were collected and processed for short-term cell culture and cytogenetic analysis. Combined immunofluorescence/fluorescence in situ hybridization was performed in cases exhibiting chromosomal gains by using antisera against intestinal (MUC2) and gastric (MUC5AC and MUC6) apomucins and chromosome pericentromeric alpha satellite DNA probes for the chromosomes involved. Each case was scored for the number of spots (0, 1, 2, >2) in 200 nonoverlapping nuclei. Columnar and goblet cells were separately assessed. Short-term cell cultures were achieved in 40/60 cases (67%). There were clonal abnormalities in 27/40 cases (68%) and tetraploid (4n) clones in 10/40 (25%). Structural alterations were detected in 14/40 (35%) with recurrent breakpoints at 1q21, 15q15 and 15q22. Numerical changes (trisomies 7 and 18 and loss of Y) occurred in 16/40 (40%). Gains of chromosomes 7 and 18 were more frequent in columnar than in goblet cells (9.8% vs 0.7% (P<0.05)) and (7.9 vs 1.9% (P<0.05)) respectively. These alterations were detected in cells exhibiting gastric as well as intestinal features and were more frequent in cells without apomucin production. Conclusions: (1) chromosomal instability is a common finding in Barretts esophagus without neoplasia. (2) The two metaplastic populations are committed, chromosomal gains being more frequent in columnar nongoblet than in goblet cells. (3) The two metaplastic phenotypes, gastric and intestinal, are equally involved.

APC or MUTYH mutations account for the majority of clinically well-characterized families with FAP and AFAP phenotype and patients with more than 30 adenomas

Patients presenting familial adenomatous polyposis (FAP), attenuated familial adenomatous polyposis (AFAP) or multiple colorectal adenomas (MCRAs) phenotype are clinically difficult to distinguish. We aimed to genetically characterize 107 clinically well‐characterized patients with FAP‐like phenotype, and stratified according to the recent guidelines for the clinical management of FAP: FAP, AFAP, MCRA (10–99 colorectal adenomas) without family history of colorectal cancer or few adenomas (FH), MCRA (10–99) with FH, MCRA (3–9) with FH. Overall, APC or MUTYH mutations were detected in 42/48 (88%), 14/20 (70%) and 10/38 (26%) of FAP, AFAP and MCRA patients, respectively. APC and MUTYH mutations accounted for 81% and 7% of FAP patients and for 30% and 40% of AFAP patients, respectively. Notably, MCRA patients did not present APC mutations. In 26% of these patients, an MUTYH mutation was identified and the detection rate increased with the number of adenomas, irrespectively of family history, being significantly higher in MCRA patients presenting more than 30 adenomas [7/12 (58%) vs 2/14 (14%), p = 0.023]. We validate the recently proposed guidelines in our patients cohort and show that APC or MUTYH germline defects are responsible for the majority of clinically well‐characterized patients with FAP and AFAP phenotype, and patients with more than 30 colorectal adenomas. The different mutation frequencies according to family history and to the number of adenomas underscore the importance of an adequate familial characterization, both clinically and by colonoscopy, in the management of FAP‐like phenotypes. The phenotypes of the mutation‐negative patients suggest distinct etiologies in these cases.

The muir-torre syndrome: a rare variant of hereditary nonpolyposis colorectal cancer associated with hmsh2 mutation

The Muir-Torre syndrome is a rare autosomal dominant disorder characterized by the association of visceral malignancies with typical skin lesions. This syndrome is now considered a subtype of the more common hereditary nonpolyposis colorectal cancer syndrome (HNPCC). This last condition has been ascribed to mutations in four mismatch repair genes, and similar mutations, mostly located at hMSH2 gene, are now being described in some Muir-Torre patients. We describe the case of a 64-yr-old woman with no family history of colorectal cancer, who developed two visceral malignancies belonging to the usual spectrum of hereditary nonpolyposis colorectal cancer (colon and stomach), beginning at age 41. She additionally developed several skin tumors, including multiple keratoacanthomas, thus fulfilling Muir-Torre diagnostic criteria. Because of her cutaneous phenotype, she was screened for DNA mismatch repair gene mutations by in vitro synthetized protein assay (IVSP) and a truncating mutation was identified at hMSH2. We further discuss the clinical significance of the Muir-Torre phenotype, the association of this syndrome with hMSH2 mutations and the important implications of genetic diagnosis for the patient and her offspring.

Losses of Heterozygosity on Chromosomes 9p and 17p Are Frequent Events in Barrett’s Metaplasia Not Associated With Dysplasia or Adenocarcinoma

OBJECTIVE:Losses of heterozygosity (LOH) on chromosomes 9p and 17p frequently accompany malignant transformation of Barrett’s esophagus (BE). They have been reported in adenocarcinoma, dysplasia, and adjacent metaplasia of patients with long-segment BE (LSBE). This study aimed to evaluate and compare the frequency of LOH on 9p and 17p in patients with long- and short-segment BE (SSBE) without dysplasia or adenocarcinomaMETHODS:Matched metaplasia and blood DNA were evaluated for LOH on chromosomes 9p and 17p in patients with a previous diagnosis of BE and no dysplasia or cancer.RESULTS:We included 18 patients (12 long-segment BE and six short-segment BE). The overall prevalence of LOH was 61% (10 of 18), with no significant difference between LSBE (58%) and SSBE (50%). The frequencies of LOH on 9p and 17p were similar (35% and 39%, respectively), with 18% of the patients showing losses at both chromosomes.CONCLUSIONS:LOH on 9p and 17p are highly frequent events in BE, even in the absence of dysplasia and adenocarcinoma. The presence of these abnormalities in non-neoplastic epithelium suggests they might be useful markers for risk stratification within endoscopic surveillance programs.

Folate status, DNA methylation and colon cancer risk in inflammatory bowel disease

*Servi¢os de Gastrenterologia e Laborat6rio de Biologia Molecular ~Servi¢o de Patologia Morfol6gica, Instituto Portugu#s de Oncologia Francisco Gentil, Lisboa Portugal, tServi¢o Universit~rio de Medicina Interna e de Gastrenterologia e do Hospital Pulido Valente, Portugal (Correspondence to MC, Servigo de Gastrenterologia, Instituto Portugu#s de Oncologia Francisco Gentil, Rua Prof. Lima Basto, 1093 Lisboa Codex, Portugal)

DNA methylation and subclinical vitamin deficiency of folate, pyridoxal-phosphate and vitamin B12 in chronic alcoholics

Alcohol abuse is known to adversely affect folate, vitamin B12 and pyridoxal-phosphate metabolism, which are required for de novo synthesis of methionine. Methionine is the precursor of S-adenosylmethionine, the principal methylating agent in the organism, including DNA. The objective of this study was to measure DNA methylation in peripheral lymphocytes and the circulating concentrations of these three vitamins in chronic alcoholics. DNA methylation was assessed by measuring DNA methyl accepting capacity in the presence of Ssauthor methylase. Serum pyridoxal-phosphate and red blood cell folate concentrations were significantly depressed in alcoholics (P<0.0001 and P=0.02, respectively). DNA from patients who consumed 3.0 g/ethanol/kg/day or more, incorporated significantly more (3H) methyl groups, which reflects a lower state of intrinsic methylation (P=0.01). DNA hypomethylation was also more pronounced in malnourished alcoholics (P=0.03). We conclude that heavy ethanol consumption is associated with folate and vitamin B6 depletion, which may interfere with DNA methylation status by impairing de novo methionine synthesis.

Adenocarcinoma of the esophagogastric junction: could the characteristics of adjacent intestinal metaplasia help in the understanding of biopathogenesis?

We report a case of early adenocarcinoma arising in foci of intestinal metaplasia (IM) at a normal-appearing gastroesophageal junction (GEJ). The tumor infiltrated the submucosa without nodal involvement (T1N0). Non-neoplastic mucosa adjacent to neoplasia had foci of incomplete IM with a band-like CK20 positivity of the surface epithelium and a diffuse CK7 staining of both superficial and deep glands. There were histological features of reflux esophagitis as well as chronic non-atrophic, Helicobacter pylori-related pangastritis, without IM, at the extensively assessed gastric mucosa. In this case, the CK7/20 pattern of IM adjacent to neoplasia, the demonstration of reflux esophagitis, and the absence of IM in the stomach favor the theory that the pathogenesis of IM and associated adenocarcinoma of the GEJ is related to gastroesophageal reflux rather than H. pylori infection.

Brief Case ReportsColonic cancer in a 34-yr-old woman: should it prompt microsatellite instability studies and mismatch repair gene testing?

It remains debatable whether young patients with colorectal tumors should undergo genetic testing with the aim of identifying new hereditary nonpolyposis colorectal cancer families. We describe a case of a young woman with colon cancer with no clinical criteria of hereditary nonpolyposis colorectal cancer, whose genetic analysis showed that the tumor displayed microsatellite instability, and in whom a truncated protein in hMSH2 gene was found, which was also present in two at-risk relatives.