Marília Cravo

DNA methylation as an intermediate biomarker in colorectal cancer: modulation by folic acid supplementation.

Several studies have suggested that DNA hypomethylation is an early step in colorectal carcinogenesis. However, it is not clear at which stage in carcinogenesis this hypomethylation occurs, what promotes it, the extent to which it can be reversed and the consequences of such reversal in affecting tumour development. In an attempt to address some of these questions, we studied three groups of subjects with similar age and gender distributions: a group of 12 patients with colorectal carcinomas; a group of 12 patients with colorectal adenomas; and a group of eight healthy control subjects. Two experimental protocols were employed. In the first protocol, intrinsic DNA methylation was evaluated in neoplastic and in normal-appearing rectal mucosa of patients with colonic carcinomas or adenomas, compared with a group of healthy controls. In the second protocol, we examined, in a prospective and controlled fashion, the effect of folic acid supplementation (10 mg/day) on the degree of DNA methylation of rectal mucosa from those same patients after removal of the neoplasms. The degree of intrinsic DNA methylation was assessed on the basis of the capacity of the DNA isolates to serve as methyl acceptors in in vitro incubations that contained DNA methylase and [3H-methyl] S-adenosylmethionine. Intrinsic DNA methylation was significantly lower in carcinomas than in adenomas (P < 0.005). In addition, normal-appearing rectal mucosa from patients with carcinomas was significantly less methylated than in healthy controls (P < 0.005); the mean value found in the latter was also greater than the value observed in patients with adenomas, but not significantly so (P > 0.05). Patients with resected neoplasms who received folk acid supplementation for 6 months had a marked increase in the degree of intrinsic DNA methylation in the rectal mucosa (P < 0.002). Three months after cessation of treatment, DNA methylation decreased substantially (P < 0.05), but remained significantly greater than baseline values (P < 0.02). In contrast, DNA methylation values remained stable throughout the study in placebo-treated patients (P = 0.40). Our study demonstrates that global DNA hypomethylation occurs in normal rectal mucosa from patients with colorectal neoplasms as compared with controls, and that it was significantly reduced with pharmacological doses of folk add. It remains to be determined whether the risk of tumour recurrence is affected as well.

Global DNA hypomethylation occurs in the early stages of intestinal type gastric carcinoma.

BACKGROUND: Global DNA hypomethylation has been found in the premalignant stages of some neoplasms and has been implicated as an important factor for tumour progression. AIMS: The aim of this study was to evaluate whether DNA hypomethylation occurs during the process of gastric carcinogenesis. METHODS: Gastric specimens were obtained from 49 patients and histologically classified as: normal 10, superficial gastritis 14, chronic atrophic gastritis with intestinal metaplasia 15, and intestinal type of gastric carcinoma 10. Global DNA methylation was assessed by incubating DNA with (3H)-S-adenosylmethionine and Sss1 methylase. A higher incorporation of (3H) methyl groups reflects a lower degree of intrinsic methylation. RESULTS: A graduated increase in (3H) methyl group incorporation into DNA was found over the range extending from normal gastric mucosa, to superficial gastritis and to chronic atrophic gastritis (136,556 (24,085) v 235,725 (38,636) v 400,998 (26,747 dpm/micrograms/DNA respectively; p = 0.0002). No further increase was found in specimens from patients with carcinoma. No differences were found between extent of DNA methylation in neoplastic or non-neoplastic mucosa from patients with gastric carcinoma. Hypomethylation of DNA increased substantially with severe atrophy (p = 0.01) or with type III intestinal metaplasia (p = 0.15). CONCLUSIONS: Global DNA hypomethylation occurs in the early stages of gastric carcinogenesis, and it may be a novel biomarker of gastric neoplasia, useful in monitoring the response to chemopreventive agents.

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.

Hyperhomocysteinemia in chronic alcoholism: relations to folic acid and vitamins b6 and b12 status

The objective of this review is to present and discuss the current perspectives of homocysteine and one carbon metabolism in chronic alcoholism. Chronic alcoholics frequently suffer from specific micronutrient deficiencies, including vitamins involved in one carbon metabolism, i.e., folate, vitamin B(6) and vitamin B(12). The possible link between homocysteine and alcoholism stems from the fact that homocysteine metabolism is closely linked to the metabolism of these three vitamins. In fact, homocysteine stands at the intersection of two pathways: methylation and transsulfuration. In methylation, homocysteine acquires a methyl group from N-5-methyltetrahydrofolate in a vitamin B(12) dependent reaction, whereas in the transsulfuration pathway, homocysteine condenses with serine to form cystathionine in an irreversible reaction catalyzed by the pyridoxal-5-phosphate-containing enzyme, cystathionine-beta-synthase. Due to these relationships, nutritional deficiency of one of these vitamins, as a consequence of chronic alcohol intake, could lead to metabolic disruption and potentially to hyperhomocysteinemia. Consistent with an interference of alcohol in these metabolic pathways, a previous study performed in chronic alcoholics in whom hyperhomocysteinemia was observed along with disturbed vitamin status, DNA hypomethylation in peripheral lymphocytes was demonstrated as well. Because all these alterations were observed in the absence of clinically overt disease, one might speculate whether these metabolic abnormalities could be involved in the pathogenesis of organic diseases associated to chronic alcoholism.

Immunohistochemical detection of mismatch repair gene proteins as a useful tool for the identification of colorectal carcinoma with the mutator phenotype

There are two well‐defined pathways for colorectal carcinogenesis, the suppressor and the mutator pathways. The latter is characteristic of hereditary non‐polyposis colorectal cancer (HNPCC), but can also be found in a subset of sporadic colorectal cancer (SCC) possessing distinctive clinical and pathological features, namely early age of onset, location in the right colon, poor differentiation, and a predominant mucinous component. This mutator pathway results from inactivation of mismatch repair (MMR) genes, namely MSH2 and MLH1. The aim of this study was to ascertain if abnormal MMR protein gene expression is a good indicator for identifying tumours from the mutator pathway. Seventy‐six cases of SCC were studied by immunohistochemistry using two monoclonal mouse antibodies that react against MSH2 and MLH1 protein gene products. Immunoexpression was assessed both in tumour and in non‐neoplastic, adjacent and distant mucosa. Microsatellite instability (MSI) was detected by evaluating the length of poly(CA) repeated sequences at seven loci, or by the detection of small unstable alleles in a poly(A) repeat – BAT‐26. Except for BAT‐26, in which only tumour DNA was used, MSI analysis was performed in both tumour and normal mucosal DNA. MSI was classified as high (MSI‐H), low (MSI‐L) or stable (MSS). Abnormal protein expression was found in 9/76 (12%) tumours. Immunohistochemistry for hmlh1 and hmsh2 detected 75% of MSI‐H. There was also a highly significant correlation between the observed immunoexpression and several clinical and pathological characteristics described as the phenotypic profile of the mutator pathway, such as right‐sided location (p=0.003), mucin production (p=0.008), and a peritumoural lymphoid infiltrate (p=0.009). Non‐neoplastic adjacent mucosa showed normal hMSH2 expression in all cases, but in ten cases there was no hMLH1 expression in this transitional mucosa, which is known to display an alterated mucin pattern and a high proliferative rate. These results demonstrated a good correlation between hMLH1 and hMSH2 gene immunoexpression and the clinico‐pathological features characteristic of the mutator phenotype and support the use of this method as a rapid and efficient way to detect tumours arising from this pathway. Copyright

Management of Portuguese Patients with Hyperplastic Polyposis and Screening of At-Risk First-Degree Relatives: A Contribution for Future Guidelines Based on a Clinical Study

BACKGROUND:Hyperplastic polyposis (HP) is a rare condition characterized by the presence of multiple hyperplastic polyps in the colon, which has been associated to an increased risk of colorectal cancer (CRC). Guidelines for management of this disease remain, so far, undefined.AIMS:To evaluate, in symptomatic patients with HP, phenotypic characteristics as well as results of a screening program in their at-risk first-degree relatives.PATIENTSPedigree information and clinical and endoscopic data of 14 patients with HP was studied. SeventeenAND METHODS:at-risk first-degree relatives from six families were also invited to perform screening colonoscopy.RESULTS:Twelve of fourteen (86%) patients had fewer than 100 colorectal polyps. Polyps sizes ranged from 2 to 25 mm and were uniformly distributed through the whole colon in 43% of the patients. Hyperplastic polyps predominated, but 11/14 (79%) patients also harbored serrated as well as classic adenomatous polyps. CRC was present in 6/14 (43%) of the patients at the time of diagnosis. Familial history of CRC/polyps was positive in 6/12 (50%) of cases. Colonoscopy in at-risk relatives disclosed polyps in 10/17 (59%) of cases with at least one additional patient having criteria for HP.CONCLUSIONS:Although small, this series demonstrates that a high level of suspicion is needed to diagnose the HP syndrome, in which serrated adenomas seem to be the hallmark. Although an elevated percentage of CRC was observed in this series of symptomatic patients with HP, prospective studies in asymptomatic individuals are needed to clearly quantify the risk of CRC in patients with HP. Because familial aggregation of HP was present in 3/12 (25%) of kindreds, screening colonoscopy should be offered to first-degree relatives.

Treatment of gastric MALT lymphoma by Helicobacter pylori eradication: a study controlled by endoscopic ultrasonography

Objective:Previous studies have demonstrated a link between Helicobacter pylori infection and low grade B-cell gastric MALT lymphoma. The aim of this study was to evaluate the effect of Helicobacter pylori eradication in 17 patients with low grade B-cell gastric MALT lymphoma stage EI.Methods:For disease staging EUS and CT scan were systematically performed. Eight patients were excluded from the present series because stage EII disease was diagnosed. To demonstrate B-cell monoclonality, immunohistochemistry and polymerase chain reaction were used. H. pylori eradication was performed with triple therapy.Results:H. pylori was eradicated in all patients after first (n = 15) or second line (n = 2) treatment. Histologic regression of lymphoma was observed in all patients after a median period of 2 mo. Disappearance of monoclonality according to polymerase chain reaction took significantly longer (7 mo). At the end of the study, four of 16 patients still exhibited persistent monoclonal bands. Relapse of lymphoma occurred in two patients associated with H. pylori reinfection/recrudescence.Conclusions:Eradication of H. pylori seems to be an effective therapy in patients with stage EI gastric MALT lymphoma, although long-term results are still uncertain. Endoscopic ultrasonography is useful for a more accurate staging of the disease. The clinical significance of detecting monoclonality by polymerase chain reaction remains to be determined.

Global DNA hypomethylation in breast carcinoma

The global DNA methylation of 136 breast lesions (117 primary invasive carcinomas, 5 benign phyllodes tumors, 11 fibroadenomas, and 3 sclerosing adenosis) and their respective adjacent parenchyma was analyzed using an in vitro enzyme assay.

A Comprehensive Approach to Evaluate Nutritional Status in Crohn's Patients in the Era of Biologic Therapy: A Case-Control Study

OBJECTIVES:Evaluate the nutritional status of patients with inactive or mildly active Crohns disease (CD), and identify possible causes for potential deficiencies.METHODS:A total of 78 CD patients and 80 healthy controls were evaluated in respect of nutritional status, dietary intake, and life styles factors.RESULTS:These 73/78 CD patients were on immunomodulating therapies. Mean body mass index (BMI) was lower in patients as compared to controls (P = 0.006) but 32% of CD patients and 33.8% of controls had a BMI > 25, whereas 8% and 23.8% in each group, respectively, were obese (BMI > 30Kg/m2). Fat free mass was significantly decreased in both genders (P < 0.05) whereas fat mass was decreased only in males (P = 0.01). Energy intake was significantly lower in CD patients (P < 0.0001) and we observed significantly lower adjusted mean daily intakes of carbohydrates, monounsaturated fat, fiber, calcium, and vitamins C, D, E, and K (P < 0.05). 29% of patients had excluded grains from their usual diet, 28% milk, 18% vegetables, and 11% fruits. Milk exclusion resulted in a significantly lower consumption of calcium and vitamin K (P < 0.001) and the exclusion of vegetables was associated to a lower consumption of vitamins C and E (P < 0.05). Physical activity was significantly lower in CD patients (P = 0.01) and this lack of physical activity was inversely correlated with increased fat mass percentage (r = −0.315, P = 0.001).CONCLUSIONS:Results showed that the most prevalent form of malnutrition in CD patients was an excess of body weight, which was concomitant with an inadequate dietary intake, namely micronutrients, clearly related to dietary exclusion of certain foods.

BAT-26 identifies sporadic colorectal cancers with mutator phenotype: a correlative study with clinico-pathological features and mutations in mismatch repair genes

Microsatellite instability (MSI) is present in most colorectal cancers (CRC) associated with hereditary nonpolyposis colorectal cancer (HNPCC). MSI testing in so‐called sporadic forms of CRC may become a useful tool in identifying new HNPCC kindred. The aim of this study was to analyse the utility of BAT‐26 as a marker to identify CRCs with MSI and to investigate whether sporadic CRCs with MSI have a phenotypic expression similar to HNPCC cases. MSI was detected using two methods, an association of 7 poly(CA) repeats and a poly(A) repeat alone, BAT‐26, in a series of 62 patients with apparently sporadic forms of CRC. Germ‐line and somatic mutations in the hMSH2, hMLH1, and hMSH6 genes were analysed in patients with MSI+ tumours. Patients with MSI+ at poly(CA) loci and at BAT‐26 were younger (pu2005=u20050·024 and pu2005=u20050·002), had tumours more frequently right sided (pu2005=u20050·017 and pu2005=u20050·0001) and more often mucinous (pu2005=u20050·037 and pu2005=u20050·005, respectively) than patients with MSI negative tumours. Mutation analysis allowed the identification of two patients carrying germ‐line mutations in the hMLH1 gene (both were BAT‐26+) and two other patients who had somatic mutation in the hMSH2 and in hMLH1 genes. In conclusion, the detection of MSI using poly(CA) repeats or BAT‐26 alone allowed the identification of a subset of patients with clinico‐pathological characteristics similar to those associated to HNPCC. BAT‐26 has the advantage of being a simple and less expensive method that might be used as a screening procedure before mutation analysis. Copyright