Carlos Nobre Leitão

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.

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

Short segments of Barrett’s epithelium and intestinal metaplasia in normal appearing oesophagogastric junctions: the same or two different entities?

Background—Endoscopic diagnosis of short segments of Barrett’s epithelium (SSBE) is difficult and its meaning in terms of the presence of specialised columnar epithelium (SCE) has not been prospectively evaluated. Aims—To evaluate the prevalence of SCE in patients with an endoscopic diagnosis of SSBE and in individuals with normal appearing oesophagogastric junctions, and to compare the clinical characteristics of these two groups. Patients—Thirty one patients with an endoscopic diagnosis of short Barrett’s oesophagus, less than 3 cm in length (group A), and 44 consecutive patients with normal appearing oesophagogastric junctions (group B). Methods—Multiple biopsies were performed in suspicious epithelium and at the oesophagogastric junction in groups A and B, respectively. Results—Age and sex distribution were similar in both groups. Reflux symptoms were more frequent in group A (p<0.001), as were endoscopic and histological signs of oesophagitis (p<0.0001 and p=0.001, respectively). SCE was found in 61.3% of group A patients compared with 25% in group B (p<0.002), with men predominating in group A while women were more frequent in group B (p=0.02). The differences in reflux symptoms and endoscopic/histological oesophagitis remained significant. Conclusions—These results show that endoscopic diagnosis of SSBE is associated with a high prevalence of SCE, significantly higher than that found in normal appearing oesophagogastric junctions. Differences between patients with SCE in the two groups suggest they may represent two different entities.

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.

Short chain fatty acids are effective in short-term treatment of chronic radiation proctitis

PURPOSE: Short chain fatty acids are the main energy source of coloncytes and their use may be impaired in chronic radiation proctitis. The aim of the present study was to evaluate the therapeutic effect of short chain fatty acid enemas in patients with chronic radiation proctitis. METHODS: A prospective, randomized, double-blind trial comparing short chain fatty acid enemas with placebo was conducted in 19 patients with chronic radiation proctitis. Short chain fatty acid enemas contained 60 mM sodium acetate, 30 mM sodium propionate, and 40 mM sodium butyrate. The treatment period lasted five weeks and patients were followed up for six months. RESULTS: On admission, both groups were similar regarding all parameters evaluated. After five weeks short chain fatty acid-treated patients showed a significant decrease in the number of days with rectal bleeding from the previous week (4.4±1.8 to 1.4±2.2;P=0.001) and an improvement of endoscopic score (4.8±1.4 to 2.2±1.2;P=0.001). Hemoglobin values were also significantly higher in short chain fatty acid-treated patients (13.1±0.9 g/dlvs. 10.7±2.1 g/dl;P=0.02). Mucosal DNA and protein concentrations decreased in both groups but significantly so only in placebotreated patients (P=0.05). Changes in histologic parameters were not significant in either group. Although short chain fatty acid-treated patients did not get worse in the next six months, placebo-treated ones gradually improved, and at the end of six months, differences between the two groups were no longer observed. CONCLUSIONS: Short chain fatty acid enemas can accelerate the process of healing in chronic radiation proctitis, but treatment has to be continuous if a complete and sustained clinical, endoscopic, and histologic response is to be obtained.

Colorectal cancers show distinct mutation spectra in members of the canonical WNT signaling pathway according to their anatomical location and type of genetic instability.

It is unclear whether the mutation spectra in WNT genes vary among distinct types of colorectal tumors. We have analyzed mutations in specific WNT genes in a cohort of 52 colorectal tumors and performed a meta‐analysis of previous studies. Notably, significant differences were found among the mutation spectra. We have previously shown that in familial adenomatous polyposis, APC somatic mutations are selected to provide the “just‐right” level of WNT signaling for tumor formation. Here, we found that APC mutations encompassing at least two β‐catenin down‐regulating motifs (20 a.a. repeats) are significantly more frequent in microsatellite unstable (MSI‐H) than in microsatellite stable (MSS) tumors where truncations retaining less than two repeats are more frequent (P = 0.0009). Moreover, in cases where both APC hits are detected, selection for mutations retaining a cumulative number of two 20 a.a. repeats became apparent in MSI‐H tumors (P = 0.001). This type of mutations were also more frequent in proximal versus distal colonic tumors, regardless of MSI status (P = 0.0008). Among MSI‐H tumors, CTNNB1 mutations were significantly more frequent in HNPCC than in sporadic lesions (28% versus 6%, P < 10‐6) and were preferentially detected in the proximal colon, independently of MSI status (P = 0.017). In conclusion, the observed spectra of WNT gene mutations in colorectal tumors are likely the result from selection of specific levels of β‐catenin signaling, optimal for tumor formation in the context of specific anatomical locations and forms of genetic instability. We suggest that this may underlie the preferential location of MMR deficient tumors in the proximal colon.

High Prevalence of Human Papillomavirus in Squamous Cell Carcinoma and Matched Normal Esophageal Mucosa Assessment by Polymerase Chain Reaction

Background. Studies using DNA technology have reported the presence of human papillomavirus (HPV) DNA in esophageal carcinomas, suggesting that it could play a role in the pathogenesis of this tumor. In the present study, in addition to DNA from neoplasms, normal mucosa was screened for viral DNA, assuming that this would increase HPV detection substantially.

Recurrent columnar-lined esophageal segments--study of the phenotypic characteristics using intestinal markers.

SUMMARY.  Barretts metaplasia is recognized by specialized columnar epithelium on the distal esophagus. The events involved in the transformation from squamous to Barretts epithelium remain unclear. The present study describes the characteristics observed during the recurrence of four cases of columnar-lined esophagus. Red velvet, gastric-like, esophageal mucosa was observed to develop above the anastomosis during follow-up of four patients submitted to surgery for esophageal and junctional adenocarcinoma. The areas of recurrence were associated with reflux symptoms and inflammation, with ulceration in two cases. Biopsies from the upper gastrointestinal endoscopies were examined histologically using periodic acid–Schiff/Alcian blue to detect acid mucins and a monoclonal antibody raised against the enterocytic enzyme sucrase–isomaltase. In all cases the recurrent columnar-lined segments displayed intestinal features recognized morphologically, histochemically, and/or immunohistochemically. There was no evidence of specialized columnar epithelium in three cases. The fourth patient developed specialized columnar epithelium during the tenth year of surveillance. The presence of AB-positive columnar cells was a frequent and early event. Columnar cells with unequivocal apical sucrase–isomaltase were observed only in association with specialized columnar epithelium. Four conclusions were reached: that the development of columnar-lined mucosa without specialized columnar epithelium may be the earliest event in Barretts metaplasia; that histochemistry is a useful method of recognizing a population with cryptic intestinal features; that acid mucin secretion precedes the production of enterocytic enzymes by columnar cells; and that a cell population with enterocytic differentiation, as assessed by sucrase–isomaltase expression, is associated with the development of specialized columnar epithelium. These characteristics of Barretts esophagus development are clinically relevant as they suggest that patients with columnar-lined esophagus without specialized columnar epithelium may acquire ‘true’ intestinal phenotype, justifying them being considered as high- risk patients.

Flow cytometric DNA ploidy and S-phase fraction correlate with histopathologic indicators of tumor behavior in colorectal carcinoma

BACKGROUND: The clinical behavior of colorectal carcinoma is highly variable without reliable predictive biomarkers. Previous reports have shown that flow cytometric DNA analysis may provide valuable prognostic information in these tumors. PURPOSE AND METHODS: This study evaluates the DNA ploidy and the S-phase fraction (SPF) on frozen samples obtained from 61 patients with colorectal carcinoma by using flow cytometry, and it correlates the data with histopathologic features known to affect disease prognosis. Tumors were classified using the World Health Organizations histologic criteria and were staged according the American Joint Committee on Cancers classification system. Grade of the neoplasm, vascular invasion, and perineural tumor spread were evaluated in every case. RESULTS: Fifty-nine percent of tumors were aneuploid and showed statistically significant higher S-phase values than diploid tumors (22.5vs.11.2 percent;P <0.00001). Mean SPF of the whole series was 17.9 (range, 4.2–44.2) percent. A statistically significant association was found between SPF values and histologic grade (P< 0.0016), nodal status (P<0.0007), distant metastasis(P <0.0001), tumor stage (P<0.0001), venous invasion (P< 0.0002), and lymphatic permeation (P< 0.01) but not with perineural growth and infiltration of the neoplasm through the bowel wall (T). DNA ploidy correlated positively with tumor stage (P<0.03), and the association between aneuploidy and advanced stages of the disease was statistically significant. CONCLUSIONS: These findings showed that flow cytometric DNA ploidy and SPF, evaluated in fresh samples, are potentially useful parameters to estimate colorectal carcinoma biopathology. Aneuploidy and high replicative neoplastic activity correlated with histopathologic features that are commonly associated with the prognosis of colorectal carcinoma, being SPF-related to disease dissemination and, therefore, an indicator of clinical relevance.

Aggressive phenotype of MYH-associated polyposis with jejunal cancer and intra-abdominal desmoid tumor: report of a case.

ABSTRACT: MYH-associated polyposis is an inherited autosomal recessive disease, linked to biallelic germline MYH mutations, which predisposes to the development of multiple colorectal adenomas and cancer. The colonic and extracolonic phenotype of this syndrome is very heterogeneous. We report the case of a young male patient with an aggressive MYH-associated polyposis phenotype. He presented at aged 30 years with more than 100 colonic polyps and 4 colonic adenocarcinomas. At aged 35 years, Spigelman Stage IV duodenal adenomatosis was detected. When he was 39 years old, he developed three synchronous jejunal adenocarcinomas and a mesenteric desmoid tumor. Based on this report, we believe that screening of the entire small bowel should be recommended in MYH-associated polyposis patients, especially in those with duodenal adenomas. Similar to patients with familial adenomatous polyposis, desmoid tumors also may be part of the clinical spectrum of MYH-associated polyposis and may prove to be a significant clinical problem in patients submitted to prophylactic colectomy.