Mariana Palma

Reprimo as a Potential Biomarker for Early Detection in Gastric Cancer

Purpose: Gastric cancer is a curable disease if diagnosed at early stage. However, most cases are diagnosed at advanced stage because of the lack of screening programs. Therefore, the identification of plasma biomarkers for early detection is necessary. Experimental Design: To search for these biomarkers, we evaluated the DNA methylation patterns of 24 genes by Methylation-specific PCR in primary tissues from 32 retrospectively collected gastric cancer cases (testing group). Correlation between methylation and gene expression was evaluated in the MKN-45 cell line after treatment with 5-aza-2′-deoxycytidine. The most frequently hypermethylated genes were next evaluated in primary tissues and plasma samples from 43 prospectively collected gastric cancer cases as well as plasma samples from 31 asymptomatic age- and gender-matched controls (validation group). Results: In the testing group, 11 genes were hypermethylated in at least 50% of cases (APC, SHP1, E-cadherin, ER, Reprimo, SEMA3B, 3OST2, p14, p15, DAPK, and p16). Eight genes (BRCA1, p73, RARβ, hMLH1, RIZI, RUNX3, MGMT, and TIMP3) were statistically associated with a particular variant of gastric cancer, the signet-ring cell type (P = 0.03). Seven genes (APC, SHP1, E-cadherin, ER, Reprimo, SEMA3B, and 3OST2) were next evaluated in the validation group. We confirm the high frequency of methylation in primary tumors for all seven genes. However, only APC and Reprimo were frequently methylated in pair plasma samples. In asymptomatic controls, only Reprimo was infrequently methylated in comparison with plasma from gastric cancer cases (P < 0.001). Conclusion: Our results identified specific methylation profile associated to signet-ring cell-type histology and aberrant hypermethylation of Reprimo as a potential biomarker for early detection of gastric cancer.

Epstein-Barr virus in gastric carcinoma is associated with location in the cardia and with a diffuse histology: A study in one area of Chile

Epstein‐Barr virus (EBV) has been associated with the most common form of stomach neoplasms, the gastric carcinoma (GC). The presence of EBV‐encoded small RNAtype‐1 (EBER‐1), a marker for EBV infection was analyzed by in situ hybridization (ISH) in 185 formalin‐fixed and paraffin‐embedded cases of GC from a high risk region. We found 31 (16.8%) EBV‐positive cases with no relationship to age. Although male predominance (19% in males and 12.5% in females) was observed, the gender difference did not achieve statistical significance. Odds ratio (OR) for cardia location was 5.4 (95% CI 1.7–17.3) when antrum was used as referent category and the effects of gender and age were taken into account. The proportion of EBV‐positive cases in diffuse histology was higher than intestinal type (OR = 4.8, 95% CI = 2.0–11.1). Our findings are contrary to a previously accepted hypothesis, that high‐risk countries for GC have low rates of EBV‐associated GC. In addition, our findings regarding location, histology and weak male predominance are different from what has been described in Asian and European countries, but similar to those described in Mexico and Mexican descendants living in the U.S. suggesting unique characteristics of EBV‐associated GC in Latin‐America.

Association of a distinctive strain of Epstein‐Barr virus with gastric cancer

Epstein‐Barr virus (EBV) has been linked to gastric carcinoma (GC) with worldwide geographical variations attributable to types and variants of EBV. Here, we compare EBV strains between EBVaGC and healthy donors in Latin America, a high frequency area for EBVaGC. Tumor samples from 73 EBVaGC cases and throat washings from 329 healthy adults were examined for types 1 and 2 EBV and polymorphism at BamHI‐F and BamHI‐W1/I1 boundary regions and XhoI restriction site in LMP1 gene. Type 1 and prototype F of BamHI‐ F polymorphism accounted 59 (81%) and 69 (95%) of EBVaGC cases and 257 (78%) and 267 (81%) of healthy donors, respectively. Types I and “i” of BamHI W1/I1 polymorphism accounted 2 (3%) and 62 (85%) of EBVaGC and 85 (26%) and 170 (52%) of healthy donors, respectively (p<0.001). XhoI+ and − polymorphism accounted 60 (82%) and 4 (5%) of EBVaGC and 142 (43%) and 92 (28%) of healthy donors, respectively (p<0.001). Cosegregation analysis demonstrated that most of the 62 type “i” EBVaGC cases harbor XhoI+ strain (81%). However, among 143 type “i” healthy adults, both XhoI polymorphism were present in relatively similar frequencies (XhoI+ 58% and XhoI− 42%) (OR 9.0; 95% CI 1.2–69). Our findings are against to the proposed hypothesis that EBV strains are geographically but not disease‐restricted. We conclude that most of the EBVaGC cases harbor a distinctive EBV strain (type “i”/XhoI +), but in healthy donors, this strain was as common as other strains. This finding is contrary to the proposed hypothesis that EBV strains are geographically but not disease‐restricted and identified a healthy population group that share the same strain that predominate in EBVaGC cases.

DNA methylation profile in diffuse type gastric cancer: evidence for hypermethylation of the BRCA1 promoter region in early-onset gastric carcinogenesis

Diffuse type gastric carcinoma is the most aggressive type of gastric cancer. This type of tumor is not preceded by precancerous changes and is associated with early-onset and hereditary syndromes. To test the hypothesis that DNA methylation profile would be useful for molecular classification of the diffuse type gastric carcinoma, DNA methylation patterns of the CpG Island of 17 genes were studied in 104 cases and 47 normal adjacent gastric mucosa by Methylation-specific PCR, Immunohistochemistry and Hierarchical clustering analysis. The most frequent methylated genes were FHIT, E-cadherin, BRCA1 and APC (>50%), followed by p14, p16, p15, p73, MGMT and SEMA3B (20-49%). Hierarchical clustering analysis reveals four groups with different clinical features. The first was characterized by hypermethylation of BRCA1 and younger age (<45 years old), and the second by hypermethylation of p14 and p16 genes, male predominance and Epstein-Barr virus infection. The third group was characterized by hypermethylation of FHIT and antrum located tumors and the fourth was not associated with any clinical variables. In normal adjacent mucosa only the p73 gene was significantly less methylated in comparison to tumor mucosa. DNA methylation identified subgroups of diffuse type gastric cancer. Hypermethylation of BRCA1 associated with young age suggests a role in early-onset gastric carcinoma.

Helicobacter pylori: análisis de cagA y genotipificación de vacA en Chile. Detección de una cepa s2/m1

Background: The genes cagA and vacA encode H pylori virulence factors. Aim: To genotype these genes in H pylori strains isolated from patients with upper gastrointestinal symptoms. Material and methods: We studied 50 patients who underwent an upper gastrointestinal endoscopy, with positive culture for H pylori. Detection of cagA and vacA gerotyping was done using polymerase chain reactions. Results: The gene cagA was detected in 19 samples (38%). Signal sequences s1 and s2 of vacA gene were detected in 16 samples each (32%). There was simultaneous amplification of s1 and s2 in 6 samples and they were not detected in 9 samples. The middle region of vacA was m1 in 9 samples, m2 in 29 samples and there was simultaneous amplification of m1 and m2 in 12 samples. In 16 samples (32%), more than one type of signal sequence or medial region was detected. Of those patients in whom vacA was the only genotype detected, 15 were s2/m2, 7 were s1/m1, 4 were s1/m2 and 1 was s2/m1. Conclusions: In these patients, the infection with cagA- H pylori strains, predominates, the prevalence of infection with s1 or s2 strains is similar and the predominant medial region is m2 (Rev Med Chile 2001; 129: 1147-53

Splanchnic and systemic hemodynamics in early abstinence and after ethanol administration in non-cirrhotic alcoholic patients.

Thirteen asymptomatic chronic alcoholic patients were studied to investigate the early stages of portal hypertension in alcoholic liver disease and the effects of withdrawal and ethanol on hepatic function and hemodynamic variables. None of the patients presented clinical signs of decompensated liver disease, and their liver biopsies showed normal liver or moderate alterations only. In basal conditions and after an intravenous ethanol infusion (1 g/kg body weight), hepatic venous pressure gradient and hepatic blood flow using indocyanine green were measured through hepatic vein catheterization. Hepatic sinusoidal vascular resistance and indocyanine green intrinsic clearance were also calculated. Portal blood flow measurements were obtained by Doppler ultrasound. No correlation was observed between hepatic venous pressure gradient and histologic features, (steatosis, necrosis, fibrosis, inflammation and hepatocyte surface area). In basal conditions, portal hypertension was not found in any case. After ethanol, portal pressure increased significantly (p < 0.001); in four cases it rose to or above 5 mmHg. Portal blood flow, hepatic blood flow and hepatic vascular resistance also increased significantly. Intrinsic indocyanine green clearance decreased slightly but significantly. No significant correlations were found between portal pressure, hepatic resistance and the histologic parameters. It was concluded that alcoholic patients, without clinical or laboratory evidence of liver failure and with minimal or moderate histologic alterations, have normal portal pressures. After an intravenous ethanol load, however, four out of 13 patients (31%) reached levels of 5 mmHg or more, irrespective of their liver histology.

Identificación de asociaciones clínico-patológicas e hipermetilación de genes supresores de tumores en cáncer gástrico difuso a través de análisis de Hierarchical clustering

Eighty three patients with diffuse gastric cancer with information about survival andinfection with Epstein Barr virus, were studied. DNA was extracted from pathological slides and themethylation status of genes p14, p15, p16, APC, p73, FHIT, E-caderin, SEMA3B, BRCA-1, MINT-2 yMGMT, was studied using sodium bisulphite modification and polymerase chain reaction. Resultswere grouped according to the methylation index or Hierarchical clustering (TIGR MultiExperimentViewer).

Características clínico-moleculares del cáncer gástrico cardial asociado al virus Epstein Barr

BACKGROUND Mortality caused by cardial gastric cancer in Chile, is increasing. Previously we demonstrated an association between Epstein Barr virus and this specific location of gastric cancer. AIM To perform a clinical and molecular characterization of cardial gastric cancer associated to Epstein Barr virus. MATERIAL AND METHODS Epstein Barr virus was identified in 93 cardial gastric tumors, by in situ hybridization. Clinical and pathological features, survival and expression of p53 and c-erbB2 were compared between tumors with or without the presence of the virus. RESULTS Twenty two (23.6%) tumors expressed Epstein Barr virus. No difference in sex or age of patients with tumors positive or negative for the virus was observed. Epstein Barr positive tumors had a tendency to have a higher frequency of Bormann III endoscopic appearance and a lower frequency of p53 accumulation (p=0.06). Five years survival was 67% and 42% of tumors positive and negative for the presence of the virus, respectively (p=0.57). CONCLUSIONS Our results, although not significant, show a tendency towards unique characteristics of cardial gastric tumors associated to Epstein Barr.

Efectos de la infusión aguda de octreotido sobre la función renal en pacientes con cirrosis hepática e hipertensión portal

BACKGROUND Octreotide is used in the treatment of acute variceal bleeding, based on its inhibitory effects of post-prandial splanchnic hyperemia and splanchnic venoconstriction. The consequences of these haemodynamic changes on renal circulation are not well known in cirrhotic patients. AIM To evaluate the effects of acute octreotide administration on several parameters of renal function, including free water clearance, in patients with cirrhosis with or without ascites. PATIENTS AND METHODS Twenty cirrhotic patients, Child-Pugh A orB, with or without ascites, with esophageal varices, normal renal function and free of medications (vasoactive drugs or diuretics) were assigned to 2 different protocols. Protocol 1: 10 patients were randomized to receive octreotide or placebo, as a bolus followed by a continuous infusion. Glomerular filtration rate (GFR) and renal plasma flow (PRF) were measured, in basal conditions and during the drug or placebo administration. Protocol 2: 10 additional patients were randomized in the same way and free water clearance and urinary sodium excretion were again measured in the basal period and during the drug or placebo infusion. RESULTS After octreotide or placebo administration no significant changes were observed neither in GFR nor in PRF. The free water clearance decreased significantly during octreotide administration (3.12 ml/min+/-1.04 SE vs 0.88+/-0.39, p<.03). In both protocols no changes in mean arterial pressure were observed. CONCLUSIONS Acute administration of octreotide to cirrhotic patients with portal hypertension, with or without ascites, did not produce any change in glomerular filtration rate or in estimated renal plasm blood flow. However the free water clearance decreased significantly. This effect, under chronic administration, could be clinically important and deserves further studies.