Henrique de Oliveira Costa

Evaluation of the immunoexpression of COX-1, COX-2 and p53 in Crohn's disease

BACKGROUND Crohns disease accompanied by nonspecific or idiopathic ulcerative proctocolitis corresponds to a condition called intestinal inflammatory disease. The immunoexpression of cyclooxygenase 2 (COX-2) in Crohns disease becomes more marked with progression of the disease and the presence of wild-type p53 suppresses the transcription of COX-2. AIMS To investigate the immunoexpression of cyclooxygenase 1 (COX-1), COX-2 and p53 in Crohns ileocolitis and to correlated this expression with clinical and histopathological parameters. METHODS Forty-five cases of Crohns disease, 16 cases of actinic colitis (diseased-control group) and 11 cases without a history of intestinal disease (normal control group) were studied. Hematoxylin-eosin-stained sections were submitted to histopathological analysis and the immunohistochemical expression of COX-1, COX-2 and p53 was evaluated by the streptavidin-biotin-peroxidase method. RESULTS Sixty percent of the Crohns disease patients were women and 40% were men, with 75.5% whites and 25.5% non-whites. The disease involved the terminal ileum in 44.5% of cases, ileum in 33.3%, colon in 20% and duodenum-ileum in 2.2%. A significant association was observed between COX-2 immunoreactivity and age < or =40 years. Histopathological analysis of Crohns disease samples showed mild or moderate crypt distortion (57.8% and 35.6% of cases), atrophy (6.6%), mild, moderate and marked chronic inflammation (46.7%, 26.7% and 20%), acute inflammatory activity (93.3%), ulceration (24.4%), mucin depletion (37.8%), Paneths cells (24.4%), intraepithelial lymphocytes (93.3%), and subepithelial collagen (6.7%). In the CD group, COX-1 immunoreactivity in epithelial and inflammatory cells was observed in 26.7% and 22.2% of cases, respectively. COX-2 immunoreactivity was detected in epithelial cells in 68.9% of cases and in inflammatory cells in 46.7%. A marginal difference in COX-2 reactivity was observed between epithelial and inflammatory cells in association with acute inflammatory activity and increase in intraepithelial lymphocytes. Comparison of the date among the threes groups (Crohns disease, actinic colitis and normal controls) showed a higher proportion of cases presenting COX-2 immunoreactivity in inflammatory cells in the Crohns disease group. No p53 reactivity was observed in all cases. CONCLUSIONS COX-2 immunoexpression is high in Crohns disease, which suggest a possible role of the protein in the pathogenesis of the inflammation. The absence of epithelial dysplasia in all Crohns disease samples was correlated with the lack of expression of p53.

The intrinsic apoptotic signaling pathway in gastric adenocarcinomas of Brazilian patients: Immunoexpression of the Bcl-2 family (Bcl-2, Bcl-x, Bak, Bax, Bad) determined by tissue microarray analysis

This study was undertaken to investigate the immunoexpression of Bcl-2 family proteins (Bcl-2, Bcl-x, Bax, Bak and Bad) and to evaluate the correlation between the immunoexpression of these proteins and that of cleaved caspase 3, Ki-67 and p53. A tissue microarray (TMA) paraffin block was constructed using gastric carcinoma tissue (test group) and adjacent normal gastric mucosa (control group) from 87 patients who had not previously undergone radiotherapy or chemotherapy. Sections from the TMA block (4 µm) were subjected to immunohistochemistry for Bcl-2, Bcl-x, Bax, Bak, Bad, p53, Ki-67 and cleaved caspase-3. The slides were evaluated by the semi-quantitative method, and the scores obtained (intensity vs. percentage of staining) were correlated with one another and with the apoptotic index, cellular proliferation and data regarding patient survival. The studied proteins were present in the tumor tissue and in the normal gastric mucosa, but at different intensities and with differences in the number of positive cells. There was an association between tumor size and p53 expression, and intestinal type adenocarcinoma was positively correlated with the expression of Bax, Bad and Ki-67. The immunoexpression of Bcl-x, Bak, Bad, p53 and Ki-67 showed statistically significant differences between the tumor tissue and the adjacent normal gastric mucosa. There was an association between the expression of Bax, Bak and Bad in the normal gastric mucosa. No correlation between patient survival and the expression of these proteins was observed. Overexpression of the Bcl-x protein in the adenocarcinomas and the difference in Bcl-x expression between the test and the control group may be related to the anti-apoptotic effect of this protein. The reduced expression of Bak and Bad and the increased expression of p53 and Ki-67 in the adenocarcinomas demonstrate the imbalance between apoptosis and cellular proliferation, which results in uncontrolled tumor cell proliferation.

Pseudomyxoma peritonei associated with primary mucinous borderline tumor of the renal pelvicalyceal system

Primary mucinous cystic neoplasms are extraordinarily rare tumors of the kidney. Herein, we present a case of a 52-year-old man who presented with painless hematuria and mucusuria. The nephrectomy showed a markedly enlarged kidney replaced by a large cystic mass filled with mucin and with almost complete destruction of the renal parenchyma. Histologically, the mass was lined primarily by simple mucinous epithelium and showed foci of adenomatous (borderline) change with focal areas exhibiting intraepithelial carcinoma. No invasion was documented after extensive sampling of the tumor. A pathologic diagnosis of intraepithelial carcinoma arising in a mucinous neoplasm of borderline malignancy was rendered. One year later, the patient presented with pseudomyxoma peritonei. This case illustrates that, in a patient with pseudomyxoma peritonei, the absence of an appendiceal, gastrointestinal, or ovarian primary tumor raises the possibility of a primary neoplasm at an unusual site such as the kidney.

Hepatocellular carcinoma in association with bile duct hamartomas: report on 2 cases and review of the literature

Biliary hamartomas are hepatic lesions related to defects in the ductal plate and are part of the spectrum of adult polycystic disease. Microscopically, the lesions are small and consist of irregular bile ducts amidst a fibrous stroma. There are reports on the malignant transformation of biliary hamartomas, particularly on cholangiocarcinoma. Case 1: a 19-year-old woman presented with increased abdominal volume, lumbar pain, jaundice, choluria, cachexia, and progression to hepatic insufficiency. At autopsy, the liver weighed 8850 g and showed a 20.5-cm mass in the right lobe and nodules in the left lobe. Case 2: a 39-year-old man presented with an asymptomatic nodule in the right hepatic lobe. A 6.5-cm liver segment containing a 3.0-cm nodule was excised. Histopathology of both cases revealed hepatocellular carcinoma associated with multiple bile duct hamartomas (von Meyenburg complex). Bile duct hamartoma is generally an incidental finding at laparotomy and autopsy because its course is asymptomatic. The literature has documented its possible progression to malignant neoplasia. However, this relationship has only been established with cholangiocarcinomas. We report herein for the first time on the association of the lesion with hepatocellular carcinoma.

Correlation between the immunohistochemical expressions of MMP-1, MMP-7 and VEGF and prognostic factors in colorectal adenocarcinoma

PURPOSE To analyze the expression of metalloproteinase-1, metalloproteinase-7 and vascular endothelial growth factor (VEGF) in colorectal adenocarcinoma, and to correlate these with the clinical-pathological prognostic factors. METHODS Tumor tissue from 82 patients was fixed in formalin and embedded in paraffin blocks. These samples were analyzed by means of the streptavidin-biotin immunohistochemical method, using the tissue microarray technique. Marker positivity was evaluated using categorical scores that determined cutoff percentages of stained tumor cells. Protein tissue expression was correlated with the variables of degree of cell differentiation, staging, disease-free interval, recurrence, survival and specific mortality. The Fisher exact and Kaplan-Meier tests were used to assess associations between the markers and the study variables. The log-rank and Wilcoxon tests were used to assess the significance of differences between curves of disease-free interval and survival. RESULTS All tumors were positive for metalloproteinase-1; 50 (61%) were positive and 32 (39%) were negative for metalloproteinase-7; and 60 (74.1%) were positive and 21 (25.9%) were negative for VEGF. Correlation of marker expression, both in groups and individually, did not show statistical significance in relation to the degree of cell differentiation, staging, disease-free interval, survival or specific mortality. Recurrence showed a statistically significant correlation with positive expression of the three markers, when analyzed as a group (p = 0.038). CONCLUSION The associated expression of metalloproteinase-1, metalloproteinase-7 and VEGF in colorectal adenocarcinoma is related to the incidence of disease recurrence.

The extrinsic apoptotic signaling pathway in gastric adenocarcinomas assessed by tissue microarray

The purpose of this investigation was to analyze the immunoexpression of FasL, Fas, FADD, cleaved caspase 8, and cleaved caspase 3 in gastric cancer. Formalin-fixed and paraffin-embedded gastric adenocarcinoma tissues from 87 patients, including adjacent normal tissues, were included on tissue microarray by immunohistochemistry. The tumor and the adjacent normal tissues were positive for FasL in 66.7% and 90.6%, for Fas in 52.8% and 52.4%, for FADD in 67.4% and 82.3%, for cleaved caspase 8 in 27.9% and 37.7%, and for cleaved caspase 3 in 33.7% and 8.3%, respectively. FasL and the FADD from tumor were statistically different in relation to the histological type. Cleaved caspase 8 was statistically different in relation to clinical stage (p=0.031). The FADD from normal tissue was statistically different in relation to age (p=0.039), sex (p=0.055), clinical stage (p=0.019), and Fas was different in relation to tumor size (p=0.012). In the tumor, we observed a correlation between FasL and Fas, FasL and FADD, and FasL and cleaved caspase 3. In the adjacent normal tissue, a correlation was observed between FasL and Fas, FasL and FADD. There was no association of another marker with sex, age, clinical stage, and survival. Our results suggest that these proteins mediate the early extrinsic apoptotic pathway in gastric cancer and adjacent normal mucosa. FasL protein binds to Fas protein and subsequently binds to death receptor FADD signaling activation of the extrinsic apoptotic pathway. In this phase, there was inhibition of caspase 8 and, consequently, decreased apoptosis.

Perda de amostras em tissue microarray: comparação entre técnicas com uso de fita adesiva comercial, lâminas silanizadas pelo método tradicional ou por método modificado

INTRODUCTION/OBJECTIVE: The tissue microarray (TMA) technique allows the evaluation of multiple tissue samples in a single block. One of the problems of TMA is the ungluing of tissue sections, thus commercial adhesive tape has been used to reduce this loss. There are no reports comparing the use of the commercial adhesive tape with the use of the modified silane-coated technique. The objective of this study was to compare section loss in slides using commercial adhesive tape, silane-coated microslides with the conventional technique or with the modified technique. MATERIAL AND METHOD: The TMA was constructed with hepatic tissue blocks embedded in paraffin, using a fixed base device, placing 32 cylinders of 2 mm in diameter in duplicate and 2.2 mm apart from each other. Fifteen 4-µm sections were placed on conventional silane-coated microslides at 4% (Group 1), 15 on silane-coated microslides with a modified technique (6% of silane and minimum use of acetone) (Group 2), and 15 on slides using commercial adhesive tape, according to the manufacturers recommendations (Group 3). All microslides were processed by immunohistochemistry for cytokeratin 18, with antigen retrieval accomplished by incubation with citrate buffer pH 6.0 with microwave enhancement. Samples loss was quantified and expressed as: total (> 80%), almost complete (75% to 79%) or partial (50% to 74%). RESULTS: The loss of sections was similar in all three groups (4.9 vs. 3.1 vs. 8.1, respectively) (analysis of variance [ANOVA], p = 0.3654). One slide using commercial adhesive tape showed artifactual ungluing of all sections and another one showed loss of 20 samples on one side of the slide. None of the silane-coated microslides showed such artifact. CONCLUSIONS: Silane-coated microslides show adequate results, require less technical training and reduce the cost of TMA procedure, what justifies their use in research. Moreover, the use of the modified silane-coating technique, with the reduction of acetone volume, lowers the costs and reduces chemical residues.

Loss of samples in the tissue microarray technique: comparison between slides using adhesive tape and silanized slides

Abstract The tissue microarray (TMA) technique allows multiple tissue samples in a single block. Commercial adhesive tape is used to avoid the loss of tissue samples during the immunostaining process. Few reports exist in the literature comparing the use of these adhesive tapes to other adhesive techniques. The objective of this study was to compare loss of sections adhered to slides using commercial adhesive tapes versus using silanized only slides. TMA was constructed with varying tissues using a fixed-base device (Beecher Instruments), placing 108 cylinders of 1 mm diameter in duplicate, spaced 1·2 mm apart. Section of 4 μm were cut from the TMA block and adhered to 30 silanized slides and 30 commercial glass slides using adhesive tape, according to manufacturer’s recommendations. Vimentin immunoexpression was evaluated by immunohistochemistry. Antigenic recovery was realized in citrate buffer using a microwave oven. Cylinder loss in the immunohistochemical process was quantified and expressed as: total (>80%), almost complete (75–79%), or partial (50–74%). The commercial adhesive tape group presented lesser total loss (1·1 versus 6·4%), almost complete loss (2·2 versus 3·5%), and partial loss (2·1 versus 3·8%) than the silanized slide group (ANOVA, P<0·05). The sum of total and almost complete losses in the silanized slide group was 9·9%, greater than the losses in slides using commercial adhesive tapes (3·3%) and less than reported and considered acceptable in the literature (10–30%). In conclusion, the use of silanized only slides presents very satisfactory results, requires less training, and reduces costs significantly, thus justifying their use in research.