Expression of Mcl-1 and Ki-67 in Papillary Thyroid Carcinomas.

UNLABELLED: Studying molecules that are differentially expressed in cancers as well as benign and normal tissues is crucial for identifying novel biomarkers for cancer immunotherapy. This study aimed to investigate the clinical utility of the immunochemical expression of the proliferative cell marker Ki-67 and the apoptotic blocker Mcl-1 in papillary thyroid carcinoma (PTC). METHODS: We built a tissue microarray with 282 thyroid specimens. There were 59 PTCs including 35 classic (CPTC), 3 tall cell (TCPTC) and 21 follicular variants (FVPTC); 79 benign thyroid diseases (22 follicular adenomas; 57 adenomatoid hyperplasia); 33 Hashimoto's thyroiditis (HT) specimens; and 111 normal thyroid tissues. Clinical history and ultrasound data were retrospectively obtained by chart review. RESULTS: Mcl-1 overexpression was evident in 66.7% of the PTC tissues compared to 32% of the benign thyroid diseases. Mcl-1 strong staining distinguished benign from malignant thyroid lesions (sensitivity=61.3%; specificity=72.8%; negative predictive value, NPV=68%; positive predictive value, PPV=66.7% and 67.5% accuracy). Positive nuclear Ki-67 staining was observed in 34% of PTCs vs. 19% of thyroid adenomas (P=0.031). Strong Mcl-1 and Ki-67 co-expression was identified in 57.5% of PTCs with a higher PPV (75.8%). Mcl-1 and Ki-67 expression was not associated with any clinicopathological feature of malignancy. No deaths occurred during the follow-up. CONCLUSIONS: Mcl-1 immunochemical overexpression allowed differentiating low-risk PTC from the benign thyroid lesions. We suggest that Mcl-1 expression may help differentiate follicular patterned thyroid lesions. The influence of the Mcl-1 expression on several features of tumor aggressiveness has to be studied in large series of high-risk thyroid carcinomas.

Stromal cells play a role in cervical cancer progression mediated by MMP-2 protein.

Metalloproteinases, especially metalloproteinase-2 (MMP-2), are known for their role in the degradation of the extracellular matrix. Nevertheless, a thorough understanding of MMP-2 expression in neoplastic lesions of the uterine cervix has yet to be accomplished. This study aimed to analyze the MMP-2 expression in cervical intraepithelial neoplasia III (CIN3) and in cervical squamous cell carcinoma, in tumor cells and adjacent stromal cells. MMP-2 expression was assessed by an immunohistochemical technique. MMP-2 expression was greater in the stromal cells of invasive carcinomas than in CIN3 (p < 0.0001). MMP-2 expression in stromal cells correlates with the clinical stage, gradually increasing as the tumor progresses (p = 0.04). This study corroborates that stromal cells play an important role in tumor invasion and progression, mediated by the progressive enhancement of MMP-2 expression from CIN3 to advanced invasive tumor. The intense MMP-2 expression most probably is associated with poor tumor prognosis.

Cell proliferation activity unrelated to COX-2 expression in ovarian tumors.

The objective of this study was to assess the expression of Cyclooxygenase-2 (COX-2) and cell proliferation activity (Ki67 expression) in benign, borderline, and malignant serous and mucinous ovarian tumors. Expression of COX-2 and Ki67 proteins were evaluated by immunohistochemistry, in paraffin-embedded sections of ovarian epithelial tumors. The study included 113 serous (67 benign, 15 borderline, and 31 malignant) and 85 mucinous (48 benign, 28 borderline, and 9 malignant) tumors, removed from women who underwent laparotomy between January 1997 and December 2003. From benign to malignant tumors, there was a progressive positive trend in COX-2 expression in both serous and mucinous tumors, more evident in mucinous ones (P < 0.001). Comparing histologic types, COX-2 expression was more prominent in serous than in mucinous benign tumors (P < 0.01), but this difference was not significant in the borderline (P= 0.11) or malignant categories (P= 0.71). There was a progressive Ki67 positivity in line with the tumor histologic gradient for both serous (P < 0.01) and mucinous lesions (P < 0.01), but this increasing expression did not correlate with COX-2 expression in the present series (P= 0.78). There was a higher COX-2 expression in serous ovarian adenomas than in mucinous ones. COX-2 positivity increases in line with the morphologic gradient, from benign to malignant in both histologic types, but it was more prominent in mucinous lesions, pointing to different oncogenic pathways related to different histologic types. A correlation between the expression of COX-2 and Ki67 was not found, suggesting that COX-2 may be required for carcinogenesis, but this pathway is not responsible for cell proliferation in ovarian tumors.

Comparison of immunoexpression of 2 antibodies for estrogen receptors (1D5 and 6F11) in breast carcinomas using different antigen retrieval and detection methods.

The importance of in situ immunodetection of hormone receptors for therapy planning and prognostic evaluation in patients with breast carcinoma is well established. Sensitive detection methods are of utmost importance, especially in poorly fixed tissues, which are not uncommon in routine pathologic practice. The purpose of the present study is to compare immunoexpression of estrogen receptors in 20 cases of invasive ductal carcinoma using two antibodies, 1D5 and 6F11, and to verify the effect of different antigen retrieval solutions and detection systems. Immunoperoxidase was performed on paraffin sections using 1D5 and 6F11 as primary antibodies. Heat-induced antigen retrieval was performed using citrate buffer (pH 6.0) or Tris-EDTA buffer (pH 8.9). Detection was achieved using the following systems: EnVision, EnVision Plus, and labeled streptavidin-biotin peroxidase complex. Reaction was semiquantified from 0 to 4. There were no differences between the two markers, 1D5 and 6F11, except when 6F11 was used with EnVision and citrate buffer, in which case weaker reactivity was observed. Only in this combination (6F11/EnVision) was EDTA buffer significantly better than citrate. Labeled streptavidin-biotin peroxidase complex presented the best results, followed by EnVision Plus.

Immunohistochemical study of the pancreatic basement membrane in non obese diabetic mice (NOD) with spontaneous autoimmune insulitis.

The role of the basement membrane as an antigenic structure in autoimmune diseases is controversial. To determine possible structural changes in the endocrine pancreatic basement membrane (PBM) in autoimmune diabetes, we studied the expression of laminin in the islets of 42 NOD mice, aged between 4 to 42 weeks, as an animal model of spontaneous diabetes. Insular lymphocytic inflammatory infiltration of variable intensity was present in 24 of these mice. An immunohistochemical staining using the streptavidin-biotin-peroxidase technique was performed on paraffin-embedded tissue sections, with a polyclonal antilaminin antibody. Staining for laminin was restricted to the basement membrane. In islets with no inflammatory infiltration, laminin was observed as a thin, continuous and uniform brown layer, covering the pericapsular basement membrane of the islets and their capillaries. The continuity of the PBM was lost in the islets with insulitis and the immunostaining showed clearcut interruption and destruction, particularly when the islets were in contact with inflammatory infiltrate. Our findings suggest that the loss of integrity of the PBM in islets with inflammatory infiltrate could facilitate antigenic exposure contributing towards the start o f autoimmune DM in NODmice.

The EBV action in tonsils and adenoids.

The bacteria involved in tonsil disease have been well studied, but we cannot say the same for the viruses. The method to detect virus make this approach difficult to study. Epstein-Barr Virus (EBV) infection usually occurs in early childhood and can persist in palatine and pharyngeal tonsil lymphocytes. EBV has been closely associated with the undifferentiated form of nasopharyngeal carcinoma (NPC) in its effect. Nevertheless, the presence of EBV in non-neoplastic lymphoid tissue of the nasopharynx and tonsil has rarely been investigated. Our objective was to study the frequency of EBV in tonsils and adenoids and to define the correlation between EBV and adenoid hyperplasia. In this study, we looked for EBV in adenoid and tonsil tissue of 165 patients (2 and 15 years old ) by in situ hybridization (ISH) for EBER 1/2 RNA. Resection of the adenoids was done for relief of upper respiratory tract obstruction, and the tonsils were resected because of recurrent tonsillitis and/or hyperplasia with upper airway obstruction. We divided the adenoid samples in two groups: one group 12-24 months old (average 18 months old) and the second group, 25 months to 15 years old. Tonsils were obtained from 85 patients, 3-13 years old (mean age 5.6 years) who underwent surgery due to recurrent tonsillitis or hyperplasia. EBV was demonstrated in lymphoid cells of 11 (34.3%) out of 32 adenoids for the first group and 36 (72%) out of 48 children of the second group. EBV was found in the respiratory epithelial cells of adenoid in one case. Children under 24 months of age can be infected by EBV, and this virus might be responsible for obstructive hyperplasia. Tonsils are less affected by EBV than the adenoids, suggesting that the EBV is more attracted to the adenoid tissue than the tonsillar tissue.

P53 and Bcl-2 as prognostic predictors in epithelial ovarian cancer.

The study was designed to evaluate the prognostic importance of clinical and pathologic variables with p53 and Bcl-2 in epithelial ovarian cancer using multivariate analysis. Tumor tissues from 90 patients were analyzed immunohistochemically for p53 and Bcl-2 expression. Hazard ratios were calculated in univariate and multivariate survival analyses. Forty-two (47%) were considered positive for p53 expression and 18 (20%) were positive for Bcl-2. Positive expression for p53 was less frequent in patients in FIGO stage I (22%). Positive staining for Bcl-2 correlated significantly with the histologic type (P < 0.01). No direct correlations could be demonstrated between p53 and Bcl-2 expression and age or histologic grade. In univariate analysis, p53 and Bcl-2 expression were not significantly correlated with overall survival, disease-free survival, or progression time. FIGO stage III and IV and residual disease > or =2 cm3 after first surgery were significantly correlated with poor outcome in univariate analysis. FIGO stage retained their independent prognostic value in multivariate analysis. Neither p53 nor Bcl-2 had any significant influence on outcome in multivariate survival analysis. FIGO stage proved to be the only significant independent prognostic factor in epithelial ovarian cancer, although residual disease remains correlated with disease-free survival.

P53 expression as a predictor of recurrence in cervical squamous cell carcinoma.

P53 protein function is frequently down-regulated in cervical cancer by complexing with human papillomavirus (HPV) E6 protein, leading to degradation of p53, genomic instability, and mutations. Results are controversial, however, on the prognostic value of p53 protein expression in cervical cancer. In this study, a cohort of 220 Brazilian women with FIGO stage IB-III cervical squamous cell carcinoma (SCC), followed for 5 years, was analyzed for p53 protein expression using immunohistochemistry. The disease-free survival (DFS) and relapse rate were analyzed using univariate (Kaplan-Meier) and multivariable (Cox's proportional hazards model) survival analyses. P53 protein expression was detected in 35% of the patients, including 21% in stage I, 28% in stage II and 51% in stage III of disease. Of 220 women, only 116 completed one of the treatment options standardized by FIGO within 120 days. There was a higher risk of relapse in stage II and III disease, that was not modified by p53 positivity; HR 3.0 (1.3-6.5) to stage II and HR 4.0 (1.9-8.5) to stage III. The multivariate analysis evidenced that p53 expression is not an independent factor exceeding the power of FIGO stage as the single most important determinant of the hazards for disease relapse.

c-Myc protein expression is not an independent prognostic predictor in cervical squamous cell carcinoma.

The c-myc protein is known to regulate the cell cycle, and its down-regulation can lead to cell death by apoptosis. The role of c-myc protein as an independent prognostic determinant in cervical cancer is controversial. In the present study, a cohort of 220 Brazilian women (mean age 53.4 years) with FIGO stage I, II and III (21, 28 and 51%, respectively) cervical squamous cell carcinomas was analyzed for c-myc protein expression using immunohistochemistry. The disease-free survival and relapse-rate were analyzed using univariate (Kaplan-Meier) survival analysis for 116 women who completed the standard FIGO treatment and were followed up for 5 years. Positive c-myc staining was detected in 40% of carcinomas, 29% being grade 1, 9% grade 2, and 2% grade 3. The distribution of positive c-myc according to FIGO stage was 19% (17 women) in stage I, 33% (29) in stage II, and 48% (43) in stage III of disease. During the 60-month follow-up, disease-free survival in univariate (Kaplan-Meier) survival analysis (116 women) was lower for women with c-myc-positive tumors, i.e., 60.5, 47.5 and 36.6% at 12, 36, and 60 months, respectively (not significant). The present data suggest that immunohistochemical demonstration of c-myc does not possess any prognostic value independent of FIGO stage, and as such is unlikely to be a useful prognostic marker in cervical squamous cell carcinoma.

C-Myc protein expression is not an independent prognostic predictor in cervical squamous cell carcinoma

The c-myc protein is known to regulate the cell cycle, and its down-regulation can lead to cell death by apoptosis. The role of c-myc protein as an independent prognostic determinant in cervical cancer is controversial. In the present study, a cohort of 220 Brazilian women (mean age 53.4 years) with FIGO stage I, II and III (21, 28 and 51 percent, respectively) cervical squamous cell carcinomas was analyzed for c-myc protein expression using immunohistochemistry. The disease-free survival and relapse-rate were analyzed using univariate (Kaplan-Meier) survival analysis for 116 women who completed the standard FIGO treatment and were followed up for 5 years. Positive c-myc staining was detected in 40 percent of carcinomas, 29 percent being grade 1, 9 percent grade 2, and 2 percent grade 3. The distribution of positive c-myc according to FIGO stage was 19 percent (17 women) in stage I, 33 percent (29) in stage II, and 48 percent (43) in stage III of disease. During the 60-month follow-up, disease-free survival in univariate (Kaplan-Meier) survival analysis (116 women) was lower for women with c-myc-positive tumors, i.e., 60.5, 47.5 and 36.6 percent at 12, 36, and 60 months, respectively (not significant). The present data suggest that immunohistochemical demonstration of c-myc does not possess any prognostic value independent of FIGO stage, and as such is unlikely to be a useful prognostic marker in cervical squamous cell carcinoma

Antithyroid drugs inhibit in vivo HLA-DR expression in thyroid follicular cells in Graves' disease.

Antithyroid drugs have been reported to reduce the expression of HLA-DR in thyrocytes in Graves' disease, but only circumstantial evidence has been provided about their in vivo immunologic effects. This present study was designed to examine the in vivo immunologic effect of antithyroid drugs on thyroid follicular cells. The study was conducted on 25 patients who had Graves' disease in remission or in activity and who were or were not receiving treatment (7 in overt thyrotoxicosis, 6 patients in remission, and 12 patients under medication). HLA-DR expression in thyroid biopsies was verified by immunohistochemistry. The follicular cells of all patients in overt thyrotoxicosis expressed HLA-DR whereas those of patients in remission were negative for HLA-DR. HLA-DR was also not expressed in all patients under medication, but this did not correlate with the clinical evolution after thyroid drug withdrawal. In conclusion, antithyroid drugs inhibit follicular cell HLA-DR expression in Graves' disease, when thyrotoxicosis is controlled. This suggests that additional mechanisms not involving HLA-DR play a role in thyroid autoimmune disease.

Detection of Epstein-Barr virus in tonsillar tissue of children and the relationship with recurrent tonsillitis.

Recurrent tonsillitis has been the subject of much investigation. Events considered to predispose to or cause recurrent tonsillitis (RT) include the misuse of antibiotic therapy in acute bouts, alterations in the microflora, structural changes in crypt epithelium and certain viral infections. Epstein-Barr Virus (EBV) infection usually occurs in early childhood and can persist in palatine tonsil lymphocytes to induce tonsillitis at a later date. We have examined the presence of EBV in palatine tonsils in order to assess the relationship between this virus and recurrent acute tonsillitis. Tonsils were obtained from 85 patients, 2--14 years old (mean 5.6 years old) who underwent tonsils and adenoid (T&A) removal because of recurrent tonsillitis (RT) or T&A hypertrophy (TH). Tissues specimens were processed for non-isotopic in situ hybridization (ISH) using EBER 1/2 oligonucleotides (EBER RNA). The indications for surgery were RT in 42 patients and TH in 43 patients. In 25 out of 85 cases (29.4%) a positive EBER RNA reaction (15 RT and 33 TH) was found. The chi(2)-test showed no statistically significant difference in frequency of positive results between RT and TH group. We conclude that tonsils of children can be colonized by EBV and that the virus may be implicated in RT and TH.

High risk HPV and p53 protein expression in cervical intraepithelial neoplasia.

INTRODUCTION: Cervical intraepithelial lesions due to HPV infection are common in Brazil. An understanding of the mechanisms of the interaction between HPV and host factors is still incomplete. In spite of the high incidence of cervical cancer in Brazil, such studies with Brazilian patients are scarce. The purpose of this study was to correlate the presence of high-risk types of HPV and expression of p53 protein, grade of cervical lesion, age, high-risk sexual behaviors and smoking. It was also intended to establish whether p53 expression might be useful as a marker for CIN progression. METHODS: HPV detection was performed on paraffin sections using biotin-labeled probes by in situ hybridization. p53 protein expression was evaluated by immunohistochemistry. RESULTS: Seventy-eight patients with cervical dysplasia were included in the study. CIN 1 was diagnosed in 38 cases, and CIN 2+3 in 40 cases. High-risk HPV was detected in 42 patients. No correlation was found between the grade of cervical lesion or the presence of HPV and smoking, and high-risk sexual behavior. Expression of p53 was significantly higher in CIN 1, as compared with CIN 2+3, but did not correlate with HPV status. CONCLUSION: Higher expression of p53 protein in early lesions supports the hypothesis of a partially protective role of the wild-type p53 in early stages of cervical lesions.

Autoimmune and non-autoimmune thyroid diseases have different patterns of cellular HLA class II expression.

CONTEXT: Surface HLA-DR antigen is usually only expressed by antigen-presenting cells (APC). In autoimmune thyroid disease, follicle cells function as APC, thus expressing HLA-DR. However, non-autoimmune thyroid diseases may also express surface class II antigens. OBJECTIVE: To evaluate the presence and pattern of HLA class II expression in autoimmune and non-autoimmune thyroid disorders. DESIGN: Retrospective: histopathological and immunohistochemical analysis. LOCATION: Referral center, university hospital. SAMPLE: Ten histologically normal thyroids, 11 Graves' disease, 7 Hashimoto's thyroiditis, 10 atoxic multinodular goiter and 3 toxic adenomas were analyzed by immunohistochemistry, using a monoclonal antibody anti-HLA-DR. MAIN MEASUREMENTS: The presence of these antigens in thyroid follicular cells and their relation to inflammatory infiltrate was evaluated. The pattern of HLA-DR expression in thyroid follicular cells was analyzed: membrane, cytoplasmic or both. RESULTS: Although HLA-DR antigens were sparsely present in one of the 8 normal thyroids, in 6 of the 9 atoxic multinodular goiter and in 2 of the 3 toxic adenomas a net positivity could be seen in large areas. In all 5 Hashimoto's thyroiditis and in 7 of the 10 Graves' disease cases. This expression occurred in follicle cells either in contact with inflammatory cells or not. In non-autoimmune thyroid disease, HLA-DR positivity was essentially cytoplasmic, whereas in Graves' disease and Hashimoto thyroiditis it was mainly in cell membranes. CONCLUSIONS: It is suggested that the HLA class II expression on the surface of follicle cells could be related to auto-antigen presentation to the immune system by these cells, leading to inflammation.

Evidence of integrity of the follicular basement membrane in Hashimoto's thyroiditis.

The basement membrane as an antigenic structure in autoimmune diseases has been a matter of controversy. The purpose of our study was to determine possible structural changes in the follicular basement membrane (FBM) in thyroid autoimmune and non-autoimmune diseases. Immunohistochemical staining for collagen IV and laminin showed that the continuity of the FBM was preserved in toxic adenoma (three cases), atoxic multinodular goiter (nine cases) as well as in autoimmune disease. Integrity of the FBM was observed in all 11 cases of Graves' disease and Hashimoto's thyroiditis (seven cases) studied. In some instances, the FBM was thinned in areas of contact with inflammatory infiltrate. We conclude that the auto-antibodies, and possibly other factors present in autoimmune thyroid diseases, do not significantly alter the integrity of the FBM.

Alteration of the follicular basement membrane in non-obese diabetic mice with spontaneous autoimmune thyroiditis.

The follicular basement membrane (FBM) prevents thyroglobulin from escaping to the peri-follicular space, where it can act as an antigen to induce experimental thyroiditis. Laminin, a component of the FBM, is responsible for directing cell migration and stimulates greater adhesion of activated T lymphocytes. Our purpose was to study the expression of laminin in the thyroid of NOD mice, which have a propensity for autoimmune diseases, including thyroiditis. Thirty NOD mice between 3 and 42 weeks old were studied. Eight had thyroiditis and 22 showed no inflammatory infiltration. An immunohistochemical examination using the streptavidin-biotin-peroxidase technique was conducted on paraffin-embedded tissue sections, with a polyclonal antilaminin antibody. Antigen retrieval was achieved through pepsin digestion and microwave irradiation in citrate buffer. Staining for laminin was restricted to the basement membrane. In thyroids with no infiltration, laminin was shown as a fine, continuous brown line in the basement membrane. In 6 out of the 8 cases of thyroiditis, clearcut interruption and destruction of the FBM was observed, particularly when the follicles were located in lymphocyte infiltrated areas or when there was fibrosis. There were significant alterations in the pattern of the FBM with extensive areas of discontinuity in the distribution of laminin. Such discontinuities could facilitate antigen exposure, especially thyroglobulin, which may contribute to autoimmune thyroiditis in NOD mice.

Immunohistochemical study of basement membrane collagen IV in uterine cervix carcinoma.

CONTEXT: The integrity of basement membrane (BM) is damaged during the evolution of a benign or potentially malignant lesion into a malignant one, in which it may undergo several degrees of discontinuity as a necessary condition for the invasive process. Immunostaining for collagen IV, which is exclusively found in BM, has been used to evaluate its formation in neoplastic and benign lesions of several organs. OBJECTIVE: To investigate BM continuity pattern in squamous carcinoma "in situ" (CIS), microinvasive (MIC) and invasive (IC) squamous cell carcinoma of the uterine cervix, and to find out if BM expression could be useful in the diagnosis of early stromal invasion (MIC). DESIGN: Archival material between 1988 and 1993 was studied at the Pathological Anatomy Department-Unicamp. PROCEDURES: The selected cases, previously formalin fixed and paraffin embedded, were reviewed retrospectively by submitting them to immunohistochemical study via the avidin-biotin-peroxidase method using a monoclonal antibody anticollagen IV. RESULTS: In all, 17 cases of CIS, 16 of MIC and 21 of IC were evaluated. All IC cases showed evident BM discontinuity, either focal or diffuse. In the CIS group, a continuous BM pattern was predominant, being focally disrupted in only 2/17 cases (11.8%). The MIC group showed an intermediate pattern, but with a clear tendency to BM discontinuity in 10/16 cases (62.5%). Inflammatory infiltrate, a variable also studied, cannot be considered responsible for BM discontinuity, since there was no statistical correlation between them. CONCLUSION: We conclude that immunostaining for collagen IV may contribute to the diagnosis of stromal invasion by BM discontinuity.

Results of transhiatal esophagectomy in cancer of the esophagus and other diseases.

We present the results we obtained during the last 12 years with transhiatal esophagectomy. Two hundred and eighty-three patients were operated on using this procedure; 171 of them underwent the operation for cancer of the esophagus, 73 for cancer of the cardia, and 11 for cancer of the hypopharynx. The tumor stage, the operative technique, and the type of esophageal replacement (stomach: 62.9%; colon: 37.1%) are described. Overall operative mortality was 5.6%, mainly as a result of respiratory insufficiency. Long-term survival was 11.9% at five years for cancer of the esophagus, much lower than the 48.3% for cancer of the cardia.