M. Mitzi Brentani

Expression of gelatinases A and B, stromelysin-3 and matrilysin genes in breast carcinomas: clinico-pathological correlations.

The purpose of this study was to investigate the association among matrix metalloproteinases (gelatinases A and B, stromelysin-3 (ST3) and matrilysin) mRNAs expressed in primary breast carcinomas and stan-dard prognostic parameters and clinical outcome. mRNA levels were determined by Northern analysis in samples of 81 breast cancer patients (median follow-up, 40 months) and 27 samples of uninvolved adjacent breast tissue. Proteases were expressed by the majority of the tumors and normal breast tissues examined. ST3, gelatinase A and matrilysin mRNAs were more often expressed at high levels in carcinomatous than in normal breast tissues. Differences in the distribution of gelatinase B mRNA were not found. However, paired normal tissues generally produced weaker signals when compared to matched tumor samples. Univariate analysis showed no significant association of gelatinase A and matrilysin mRNAs with the classical prognostic markers (age, menopausal status, stage, size, nodal status, vascular infiltrate, necrosis, steroid receptors, metastasis and survival). Overexpression of ST3 was more frequently found in tumors of post-menopausal women (P < 0.022). Elevated expression of gel B mRNA was associated with the presence of vascular infiltrate (P < 0.026), necrosis (P < 0.039), PR negative tumors (P < 0.014) and inversely corre-lated to the number of survivors (P < 0.021). Multivariate analysis including 68 patients for whom all information was available indicated that neither stromelysin correlated significantly with pathological, clin-ical or biochemical features. High levels of gelatinase A and B mRNAs were inversely associated with the number of survivors. Our findings suggest that measurements of gelatinase A and B mRNAs expression in breast carcinoma may help to identify patients with an agressive form of the disease. ©Lippincott Williams & Wilkins

Genetic alterations in juvenile nasopharyngeal angiofibromas

Juvenile nasopharyngeal angiofibroma (JNA) is a rare benign neoplasm of the nasopharynx that accounts for 0.5% of all head and neck tumors. Although histologically benign in appearance, JNAs are locally aggressive and destructive, spreading from the nasal cavity to the nasopharynx, paranasal sinuses, and orbit skull base with intracranial extension. The gender selectivity of JNA and the relatively young age at diagnosis suggest hormone‐dependent development. Hormonal disorders have been reported in patients with JNA, and androgen and estrogen receptors have been identified in tumor tissue; however, a hormonal influence on JNA is controversial. Recent studies have attempted to further delineate the pathogenesis of JNA through analysis of genetic and molecular changes. Understanding of the molecular mechanisms involved in JNA might improve prevention, prognosis, and treatment of this tumor. In this review, we discuss published studies addressing the possible molecular pathways that might be involved in the development of JNA.

Expression of growth factors, proto-oncogenes, and p53 in nasopharyngeal angiofibromas

Biopsies from 25 juvenile nasopharyngeal angiofibromas (JNAs) and respective normal inferior turbinates were examined and compared. The expression patterns of the messenger RNAs (mRNAs) for various growth factors possibly involved in the growth of mesenchymal cells, as well as angiogenesis and fibrosis, were also compared. These growth factors included insulin‐like growth factor II (IGF‐II), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), transforming growth factors‐β1 (TGF‐β1) and platelet‐derived growth factors (PDGF‐A and PDGF‐B). Quantification of mRNA coding for proto‐oncogenes and suppressor genes related to proliferation (i.e., c‐myc, c‐fos, p53) was also undertaken.

Multiple steroid receptors in nasopharyngeal angiofibromas

Cytosols from 12 nasopharyngeal angiofibroma tissues were analyzed for the presence of estrogen (ER), progesterone (PR), androgen (AR), and glucocorticoid (GR) receptors, by using a dextran charcoal assay. Progesterone receptors (58%) clearly predominate over ER and AR (25%). Thus, nasoangiofibromas can be classified as ER‐PR+ tumors. Glucocorticoid receptors were found in 84% of the tumors. Steroid receptor levels, although slightly higher than those determined in three specimens of normal turbinate, were quantitatively low. Positive PR and AR tumors appeared to correlate with high density of fibroblasts and endothelial cells.

Prognostic significance of the combined expression of matrix metalloproteinase-9, urokinase type plasminogen activator and its receptor in breast cancer as measured by Northern blot analysis.

Using Northern blot analysis we have measured the co-expression of the matrix metalloprotease MMP-9, plasminogen activator urokinase type (uPA) and its receptor (uPAR) mRNAs in 81 biopsies of breast carcinomas with the objective of analyzing the impact of these factors on the overall survival probability of the patients (median follow-up time: 4 years). Individual mRNA levels of either uPA or uPAR showed parallel variations with MMP-9 mRNA, suggesting a coordinate transcription of these markers. When median values were used as cutoff points to discriminate between high and low factor expression, no association was found with patient outcome and MMP-9 or uPA mRNA distribution. However, increased uPAR mRNA levels were associated with poor prognosis (p = 0.01). The combination of MMP-9 and uPAR mRNA measurements has not enhanced prognostic information compared to information supplied by the receptor alone (p = 0.01). The combination of MMP-9 and high levels of uPA mRNA led to a significant association with poor outcome (p = 0.03). Multivariate analysis supported the notion that increased uPAR mRNA production in primary breast cancer may be a predictor of overall survival.

Relaxation of imprinting of IGFII gene in Juvenile Nasopharyngeal Angiofibromas

IGFII and H19 genes are expressed only from one allele due to genomic imprinting, biallelic expression (loss of imprinting) being associated with the tumorigenic process of different types of tumors. The mechanism responsible for genomic imprinting is not yet determined, although DNA methylation has been considered the main genetic event for an imprinted mark. In the current study, the authors analyzed the imprinting status and expression levels of the IGFII and H19 genes in 27 cases of Juvenile Nasopharyngeal Angiofibroma (JNA) using RFLPs, RT-PCR, and Southern and Northern Blots. The authors found that four out of eight informative cases (50%) for ApaI/IGFII polymorphism showed biallelic expression of IGFII, whereas none of the nine informative cases for the RsaI/H19 polymorphism showed biallelic expression of the H19 gene. Overexpression of IGFII was observed in 8 out of 22 cases (36.4%), and 7 out of 19 cases (36.8%) showed H19 overexpression. Hypomethylation was found only in the H19 gene in six out of eight cases analyzed. Therefore, our results demonstrate that alterations in the IGFII/H19 imprinted region occur in JNA.

Five distinct deleted regions on chromosome 17 defining different subsets of human primary breast tumors.

In this study, we analyzed 105 paired sporadic primary breast tumor and normal tissue samples for loss of heterozygosity (LOH) on chromosome 17, using 12 polymorphic markers. We have identified partial or interstitial LOH in five separate regions of chromosome 17. Two of the deleted regions lie on the short arm of the chromosome, the first (region I, D17S5) in the telomeric part, distal to TP53 and the second spanning the TP53 gene (region II). Three of the five deleted regions lie on the long arm of chromosome 17: region III, on the proximal long arm between D17S250 and THRA1; region IV, between D17S776 and D17S579, including the BRCA1 gene, and region V, located distal to D17S733. No statistically significant correlations were observed between clinicopathological characteristics or steroid hormone receptor status and deletion of either region I or II. However, patients whose tumors had LOH for region I showed relapse or death more frequently than patients with tumors informative for this region but without LOH (p = 0.002). Statistically significant correlations between LOH at each of the three deleted regions of 17q and a high mitotic index were observed (region III, p = 0.005; region IV, p = 0.02, and region V, p = 0.004). In addition, LOH at region IV showed a significant association with paucity of estrogen receptors (p = 0.01). Our results show a complex pattern of LOH on chromosome 17 in breast cancer and a correlation of these events with different clinical parameters. This pattern suggests that particular subsets of allele loss may contribute specifically to different clinically defined subsets of sporadic breast tumors.

Smad2 and Smad6 as predictors of overall survival in oral squamous cell carcinoma patients

BackgroundTo test if the expression of Smad1-8 mRNAs were predictive of survival in patients with oral squamous cell carcinoma (SCC).Patients and MethodsWe analyzed, prospectively, the expression of Smad1-8, by means of Ribonuclease Protection Assay in 48 primary, operable, oral SCC. In addition, 21 larynx, 10 oropharynx and 4 hypopharynx SCC and 65 matched adjacent mucosa, available for study, were also included. For survival analysis, patients were categorized as positive or negative for each Smad, according to median mRNA expression. We also performed real-time quantitative PCR (QRTPCR) to asses the pattern of TGFβ1, TGFβ2, TGFβ3 in oral SCC.ResultsOur results showed that Smad2 and Smad6 mRNA expression were both associated with survival in Oral SCC patients. Cox Multivariate analysis revealed that Smad6 positivity and Smad2 negativity were both predictive of good prognosis for oral SCC patients, independent of lymph nodal status (P = 0.003 and P = 0.029, respectively). In addition, simultaneously Smad2- and Smad6+ oral SCC group of patients did not reach median overall survival (mOS) whereas the mOS of Smad2+/Smad6- subgroup was 11.6 months (P = 0.004, univariate analysis). Regarding to TGFβ isoforms, we found that Smad2 mRNA and TGFβ1 mRNA were inversely correlated (p = 0.05, R = -0.33), and that seven of the eight TGFβ1+ patients were Smad2-. In larynx SCC, Smad7- patients did not reach mOS whereas mOS of Smad7+ patients were only 7.0 months (P = 0.04). No other correlations were found among Smad expression, clinico-pathological characteristics and survival in oral, larynx, hypopharynx, oropharynx or the entire head and neck SCC population.ConclusionSmad6 together with Smad2 may be prognostic factors, independent of nodal status in oral SCC after curative resection. The underlying mechanism which involves aberrant TGFβ signaling should be better clarified in the future.

Expression of vitamin D receptor mRNA in the hippocampal formation of rats submitted to a model of temporal lobe epilepsy induced by pilocarpine.

Vitamin D (VD), is a steroid hormone with multiple functions in the central nervous system (CNS), producing numerous physiological effects mediated by its receptor (VDR). Clinical and experimental studies have shown a link between VD dysfunction and epilepsy. Along these lines, the purpose of our work was to analyze the relative expression of VDR mRNA in the hippocampal formation of rats during the three periods of pilocarpine-induced epilepsy. Male Wistar rats were divided into five groups: (1) control group; rats that received saline 0.9%, i.p. and were killed 7 days after its administration (CTRL, n=8), (2) SE group; rats that received pilocarpine and were killed 4h after SE (SE, n=8), (3) Silent group--7 days; rats that received pilocarpine and were killed 7 days after SE (SIL 7d, n=8), (4) Silent group--14 days; rats that received pilocarpine and were killed 14 days after SE (SIL 14d, n=8), (5) Chronic group; rats that received pilocarpine and were killed 60 days after the first spontaneous seizure, (chronic, n=8). The relative expression of VDR mRNA was determined by real-time PCR. Our results showed an increase of the relative expression of VDR mRNA in the SIL 7 days, SIL 14 days and Chronic groups, respectively (0.060+/-0.024; 0.052+/-0.035; 0.085+/-0.055) when compared with the CTRL and SE groups (0.019+/-0.017; 0.019+/-0.025). These data suggest the VDR as a possible candidate participating in the epileptogenesis process of the pilocarpine model of epilepsy.

Gene expression profiling of clinical stages II and III breast cancer

Clinical stage (CS) is an established indicator of breast cancer outcome. In the present study, a cDNA microarray platform containing 692 genes was used to identify molecular differences between CSII and CSIII disease. Tumor samples were collected from patients with CSII or CSIII breast cancer, and normal breast tissue was collected from women without invasive cancer. Seventy-eight genes were deregulated in CSIII tumors and 22 in CSII tumors when compared to normal tissue, and 20 of them were differentially expressed in both CSII and CSIII tumors. In addition, 58 genes were specifically altered in CSIII and expression of 6 of them was tested by real time RT-PCR in another cohort of patients with CSII or CSIII breast cancer and in women without cancer. Among these genes, MAX, KRT15 and S100A14, but not APOBEC3G or KRT19, were differentially expressed on both CSIII and CSII tumors as compared to normal tissue. Increased HMOX1 levels were detected only in CSIII tumors and may represent a molecular marker of this stage. A clear difference in gene expression pattern occurs at the normal-to-cancer transition; however, most of the differentially expressed genes are deregulated in tumors of both CS (II and III) compared to normal breast tissue.