Polymorphism in CYP17, GSTM1 and the progesterone receptor genes and its relationship with mammographic density

Radiologic breast density is one of the predictive factors for breast cancer and the extent of the density is directly related to postmenopause. However, some patients have dense breasts even during postmenopause. This condition may be explained by the genes that codify for the proteins involved in the biosynthesis, as well as the activity and metabolism of steroid hormones. They are polymorphic, which could explain the variations of individual hormones and, consequently, breast density. The constant need to find markers that may assist in the primary prevention of breast cancer as well as in selecting high risk patients motived this study. We determined the influence of genetic polymorphism of CYP17 (cytochrome P450c17, the gene involved in steroid hormone biosynthesis), GSTM1 (glutathione S-transferase M1, an enzyme involved in estrogen metabolism) and PROGINS (progesterone receptor), for association with high breast density. One hundred and twenty-three postmenopausal patients who were not on hormone therapy and had no clinical or mammographic breast alterations were included in the present study. The results of this study reveal that there was no association between dense breasts and CYP17 or GSTM1. There was a trend, which was not statistically significant (P = 0.084), towards the association between PROGINS polymorphism and dense breasts. However, multivariate logistic regression showed that wild-type PROGINS and mutated CYP17, taken together, resulted in a 4.87 times higher chance of having dense breasts (P = 0.030). In conclusion, in the present study, we were able to identify an association among polymorphisms, involved in estradiol biosyntheses as well as progesterone response, and radiological mammary density.

Immunohistochemical expression of oestrogen and progesterone receptors during experimental acute and chronic murine Schistosomiasis mansoni

INTRODUCTION: The responsibility of Schistosoma mansoni in female infertility is still controversial. This study was conducted to evaluate the effect of acute and chronic schistosomiasis mansoni infection on the endometrium using immunohistochemical analysis of uterine hormone receptor expression. METHODS: Twenty-four nonpregnant swiss albino mice were divided into three groups: control, noninfected; acute; and chronic Schistosoma mansoni infection. Histological sections of uterine specimens were examined by light microscope with an image analyzing system to detect structural histological, estrogen receptor (ER) and progesterone receptor (PR) expression in the endometrium. RESULTS: No secretory phase was detected in the endometrium in acute and chronic Schistosoma infection. Hormone receptor expression (ER and PR) showed statistically significant differences among the groups (p< 0.05), with significant low ER hormone expression in chronic infection, compared to control proliferative, control secretory and acute infection cases, and statistically significant high PR expression in both acute and chronic infection cases compared to the control secretory cases (p< 0.05). CONCLUSIONS: Schistosomiasis mansoni seems to have an important impact on the hormone expression of affected women. Further studies to explore the mechanism of such changes are recommended.

Comparison of nuclear grade and immunohistochemical features in situ and invasive components of ductal carcinoma of breast

PURPOSE:To compare the prognostic and predictive features between in situ and invasive components of ductal breast carcinomas. METHODS:We selected 146 consecutive breast samples with ductal carcinoma in situ (DCIS) associated with adjacent invasive breast carcinoma (IBC). We evaluated nuclear grade and immunohistochemical expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), cytokeratin 5/6 (CK5/6), and epidermal growth factor receptor (EGFR) in both components, in situ and invasive, and the Ki-67 percentage of cells in the invasive part. The DCIS and IBC were classified in molecular surrogate types determined by the immunohistochemical profile as luminal (RE/PR-positive/ HER2-negative), triple-positive (RE/RP/HER2-positive), HER2-enriched (ER/PR-negative/HER2-positive), and triple-negative (RE/RP/HER2-negative). Discrimination between luminal A and luminal B was not performed due to statistical purposes. Correlations between the categories in the two groups were made using the Spearman correlation method. RESULTS:There was a significant correlation between nuclear grade (p<0.0001), expression of RE/RP (p<0.0001), overexpression of HER2 (p<0.0001), expression of EGFR (p<0.0001), and molecular profile (p<0.0001) between components in situ and IBC. CK 5/6 showed different distribution in DCIS and IBC, presenting a significant association with the triple-negative phenotype in IBC, but a negative association among DCIS. CONCLUSIONS: Our results suggest that classical prognostic and predictive features of IBC are already determined in the preinvasive stage of the disease. However the role of CK5/6 in invasive carcinoma may be different from the precursor lesions.

Estrogen and progesterone receptors in human papilloma virus-related cervical neoplasia

Estrogen (ER) and progesterone (PR) receptors in the normal uterine cervix, cervical intraepithelial neoplasia and invasive carcinoma were studied in consecutive samples from Hospital do Câncer, São Paulo, between 1996 and 1997. Tissue was collected by removing a fragment of the tumoral area using a 5-mm diameter biopsy punch, followed by removal of a macroscopically normal area as close as possible from the tumor. Histopathological confirmation was obtained for all specimens analyzed. A total of 24 normal tissues, 17 cases of cervical intraepithelial neoplasia and 7 of invasive carcinomas were studied. The ER/PR ratio was determined by immunohistochemistry using monoclonal antibodies specific for each receptor. Adjacent tissue slides were submitted to generic PCR for human papillomavirus (HPV) DNA detection followed by typing by dot blot hybridization. About half (45.8%) of the tumors were HPV DNA positive while 29.1% of the patients were also HPV positive in their respective normal tissue. ER was negative in the tumoral epithelium of 11 HPV-positive patients (P = 0.04). There was a trend in the ER distribution in normal tissue that was opposite to that from lesions, but it was not statistically significant (P = 0.069). No difference in ER distribution in stromal tissues was observed between HPV-positive and HPV-negative tissues. PR staining was negative in the epithelium of all cases studied. The results obtained from this small number of cases cannot be considered to be conclusive but do suggest that factors related to viral infection affect the expression of these ER/PR cervix receptors.

Does the correlation between EBNA-1 and p63 expression in breast carcinomas provide a clue to tumorigenesis in Epstein-Barr virus-related breast malignancies?

Several investigators have identified Epstein-Barr virus (EBV) particles in breast carcinomas, a fact that supports a role for EBV in mammary tumorigenesis. The possible mechanism involved in this process is not clear. The present study was carried out in an attempt to determine whether there is a relationship between latent infection with EBV and p53 and p63 expression in breast carcinomas. Immunohistochemistry developed with 3.3-diaminobenzidine tetrahydrochloride was performed in 85 formalin-fixed paraffin-embedded breast carcinomas using anti-EBV EBNA-1, anti-p63, anti-p53, anti-estrogen receptor (ER) and anti-progesterone receptor (PR) antibodies. The cases were selected to represent each of the various histologic types: intraductal carcinoma (N = 12), grade I invasive ductal carcinoma (N = 15), grade II invasive ductal carcinoma (N = 15), grade III invasive ductal carcinoma (N = 15), tubular carcinoma (N = 8), lobular carcinoma (N = 10), and medullary carcinoma (N = 10). The ductal breast carcinomas were graded I, II and III based on the Scarff-Bloom and Richardson grading system modified by Elston and Ellis. One slide containing at least 1000 neoplastic cells was examined in each case. ER, PR, p63, p53 and EBNA-1 were positive in 60, 40, 11.8, 21.2 and 37.6% of carcinomas, respectively. There was a correlation between EBNA-1 and p63 expression (P < 0.001), but not between EBNA-1 and p53 (P = 0.10). These data suggest a possible role for p63 in the mammary tumorigenesis associated with Epstein-Barr virus infection.

Amplificação dos genes que codificam a endotelina-1 e seus receptores em valvas mitrais reumáticas

FUNDAMENTO: As cardiopatias são doenças de alta prevalência, sendo a cardite reumática uma doença de grande relevância em países em desenvolvimento. As alterações em câmaras cardíacas esquerdas se associam à disfunção endotelial, com aumento dos níveis de endotelina-1 (ET-1) e consequências sobre a circulação pulmonar, muitas vezes determinando a hipertensão pulmonar (HP). No entanto, a presença de ET-1 e seus receptores na própria valva mitral, promovendo alterações vasculares pulmonares e aumentando a deformação valvar reumática, ainda é um assunto não abordado na literatura. OBJETIVO: Determinar, mediante técnicas moleculares, a expressão dos genes da endotelina e dos seus receptores em valvas mitrais reumáticas. MÉTODOS: 27 pacientes submetidos à troca valvar mitral tiveram seu tecido valvar analisado, a fim de determinar a presença de genes de ET-1 e seus receptores A e B. Foram feitas análises histológica e molecular das valvas (divididas em fragmentos M1, M2 e M3) e colhidos dados clínicos e epidemiológicos dos pacientes. Foram divididos em três grupos: valvopatia mitral, mitroaórtica e pacientes reoperados. RESULTADOS: O estudo mostrou a manifestação do gene da ET-1 em 40,7% dos espécimes e de seu receptor A em todas as amostras, com manifestação minoritária do gene do receptor B (22,2%). CONCLUSÃO: Todos os pacientes expressaram a presença do gene do receptor A. Não houve diferença estatística quanto à gravidade da doença, expressa em classe funcional, e aos subgrupos estudados (valvopatas mitrais, mitroaórticos e pacientes reoperados), ou quanto à expressão dos genes da ET-1 e seus receptores entre os subgrupos estudados (valvopatas mitrais, mitroaórticos e pacientes reoperados).

Cox-2 e sua associação com fatores prognósticos e resposta à quimioterapia primária em pacientes com câncer de mama

OBJETIVO: Avaliar em pacientes com câncer de mama a expressão imunoistoquímica da cox-2 antes da quimioterapia primária com 5-fluorouracil, epirrubicina e ciclofosfamida (FEC) e a associação desta com tamanho inicial do tumor, estado linfonodal, receptores hormonais, expressão da Her-2 e com a resposta clínica e anatomopatológica. MÉTODOS: Estudo retrospectivo com 41 mulheres portadoras do diagnóstico histopatológico de carcinoma ductal de mama. Foram submetidas à quimioterapia primária com esquema FEC (5-fluorouracil, epirrubicina e ciclofosfamida) na dosagem de 500mg/m2, 75mg/m2 e 500 mg/m2, respectivamente. Os critérios de inclusão foram intervalo etário entre 30 e 70 anos, estadiamento II a IIIA, após comprovação da ausência de metástase, tumor primário de mama, único e unilateral, tipo histológico ductal invasivo e ausência de cardiopatia e gestação. Para avaliação da expressão da proteína Her 2 neuutilizaram-se anticorpos monoclonais de coelho. Para visibilizar a expressão da proteína cox-2 utilizaram-se anticorpos policlonais obtidos do soro de cabras. A avaliação da resposta clínica ao tratamento foi realizada por exame físico mensurando-se o maior eixo tumoral por paquímetro. As medidas foram realizadas à admissão e após os ciclos de quimioterapia primária. Após três sessões quimioterápicas com intervalos de 21 dias realizou-se o procedimento cirúrgico. Adotaram-se os critérios do RECIST. Após a operação foi avaliada a resposta anatomopatológica local, sendo considerada completa quando da ausência de neoplasia invasiva e do componente in situ. Na avaliação imumoistoquímica para os receptores de estrogênio utilizaram-se estrogen receptor NCL-ER6F11 e para progesterona, progesterone receptor, NCL-PGR-312 considerando positiva quando da coloração em 10% ou mais das células tumorais. RESULTADOS: A distribuição segundo estadiamento clínico UICC verificaram-se seis no estádio IIA (14,6%), 22 no estádio IIB (53,6%) e 13 estádio IIIA (31,8%). A avaliação clínica inicial do maior eixo tumoral variou de 2,5 a 15 cm e mediana de 5 cm. Foram identificadas 14 pacientes (34,1%) com estado linfonodal negativo e 27 positivo (65,9%). Observou-se que 19 (46,3%) apresentavam-se no menacme e 22 (53,6%) na menopausa. CONCLUSÃO: Houve associação da expressão da cox-2 à fatores de pior prognóstico no câncer de mama como estado linfonodal positivo, receptores hormonais negativos e expressão da Her-2.

Criteria for prediction of metastatic axillary lymph nodes in early-stage breast cancer

PURPOSE: To estimate the likelihood of axillary lymph node involvement for patients with early-stage breast cancer, based on a variety of clinical and pathological factors. METHODS: A retrospective analysis was done in hospital databases from 1999 to 2007. Two hundred thirty-nine patients were diagnosed with early-stage breast cancer. Predictive factors, such as patient age, tumor size, lymphovascular invasion, histological grade and immunohistochemical subtype were analyzed to identify variables that may be associated with axillary lymph node metastasis. RESULTS: Patients with tumors that are negative for estrogen receptor, progesterone receptor, and HER2 had approximately a 90% lower chance of developing lymph node metastasis than those with luminal A tumors (e.g., ER+ and/or PR+ and HER2-) - Odds Ratio: 0.11; 95% confidence interval: 0.01-0.88; p=0.01. Furthermore, the risk for lymph node metastasis of luminal A tumors seemed to decrease as patient age increased, and it was directly correlated with tumor size. CONCLUSION: The molecular classification of early-stage breast cancer using immunohistochemistry may help predicting the probability of developing axillary lymph node metastasis. Further studies are needed to optimize predictions for nodal involvement, with the aim of aiding the decision-making process for breast cancer treatment.

Prevalence of ERα-397 PvuII C/T, ERα-351 XbaI A/G and PGR PROGINS polymorphisms in Brazilian breast cancer-unaffected women

Polymorphisms of hormone receptor genes have been linked to modifications in reproductive factors and to an increased risk of breast cancer (BC). In the present study, we have determined the allelic and genotypic frequencies of the ERα-397 PvuII C/T, ERα-351 XbaI A/G and PGR PROGINS polymorphisms and investigated their relationship with mammographic density, body mass index (BMI) and other risk factors for BC. A consecutive and unselected sample of 750 Brazilian BC-unaffected women enrolled in a mammography screening program was recruited. The distribution of PGR PROGINS genotypic frequencies was 72.5, 25.5 and 2.0% for A1A1, A1A2 and A2A2, respectively, which was equivalent to that encountered in other studies with healthy women. The distribution of ERα genotypes was: ERα-397 PvuII C/T: 32.3% TT, 47.5% TC, and 20.2% CC; ERα-351 XbaI A/G: 46.3% AA, 41.7% AG and 12.0% GG. ERα haplotypes were 53.5% PX, 14.3% Px, 0.3% pX, and 32.0% px. These were significantly different from most previously published reports worldwide (P < 0.05). Overall, the PGR PROGINS genotypes A2A2 and A1A2 were associated with fatty and moderately fatty breast tissue. The same genotypes were also associated with a high BMI in postmenopausal women. In addition, the ERα-351 XbaI GG genotype was associated with menarche ≥12 years (P = 0.02). ERα and PGR polymorphisms have a phenotypic effect and may play an important role in BC risk determination. Finally, if confirmed in BC patients, these associations could have important implications for mammographic screening and strategies and may be helpful to identify women at higher risk for the disease.

Toll-like receptor 3 gene polymorphisms are not associated with the risk of hepatitis B and hepatitis C virus infection

INTRODUCTION: The present study investigated the prevalence of two single-nucleotide polymorphisms (SNPs) in the Toll-like receptor 3 (TLR3) gene in patients infected with hepatitis B virus (HBV) and hepatitis C virus (HCV). METHODS: Samples collected from HCV (n = 74) and HBV (n = 35) carriers were subjected to quantitative real-time PCR (qPCR) to detect the presence of the SNPs rs5743305 and rs3775291 in TLR3 and to measure the following biomarkers: alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), and prothrombin time (PT). A healthy control group was investigated and consisted of 299 HCV- and HBV-seronegative individuals. RESULTS: No significant differences in allele, genotype and haplotype frequencies were observed between the investigated groups, and no association was observed between the polymorphisms and histopathological results. Nevertheless, genotypes TA/AA (rs5743305) and GG (rs3775291) appear to be associated with higher levels of ALT (p<0.01), AST (p<0.05) and PT (p<0.05). In addition, genotypes TT (rs5743305; p<0.05) and GG (rs3775291; p<0.05) were associated with higher GGT levels. CONCLUSIONS: This genetic analysis revealed the absence of an association between the polymorphisms investigated and susceptibility to HBV and HCV infection; however, these polymorphisms might be associated with a greater degree of biliary damage during the course of HCV infection.

CPU0213, a novel endothelin type A and type B receptor antagonist, protects against myocardial ischemia/reperfusion injury in rats

The efficacy of endothelin receptor antagonists in protecting against myocardial ischemia/reperfusion (I/R) injury is controversial, and the mechanisms remain unclear. The aim of this study was to investigate the effects of CPU0123, a novel endothelin type A and type B receptor antagonist, on myocardial I/R injury and to explore the mechanisms involved. Male Sprague-Dawley rats weighing 200-250 g were randomized to three groups (6-7 per group): group 1, Sham; group 2, I/R + vehicle. Rats were subjected to in vivo myocardial I/R injury by ligation of the left anterior descending coronary artery and 0.5% sodium carboxymethyl cellulose (1 mL/kg) was injected intraperitoneally immediately prior to coronary occlusion. Group 3, I/R + CPU0213. Rats were subjected to identical surgical procedures and CPU0213 (30 mg/kg) was injected intraperitoneally immediately prior to coronary occlusion. Infarct size, cardiac function and biochemical changes were measured. CPU0213 pretreatment reduced infarct size as a percentage of the ischemic area by 44.5% (I/R + vehicle: 61.3 ± 3.2 vs I/R + CPU0213: 34.0 ± 5.5%, P < 0.05) and improved ejection fraction by 17.2% (I/R + vehicle: 58.4 ± 2.8 vs I/R + CPU0213: 68.5 ± 2.2%, P < 0.05) compared to vehicle-treated animals. This protection was associated with inhibition of myocardial inflammation and oxidative stress. Moreover, reduction in Akt (protein kinase B) and endothelial nitric oxide synthase (eNOS) phosphorylation induced by myocardial I/R injury was limited by CPU0213 (P < 0.05). These data suggest that CPU0123, a non-selective antagonist, has protective effects against myocardial I/R injury in rats, which may be related to the Akt/eNOS pathway.

Overexpression of Fc receptor-like 1 associated with B-cell activation during hepatitis B virus infection

The role of B cells in the pathogenesis of hepatitis B virus (HBV) infection has not been explored in depth. In the present study, the activation status of B cells from peripheral blood of healthy controls (N = 20) and patients with acute hepatitis B (AHB, N = 15) or chronic hepatitis B (CHB, N = 30) was evaluated by measuring the expression levels of B-cell activation markers CD69 and CD86, using quantitative real-time PCR and flow cytometry. Moreover, the potential mechanism underlying B-cell activation during HBV infection was further investigated by analyzing the expression profile of FCRL1, an intrinsic activation molecule of B cells. An elevation in the levels of B-cell activation markers including CD69 and CD86 was observed in the AHB patients (44.31 ± 9.27, 27.64 ± 9.26%) compared to CHB patients (30.35 ± 11.27, 18.41 ± 6.56%, P < 0.05), which was still higher than healthy controls (12.23 ± 7.84, 8.22 ± 3.43%, P < 0.05). Furthermore, the expression of FCRL1 was found to be similar to B-cell activation markers, which was highest in AHB patients (70.15 ± 17.11%), lowest in healthy donors (36.32 ± 9.98%, P < 0.05) and half-way between these levels in patients with CHB (55.17 ± 12.03%, P < 0.05). The results were positively associated with aberrant B-cell activation. These data suggest that B cells can play a role in HBV infection, and therefore more effort should be devoted to exploring their functions.

Effect of arginine vasopressin on the canine epicardial coronary artery: experiments on V1-receptor-mediated production of nitric oxide

OBJECTIVE: To determine whether arginine vasopressin releases endothelium-derived nitric oxide (EDNO) from the epicardial coronary artery. METHODS: We studied segments of canine left circumflex coronary arteries suspended in organ chambers to measure isometric force. The coronary artery segments were contracted with prostaglandin F2alpha (2 x 10-6M) and exposed to a unique, strong arginine vasopressin concentration (10-6M) or titrated concentrations (10-9 a 10-5 M). RESULTS: The unique dose of arginine vasopressin concentration (10-6M) induced transient, but significant (p<0.05), relaxation in arterial segments with endothelium, and an increase, not significant, in tension in arteries without endothelium. Endothelium-dependent relaxation to arginine vasopressin was inhibited by Ng-monomethyl-L-arginine (L-NMMA, 10-5M) or N G-nitro-L-arginine (L-NOARG) (10-4M), 2 inhibitors of nitric oxide synthesis from L-arginine. Exogenous L-arginine (10-4M), but not D-arginine (10-4M), reversed the inhibitory effect of L-NMMA on vasopressin-mediated vasorelaxation. Endothelium dependent relaxation to vasopressin was also reversibly inhibited by the vasopressin V1-receptor blocker d(CH2)5Try(Me) arginine vasopressin (10-6M) (n=6, P<0.05). CONCLUSION: Vasopressin acts through V1 endothelial receptors to stimulate nitric oxide release from L-arginine.

Benzodiazepine receptor ligand influences on learning: an endogenous modulatory mechanism mediated by benzodiazepines possibly of alimentary origin

In rats pre-but not post-training ip administration of either flumazenil, a central benzodiazepine (BSD) receptor antagonist, or of n-butyl-B-carboline-carboxylate (BCCB), an inverse agonist, enhanced retention of inhibitory avoidance learning. Flumazenil vlocked the enhancing effect of BCCB, and the inhibitory effect of the BZD agonists clonazepam and diazepam also given pre-training. Post-training administration of these drugs had no effects. The peripheral BZD receptor agonist/chloride channel blocker Ro5-4864 had no effect on the inhibitory avoidance task when given ip prior to training, buth it caused enhancement when given immediately post-training either ip or icv. This effect was blocked by PK11195, a competitive antagonist of Ro5-4864. These results suggest that ther is an endogenous mechanism mediated by BZD agonists, which is sensitive to inverse agonists and that normally down-regulates the formation of memories through a mechanism involving GABA-A receptors and the corresponding chloride channels. The most likely agonists for the endogenous mechanism suggested are the diazepam-like BZDs found in brain whose origin is possibly alimentary. Levels of these BZDs in the cortex were found to sharply decrease after inhibitory acoidance training or mere exposure to the training apparatus.