Genomic instability association with intracellular aluminum concentration in breast tumors

Orientador: Luis Otavio Zanatta SarianTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: Introdução: A hipótese de que os efeitos do alumínio em células humanas podem ter implicações clínicas tem sido levantada há algum tempo, especialmente no que concerne ao câncer de mama. As evidências laboratoriais mostrando altos níveis de alumínio nos tecidos da mama e os efeitos biológicos conhecidos sobre esse metal não são suficientes para estabelecer uma relação causal entre a exposição ao alumínio e o risco aumentando para o desenvolvimento do câncer de mama. O objetivo deste estudo foi estabelecer a concentração de alumínio nas áreas centrais e periféricas de tumores de mama, assim como na área glandular normal da mama e correlacionar esses achados com a instabilidade dos genes ERBB2, C-MYC e CCND1 e a aneuploidia dos cromossomos que contêm estes genes. Métodos: Para este estudo foram incluídas 176 mulheres com diagnóstico de carcinoma invasor de mama, com tumores maiores de 1cm3, sem quimioterapia neoadjuvante, operadas enter 2008 e 2010 no Hospital da Mulher Prof. Dr. José Aristodemo Pinotti - Centro de Atenção Integral à Saúde da Mulher (CAISM) - UNICAMP. Para a análise da concentração de alumínio intracelular, amostras de 150 pacientes foram consideradas viáveis; para a análise da instabilidade genômica em função da concentração de alumínio, 118 amostras foram consideradas viáveis, definindo o espaço amostral de cada um dos artigos apresentados. As amostras das áreas centrais e periféricas dos tumores de mama e das áreas glandulares normais da mama foram obtidas. A quantificação do alumínio contido nos tecidos da mama foi feita através da técnica de Espectrometria de Absorção Atômica em Forno de Grafite (GFAAS). Uma lâmina de Tissue Microarray (TMA), contendo as amostras de tumor e tecido normal foi utilizado para a realização da técnica de FISH para acessar o status dos genes ERBB2, C-MYC e CCND1 e dos centrômeros dos seus respectivos cromossomos 17, 8 e 11. Os dados clínico-patológicos foram obtidos dos prontuários de pacientes. Resultados: A média da concentração de alumínio encontrada na mama foi de 1,88 mg/kg nas áreas centrais do tumor, 2,10mg/kg nas áreas periféricas do tumor e 1,68mg/kg nas áreas de tecido glandular normal. A amplificação e/ou aneuploidia para ERBB2/CEP17, C-MYC/CEP8 e CCND1/CEP11 foi encontrada em 24%, 36,7% e 29,3% dos tumores, respectivamente. A média da concentração de alumínio nas áreas tumorais (tanto centrais quanto periféricas) não foi significativamente diferente daquela nas áreas de tecido normal. A concentração de alumínio também não foi significativamente associada a nenhum status de amplificação e/ou aneuploidia para os genes/cromossomos em questão. Conclusões: Consideramos importante que estudos experimentais in vitro continuem sendo realizados para elucidar os possíveis efeitos do alumínio nos tumores de mama, quer sejam esses efeitos relacionados ao microambiente tecidual ou mesmo a outras vias de estabilidade genômicaAbstract: Introduction: It has long been hypothesized if the effects of aluminum on human cells may have clinical implications, especially regarding to breast cancer. The current laboratorial evidence showing higher levels of aluminum in breast tissues and the known biological effects of this metal, are not sufficient to establish a causal relationship between aluminum exposure and increased risk of developing breast cancer. The objective of this study was to establish the aluminum concentration in the central and peripheral areas of breast tumors as well as in normal glandular area of the breast and to correlate these findings with the instability of ERBB2, C-MYC and CCND1, and aneuploidy of chromosomes harboring these genes. Methods: This study included 176 women diagnosed with invasive breast carcinoma with tumors larger than 1cm3 without neoadjuvant chemotherapy, operated between 2008 and 2010 at the Women's Hospital Professor. Dr. José Aristodemo Pinotti - Centro de Atenção Integral à Saúde da Mulher (CAISM) - UNICAMP. To analyze the intracellular concentration of aluminum, samples from 150 patients were considered viable; for the analysis of genomic instability as a function of the concentration of aluminum, 118 samples were considered viable. These figures define the sample of each of the two articles that this PhD thesis comprises. Evaluation of tissue aluminum content was carried out using Graphite Furnace Atomic Absorption Spectrometry (GFAAS). A TMA slide containing the tumor and normal samples was used in FISH assays to assess ERBB2, C-MYC and CCND1 and the respective chromosomes 17, 8 and 11 centromeres status. Clinicopathological data were obtained from patients' records. Results: The average aluminum content found in breast was 1.88 mg/kg in the central tumor areas, 2.10 mg/ kg in the peripheral tumor areas and 1.68 mg/ kg in the normal tissue areas. The amplification and/or aneuploid status for the ERBB2/CEP17, C-MYC/CEP8 and CCND1/CEP11 was detected in 24%, 36.7% and 29.3% of the tumors, respectively. The average aluminum content in tumor areas (either central or peripheral) was not significantly different from that in normal tissues. We found that aluminum concentration was not related to any of the gene status. Conclusions: We consider important that in vitro experimental studies continue to be done in order to elucidate the possible effects of aluminum in the development of breast tumors, whether it is influencing the tissue microenvironment or other genome stability pathwaysDoutoradoOncologia Ginecológica e MamáriaDoutora em Ciências da Saúd

ErbB-2 expression and hormone receptor status in areas of transition from in situ to invasive ductal breast carcinoma

OBJETIVOS: avaliar a expressão de erbB-2 e dos receptores hormonais para estrógeno e progesterona (RE/RP) nas regiões de transição entre as frações in situ e invasoras de neoplasias ductais da mama (CDIS e CDI, respectivamente). MÉTODOS: oitenta e cinco casos de neoplasias mamárias, contendo regiões contíguas de CDIS e CDI, foram selecionados. Espécimes histológicos das áreas de CDIS e de CDI foram obtidos através da técnica de tissue microarray (TMA). As expressões da erbB-2 e dos RE/RP foram avaliadas por meio de imunoistoquímica convencional. A comparação da expressão da erbB-2 e dos RE/RP nas frações in situ e invasoras da mama foi realizada com emprego do teste de McNemar. Os intervalos de confiança foram determinados em 5% (p=0,05). Foram calculados coeficientes de correlação intraclasse (ICC) para avaliar a concordância na tabulação cruzada da expressão de erbB-2 e RE/RP nas frações de CDIS e CDI. RESULTADOS: a expressão da erbB-2 não diferiu entre as áreas de CDIS e CDI (p=0,38). Comparando caso a caso suas áreas de CDIS e CDI, houve boa concordância na expressão da erbB-2 (coeficiente de correlação intraclasse, ICC=0,64), dos RP (ICC = 0,71) e dos RE (ICC = 0,64). Considerando apenas tumores cujo componente in situ apresentasse áreas de necrose (comedo), o ICC para erbB-2 foi de 0,4, comparado a 0,6 no conjunto completo de casos. Os ICC não diferiram substancialmente daqueles obtidos com o conjunto completo de espécimes em relação aos RE/RP: para RE, ICC=0,7 (versus 0,7 no conjunto completo), e para RP, ICC=0,7 (versus 0,6 no conjunto completo). CONCLUSÕES: nossos achados sugerem que as expressões de erbB-2 e RE/RP não diferem nos componentes contíguos in situ e invasivo em tumores ductais da mama.PURPOSE: to evaluate the expression of erbB-2 and of the estrogen and progesterone (ER/P) hormonal receptors in the transition regions between the in situ and the invasive fractions of ductal breast neoplasia (ISDC and IDC, respectively). METHODS: Eighty-five cases of breast neoplasia, containing contiguous ISDC and IDC areas, were selected. Histological specimens from the ISDC and the IDC areas were obtained through the tissue microarray (TMA) technique. The erbB-2 and the ER/PR expressions were evaluated through conventional immunohistochemistry. The McNemar's test was used for the comparative analysis of the expressions of erbB-2 protein and the ER/PR in the in situ and invasive regions of the tumors. The confidence intervals were set to 5% (p=0.05). Intraclass correlation coefficients (ICC) were calculated to assess the cross-tabulation agreement of the erbB-2 and the ER/PR expression in the ISDC and the IDC areas. RESULTS: the erbB-2 expression has not differed between the ISDC and the IDC areas (p=0.38). Comparing the two areas in each case, there was agreement in the expression of erbB-2 (ICC=0.64), PR (ICC=0.71) and ER (ICC=0.64). Restricting the analysis to tumors with the in situ component harboring necrosis (comedo), the ICC for erbB-2 was 0.4, compared to 0.6 for the whole sample. In this select group, the ICC for PR/ER did not differ substantially from those obtained with the complete dataset: as for the ER, ICC=0.7 (versus 0.7 for the entire sample) and for PR, ICC=0.7 (versus 0.6 for the entire sample). CONCLUSIONS: our findings suggest that the erbB-2 and the ER/PR expressions do not differ in the contiguous in situ and invasive components of breast ductal tumors.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

ErbB-2 expression and hormone receptor status in areas of transition from in situ to invasive ductal breast carcinoma

OBJETIVOS: avaliar a expressão de erbB-2 e dos receptores hormonais para estrógeno e progesterona (RE/RP) nas regiões de transição entre as frações in situ e invasoras de neoplasias ductais da mama (CDIS e CDI, respectivamente). oitenta e cinco casos de neoplasias mamárias, contendo regiões contíguas de CDIS e CDI, foram selecionados. Espécimes histológicos das áreas de CDIS e de CDI foram obtidos através da técnica de tissue microarray (TMA). As expressões da erbB-2 e dos RE/RP foram avaliadas por meio de imunoistoquímica convencional. A comparação da expressão da erbB-2 e dos RE/RP nas frações in situ e invasoras da mama foi realizada com emprego do teste de McNemar. Os intervalos de confiança foram determinados em 5% (p=0,05). Foram calculados coeficientes de correlação intraclasse (ICC) para avaliar a concordância na tabulação cruzada da expressão de erbB-2 e RE/RP nas frações de CDIS e CDI. a expressão da erbB-2 não diferiu entre as áreas de CDIS e CDI (p=0,38). Comparando caso a caso suas áreas de CDIS e CDI, houve boa concordância na expressão da erbB-2 (coeficiente de correlação intraclasse, ICC=0,64), dos RP (ICC = 0,71) e dos RE (ICC = 0,64). Considerando apenas tumores cujo componente in situ apresentasse áreas de necrose (comedo), o ICC para erbB-2 foi de 0,4, comparado a 0,6 no conjunto completo de casos. Os ICC não diferiram substancialmente daqueles obtidos com o conjunto completo de espécimes em relação aos RE/RP: para RE, ICC=0,7 (versus 0,7 no conjunto completo), e para RP, ICC=0,7 (versus 0,6 no conjunto completo). nossos achados sugerem que as expressões de erbB-2 e RE/RP não diferem nos componentes contíguos in situ e invasivo em tumores ductais da mama319CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP307988/2008-22008/08536-9to evaluate the expression of erbB-2 and of the estrogen and progesterone (ER/P) hormonal receptors in the transition regions between the in situ and the invasive fractions of ductal breast neoplasia (ISDC and IDC, respectively). Eighty-five cases of breast neoplasia, containing contiguous ISDC and IDC areas, were selected. Histological specimens from the ISDC and the IDC areas were obtained through the tissue microarray (TMA) technique. The erbB-2 and the ER/PR expressions were evaluated through conventional immunohistochemistry. The McNemar's test was used for the comparative analysis of the expressions of erbB-2 protein and the ER/PR in the in situ and invasive regions of the tumors. The confidence intervals were set to 5% (p=0.05). Intraclass correlation coefficients (ICC) were calculated to assess the cross-tabulation agreement of the erbB-2 and the ER/PR expression in the ISDC and the IDC areas. the erbB-2 expression has not differed between the ISDC and the IDC areas (p=0.38). Comparing the two areas in each case, there was agreement in the expression of erbB-2 (ICC=0.64), PR (ICC=0.71) and ER (ICC=0.64). Restricting the analysis to tumors with the in situ component harboring necrosis (comedo), the ICC for erbB-2 was 0.4, compared to 0.6 for the whole sample. In this select group, the ICC for PR/ER did not differ substantially from those obtained with the complete dataset: as for the ER, ICC=0.7 (versus 0.7 for the entire sample) and for PR, ICC=0.7 (versus 0.6 for the entire sample). our findings suggest that the erbB-2 and the ER/PR expressions do not differ in the contiguous in situ and invasive components of breast ductal tumor

erbB-2 expression and hormone receptor status in the transition areas from in situ to invasive ductal carcinoma of the breast

Orientador: Luis Otavio SarianDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias MedicasResumo: Introdução: Há evidências indicando que a transição de carcinoma ductal in situ (CDIS) para carcinoma ductal invasivo (CDI) é regulada por um número restrito de genes. Ainda permanece obscuro se a expressão do receptor erbB-2 e dos receptores hormonais predizem o potencial invasivo dos CDIS. Objetivos: Avaliar a expressão dos receptores de estrógeno e progesterona (RE/RP) e da proteína erbB-2 em áreas específicas em tumores que apresentem componentes de CDIS e CDI concomitantemente, indicando uma possível transição. Materiais e Métodos: Foram selecionados 85 casos de carcinomas da mama apresentando regiões contíguas de CDIS e CDI e obtidas amostras histológicas separadas, uma da região CDIS e outra da região CDI, do bloco de parafina original do diagnóstico, para montagem de um TMA (tissue microarray). A expressão das proteínas erbB-2, RE e RP foram verificadas através da técnica convencional de imuno-histoquímica. Os dados clínicos das pacientes foram coletados por consulta ao prontuário. Resultados: Aproximadamente 35% das áreas invasivas foram classificadas como 2+ ou 3+ para o erbB-2, em contraste a 47% no componente invasivo correspondente. A expressão de erbB-2 não se mostrou diferente nos componentes in situ e invasivo (p=0,12). A expressão de RP não foi estatisticamente diferente comparando-se regiões in situ e invasivas do tumor (p=0,06). Em contraste, houve uma discreta mudança na expressão de RE (p=0,04). O coeficiente de concordância intraclasse (ICC) revelou forte concordância nas comparações entre as amostras de erbB-2 (ICC=0,61), RP (ICC=0,61) e RE (ICC=0,70), nos componentes in situ e invasivo. Conclusão: Os achados sugerem que a expressão de erbB-2, RE e RP não diferem entre os componentes in situ e invasivo do carcinoma da mama, especialmente nas regiões de transição de um componente a outro. Espera-se que as alterações moleculares ocorridas antes da mudança morfológica do tecido persistam na forma invasiva do tumor. A concomitância da atuação de fatores epigenéticos pode explicar porque alguns tumores in situ evoluem para invasivos, enquanto outros tumores in situ com características moleculares semelhantes não apresentam a mesma evolução, suportando a hipótese de uma base multifatorial para a progressão da doença.Abstract: Introduction: There is evidence indicating that the transition from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) is regulated by a restrict number of genes. It remains unknown whether erbB-2 and hormone receptor status predict the invasive potential of DCIS. Objectives: To evaluate estrogen/progesterone receptor (ER/PR) and protein erbB-2 status in the specific areas of breast tumors which share both components of DCIS and IDC, concomitantly, indicating a possible transition. Materials and methods: 85 cases of breast malignancies harboring contiguous regions of DCIS and IDC were selected. Separate histological samples, one from the DCIS and other from the neighboring IDC, from the original paraffin block used on diagnosis, were sampled using tissue microarrays (TMA). The erbB-2 and ER/PR status was assessed using conventional immunohistochemistry techniques. Clinicopathological data from the patients and from the disease were retrieved out of the patients' medical records. Results: Approximatelly 35% of the invasive areas were erbB-2 2+ or 3+, compared to 47% of their in situ counterparts. The expression of erbB-2 did not differ in the in situ and in the invasive components of the breast tumors (p=0.12). The PR status was not statistically different comparing the in situ and invasive regions of the tumors (p=0.06). By contrast, there was a slight imbalance in ER status (p=0.04). The intraclass correlation coefficient (ICC) disclosed good agreement in sample-by-sample comparisons of erbB-2 (ICC = 0.61), PR (ICC= 0.61) and ER (ICC=0.70) expression in the in situ and invasive components. Conclusion: Our findings suggest that the expression of erbB-2 and ER/PR does not differ accross the in situ and invasive components of breast malignancies, especially in the transition region from one component to the other. It is expected that the molecular alterations which take place before the morphologic tissue changes persist in the invasive forms of breast tumors. The ocurrance of concomitant epigenetic factors may explain why some in situ tumors develop to an invasive phenotype, while other in situ tumors with similar molecular characteristics do not have the same evolution, supporting the hypothesis for a multifactorial basis for disease progression.MestradoCiencias BiomedicasMestre em Tocoginecologi

Expression Of Cyclooxygenase-2 (cox-2) And P53 In Neighboring Invasive And In Situ Components Of Breast Tumors.

The aim of the study was to assess the relationship between the expression of COX-2 and p53, hormone receptors and HER-2 in the in situ (DCIS) and invasive components of ductal carcinomas (IDC) of the same breast. The expression of COX-2, p53, and hormone receptors was assessed in 87 cases of IDC with contiguous areas of DCIS. Results showed that there was no difference in COX-2 expression comparing the in situ and invasive components of the tumors. In the in situ component, there was a statistically borderline increase in p53 expression in tumors that also expressed COX-2. ER-positive specimens were more common in the group of tumors that expressed COX-2 in the invasive component. From this study we conclude that the expression of COX-2 was similar in the in situ and invasive components of the breast carcinomas. COX-2 positivity was marginally related with the expression of p53 in the in situ components, and with the ER expression in the invasive components.114226-3

Comparative Evaluation Of The Erbb2 And Hormone Receptor Status Of Neighboring Invasive And In Situ Components Of Ductal Carcinomas Of The Breast.

It remains unknown whether erbB2 expression and hormone receptor status predict the invasive potential of ductal carcinoma in situ (DCIS) of the breast. To examine erbB2 and estrogen/progesterone receptor (ER/PR) status in the precise areas where DCIS turns into invasive ductal carcinoma (IDC). Eighty-seven cases of breast malignancies harboring contiguous regions of DCIS and IDC were selected. Separate histological samples from the DCIS and the neighboring IDC were obtained using tissue microarrays. The erbB2 and ER/PR statuses were assessed using immunohistochemistry (erbB2 and ER/PR) and fluorescence in situ hybridization (FISH - only erbB2). The expression of erbB2 did not differ in the DCIS and IDC components of the breast tumors (p=0.35). There was good agreement in sample-by-sample comparisons of erbB2 (intraclass correlation coefficient [ICC]=0.78), PR (ICC=0.61) and ER (ICC=0.70) expression in the DCIS and IDC components. Our findings suggest that the expressions of erbB2 and ER/PR do not differ in the contiguous regions from DCIS to IDC.24238-4

Cyclooxygenase-2 (COX2) and p53 protein expression are interdependent in breast cancer but not associated with clinico-pathological surrogate subtypes, tumor aggressiveness and patient survival

In the last decade, different molecular subtypes of breast cancer have been proposed. Although displaying appreciable association with disease prognosis and the prognostic value of cytotoxic and endocrine therapeutic modalities, the subtypes seem to fail at completely explaining disease behavior and response to treatment. Molecules such as those of the cyclocooxigenase (COX) family, currently composed of three entities (COX 1, 2 and 3) have been shown to be associated with breast carcinogenesis, and the analysis of p53 expression in breast tumors may also offer some additional prognostic clues. Our study is aimed at assessing COX2 and p53 expression in these clinico-pathological surrogate subtypes, and to evaluate whether the expression of these molecules can help further explain the variability in prognosis still found within the clinico-pathological subtypes groups of breast cancer. Methods: A total of 183 breast cancer samples were obtained from women treated at the Womenis Hospital of Campinas State University, Campinas, Brazil, between June 2008 and January 2011. Immunohistochemistry was performed to detect the expression of ER, PR, ki67, COX2, and p53 and the HER2 status of the 183 specimens was assessed using FISH. Two COX2 staining thresholds were used to define COX2 positivity: low threshold (LT): moderate and intense staining were considered positive; high-threshold (HT): only intense staining was considered positive. Results: There was no trend in COX2 overexpression from Luminal A-like to Triple-negative subtypes. By contrast, p53 was expressed in roughly 67% of the Luminal A-like tumors, 50% of the Luminal B-like HER2 positive tumors, 60.9% of the Luminal B-like HER2 negative, approximately 82% of the HER2 positive (non-luminal) and 87% of the Triple-negative tumors (p for trends = 0.06). There was a significantly higher proportion of COX2 positive (LT) tumors (66.9%) when p53 was also positive compared to when the tumor was negative for p53 (in which case only 18.0% of the tumors were positive for COX2; p<0.001). Neither marker was found to be associated with patients' survival. Conclusions: There seems to be a positive association between the expressions of COX2 and p53. Otherwise, neither the expression of COX nor that of p53 was associated with clinico-pathological subtypes, tumor features and prognosis. It seems to be too early to elect the detection of COX2 using IHC as prognostic or predictive tool, but incipient evidence points toward a possible role for the marker1182176182FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2009/17097-