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O câncer de mama é uma das neoplasias de maior incidência no mundo, afetando uma em cada oito. Dois tipos histológicos de câncer de mama, ductal e lobular, somam aproximadamente 90% dos casos, O carcinoma ductal é o mais freqüente (80%). O câncer de mama é uma doença heterogênea e pacientes com o mesmo diagnóstico e prognóstico clínico podem apresentar diferentes evoluções da doença. Essa diferença ocorre, pela limitação da atual classificação, baseada principalmente na morfologia e em alguns marcadores moleculares, que agrupam tumores molecularmente distintos em um mesmo grupo. Nesse estudo, para identificar marcadores moleculares relacionados à progressão e metástase em câncer de mama, nós analisamos o perfil de expressão gênica de tumores primários de carcinoma ductal. As amostras foram divididas em dois grupos, amostras com e sem comprometimento de linfonodo. Para se obter uma população celular mais homogênea e um perfil de expressão mais fidedigno, as amostras foram microdissecadas a laser. Doze amostras com e 17 sem comprometimento linfonodal foram microdissecadas a laser, o RNA foi extraído, tratado com DNase e amplificado. Uma plataforma customizada de cDNA microarray contendo 1808 genes foi utilizada, sendo desses, trezentos e quarenta e nove genes pertencentes a vias de sinalização como a PI3K, Wnt e processo de transição epitélio-mesênquima (EMT), envolvidas na diferenciação embrionária, invasão e progressão do câncer. Na comparação do perfil de expressão gênica entre os dois grupos, nós identificamos 67 genes diferencialmente expressos (p<0,05), dos quais 8 (CTNNB1, DAAM2, SKP1A, WNT10B, PLCD1, PLCB4, DAAM1, KRT19) pertencem a uma das três vias de interesse. CTNNB1, DAAM2, SKP1A foram mais expressos e SKP1A, WNT10B, PLCD1, PLCB4, DAAM1 foram menos expressos no grupo com comprometimento de linfonodo. Além disso, nós analisamos a correlação entre pares de genes nos dois grupos de amostras e identificamos pares de genes com diferença de correlação significativa. Essa perda de correlação entre os dois grupos pode estar relacionada ao processo de invasão linfonodal. Esse estudo identificou alguns genes candidatos, potencialmente envolvidos no processo de invasão linfonodal no carcinoma ductal de mama e devem ser investigados mais profundamente

Breast cancer is one of the most incident neoplasia in the world, affecting one in eight women. Two histological types of breast cancer, ductal and lobular, represent approximately 90% of all cases. Ductal carcinoma is the most frequent (80%). Breast cancer is a heterogeneous disease and patients with the same diagnostic and clinical prognostic profile can have markedly different clinical outcomes. This difference is due to the limitation of the current classification based mainly on morphology and use of some molecular markers, which groups molecularly distinct diseases into the same group. In this study, to identify molecular markers related to progression and metastasis in breast tumor, we examined gene expression profile of primary ductal carcinoma samples. The samples were divided in two groups, samples without and with lymph node metastases. Laser microdissection was used to obtain homogenous cell population and therefore a cellular-based gene expression profile. Twelve samples without and 17 with limph-node metastases were laser microdissected, RNA was extracted, DNase treated and amplified. A customized cDNA microarray platform containing 1808 genes was used. Three hundred forty nine of the genes belonged to cancerrelated pathways such as PI3K, WNT signaling pathway and Epithelialmesenchimal transition (EMT). These processes are involved in embryonic differentiation and also in tumors invasion and progression. Comparison of the gene expression profiles of the 2 groups, identified 67 genes as differentially expressed (p<0,05). Eight of them (CTNNB1, DAAM2, SKP1A, WNT10B, PLCD1, PLCB4, DAAM1, KRT19), belong to one of the 3 pathways. CTNNB1, DAAM2, SKP1A were up and SKP1A, WNT10B, PLCD1, PLCB4, DAAM1 were down regulated in the group with lymph node metastases. Moreover, we analyzed the correlation between pairs of genes in the 2 sample groups and identified the gene pairs with significant difference in the correlation values between them. Some gene pairs showed loss of correlation in the 2 groups, which may be related to the lymph node invasion process. This study identified some candidate genes potentially involved in lymph node invasion process in ductal carcinoma of the breast, that have to be deeper investigated.

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Introduction: Formalin-Fixed Paraffin-Embedded Tissue samples (FFPET) represent a valuable source for studies of geneexpression comparisons, since a great number of these samples is available in archive and presents a long time of clinicalfollow-up. However, the quality of total RNA of these samples is known to be inferior to frozen samples, being many timesinadequate for studies of gene expression using conventional methodologies. Objective: This study aims to establish a protocolfor amplification of messenger RNA (mRNA) derived from FFPET samples for using in microarray experiments. Material andMethods: 4 tumoral samples of invasive ductal breast carcinoma FFPET-buffered 10% were used. Total RNA was extracted andthe mRNA was linearly amplified in two rounds based on T7 RNA polymerase methodology using different concentrations ofoligo dT-T7 Primer for first strand cDNA (1st-cDNA) synthesis. Amplified antisense RNA (aRNA) was labeled with cianine-Cy3through reverse transcription in the presence of random primers and co-hybridized with reference RNA (HB4a) labeled withcianine-Cy5 in a customized platform containing 4,608 cDNAs corresponding to human genes. Results: The amplified RNAquality was influenced by the relative amount of oligo dT-T7, showing better results for ratio of 1:0.1 (total RNA : oligo dT-T7).Hybridizations showed value of intensity signals for the most of cDNAs immobilized in the platform. Conclusion: This studyshowed that the control of the relative amounts of RNA derived from FFPET material and oligo dT-T7 is extremely important toobtain high-quality amplified RNA, allowing its use in microarray experiments.

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