RESUMO
BACKGROUND: Molecular genetic markers to identify the 13% lymph node-negative mammary carcinomas that are prone to develop metastases would clearly be of considerable value in indicating those cases in need of early aggressive therapy. METHODS: Representational difference analysis was used in an attempt to identify genetic alterations related to breast cancer metastasis by comparing genomic DNA from microdissected normal cells and from metastatic cells of ductal breast carcinoma patients. RESULTS: Representational difference analysis products yielded 10 unique metastasis-associated DNA sequences (MADS), i.e. products apparently lost in metastatic cell DNA. Of these sequences, MADS-IX was found to be lost in the transition from primary to metastasis in two out of five ductal breast carcinoma cases. This sequence was localized on chromosome 10q21 by radiation hybrid mapping and fluorescence in situ hybridization. The PTEN gene, which is also located on chromosome 10q, was detected to be present by PCR in all five cases. On the contrary, a breast carcinoma cell line, HCC-1937, which has homozygous loss of a region encompassing the PTEN gene, showed the presence of MADS-IX. PCR screening of three additional breast carcinoma cell lines with known losses in specific chromosomal regions also showed the presence of MADS-IX. CONCLUSION: These data suggest that MADS-IX possibly is part of a novel candidate metastasis-associated gene located close to the PTEN gene on chromosome 10q. The first set of PCR screening in five patient samples indicates that it could be used as a molecular marker for ductal mammary metastasis.
Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , DNA de Neoplasias/genética , Metástase Neoplásica/genética , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , DNA de Neoplasias/química , Feminino , Marcadores Genéticos/genética , Predisposição Genética para Doença/genética , Humanos , Hibridização in Situ Fluorescente , Pessoa de Meia-Idade , Metástase Neoplásica/patologia , PTEN Fosfo-Hidrolase , Monoéster Fosfórico Hidrolases/genética , Mapeamento de Híbridos Radioativos , Análise de Sequência de DNA , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/genéticaRESUMO
A dinucleotide (T-G) repeat sequence was isolated by comparing DNA from metastatic lymph node and matched normal breast samples from a ductal mammary carcinoma patient using representational difference analysis (RDA) method. Our present study used this metastasis associated DNA sequence (MADS) as a diagnostic probe to screen five patient samples by slot blot method. A new approach to isolate single cells by microdissection, namely single cell microdissection (SCM) was developed to obtain homogeneous population of tumor cells (approximately 1000) from matched primary tumors and corresponding positive lymph nodes of five patients. We isolated DNA from these homogeneous tumor cells and used for the RDA and DNA slot blot experiments. The screening of patient samples showed loss of this MADS in the transition from primary to metastasis in four out of five cases (80%) suggesting its possible role in breast metastasis.