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2.
Lab Invest ; 81(4): 565-71, 2001 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11304576

RESUMO

Ductal carcinoma in situ (DCIS), as an identifiable progenitor lesion of invasive breast cancer, represents a morphologically, biologically, and prognostically heterogeneous disease. It is not clear which molecular mechanisms are involved in progression to infiltrative growth. In this study, 83 DCIS classified according to the Van Nuys grading scheme were examined for amplification of growth regulatory genes that have been found to be amplified in invasive breast cancer (c-erbB2, topoisomerase IIalpha, c-myc, and cyclinD1 genes). Exact quantification of gene amplification was enabled by a combination of laser microdissection of paraffin-embedded tissue with real-time PCR. In DCIS, gene amplifications of all tested genes were found. The most frequently amplified gene was c-erbB2 found in 21 of 83 (25%) cases. Amplification of the other genes under investigation was observed in 4% to 6% of cases, high-grade DCIS being predominantly affected. High-grade DCIS differed significantly from low- and intermediate-grade DCIS in frequency and level of c-erbB2 amplification. In addition, high-grade DCIS revealed an accumulation of genetic aberrations. Amplification status in pure in situ lesions did not differ from intraductal carcinoma with an infiltrative component, indicating that although associated with a higher nuclear grade gene amplification might not represent an independent prognostic marker of disease progression.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/patologia , Carcinoma Intraductal não Infiltrante/genética , Carcinoma Intraductal não Infiltrante/patologia , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Neoplasias da Mama/classificação , Carcinoma Ductal de Mama/classificação , Carcinoma Intraductal não Infiltrante/classificação , Núcleo Celular/ultraestrutura , Progressão da Doença , Feminino , Genes erbB-2 , Substâncias de Crescimento/genética , Humanos , Invasividade Neoplásica
3.
Am J Pathol ; 156(6): 1855-64, 2000 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10854209

RESUMO

Gene amplification is one of the most important mechanisms leading to deregulated gene expression in cancer. The exact quantitative detection of this frequent genomic alteration in solid tumors is often hampered by an admixture of nonneoplastic bystander and stroma cells. To overcome this obstacle and to develop an objective quantitative method we have combined laser-assisted microdissection of tumor cells with the novel 5'-exonuclease-based real-time polymerase chain reaction (PCR) assay. The latter method enables the highly reproducible exact quantification of minute amounts of nucleic acids. As a model system amplification of c-erbB2/Her-2/neu gene and the adjacent topoisomerase IIalpha gene was determined in paraffin-embedded breast cancer specimens (n = 23) after immunohistochemical labeling and laser-based microdissection of tumor cells. The high sensitivity of real-time PCR enabled the reliable and objective detection of low-level amplifications in as few as 50 cells from archival tissue sections. Low-level amplifications were shown to escape from detection unless tumor cells were isolated by microdissection. In selected cases intratumor heterogeneity was demonstrated using areas of approximately 50 to 100 cells. This novel approach combining immunohistochemistry, laser microdissection, and quantitative kinetic PCR allows morphology-guided studies in archival tissue specimens and will enable the exact quantification of gene copy numbers in even small and precancerous lesions.


Assuntos
Neoplasias da Mama/genética , DNA Topoisomerases Tipo II , Dissecação , Amplificação de Genes , Lasers , Reação em Cadeia da Polimerase/métodos , Antígenos de Neoplasias , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Sistemas Computacionais , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA , Feminino , Genes erbB-2/genética , Humanos , Imuno-Histoquímica , Isoenzimas/genética , Inclusão em Parafina , Receptor ErbB-2/metabolismo , Células Tumorais Cultivadas
4.
Pathobiology ; 68(4-5): 173-9, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-11279343

RESUMO

Gene amplification is one essential mechanism leading to oncogene activation which is supposed to play a major role in the pathogenesis of invasive breast cancer. However, using standard methodologies the detection of gene amplifications has been limited especially in small-sized lesions, like pre-invasive precursor lesions. The combination of two novel technologies, laser-based microdissection and quantitative real-time PCR, facilitates the detection of low-level amplifications in morphologically defined lesions. As a model system we investigated in situ breast cancer (ductal carcinoma in situ, DCIS) classified according to the morphology-based Van Nuys grading system for amplification of growth-regulatory genes. In this study 83 formalin-fixed, paraffin-embedded archival DCIS specimens were examined after laser-based microdissection by quantitative real-time PCR using the TaqMan detection system for amplification of the c-erbB2, topoisomerase IIalpha, c-myc and cyclinD1 gene. In a subset of 17 DCIS with adjacent infiltrating tumour components we compared intraductal and invasive tumour components in parallel for differences in amplification status. The combination of these new techniques represents an excellent tool to gain new insights into carcinogenesis by analyzing genetic alterations in morphologically identified heterogeneous lesions in breast cancer progression within the very same specimen or even tissue slide.


Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Carcinoma Intraductal não Infiltrante/genética , DNA Topoisomerases Tipo II , Dissecação/métodos , Amplificação de Genes/genética , Lasers , Reação em Cadeia da Polimerase/métodos , Antígenos de Neoplasias/genética , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Carcinoma Intraductal não Infiltrante/patologia , Separação Celular/instrumentação , Separação Celular/métodos , Ciclina D1/genética , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA , Dissecação/instrumentação , Feminino , Genótipo , Humanos , Isoenzimas/genética , Fenótipo
5.
Pathobiology ; 68(4-5): 196-201, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-11279346

RESUMO

The detection of donor-derived cells in the blood and tissues of graft recipients after solid organ transplantation is a readily observed phenomenon called microchimerism. Yet very little is known about the persistence and integration of recipient-derived cells in the transplanted organ, indicating a form of intragraft chimerism. To further study this phenomenon and its possible influence on graft acceptance or rejection, we developed the following novel approach. Immunohistochemically labeled cells were isolated by means of laser-based microdissection and subsequent laser pressure catapulting from paraffine-embedded posttransplantation biopsies. The following use of a highly sensitive PCR assay analyzing one polymorphic short tandem repeat (STR) marker enabled us to clearly identify the genotypes in samples containing as little as 10 isolated cells. The combination of laser-based microdissection and STR-PCR thus provides a powerful tool for the genotyping of even very few cells isolated from routinely processed biopsies after solid organ transplantation.


Assuntos
Separação Celular , Dissecação , Lasers , Reação em Cadeia da Polimerase/métodos , Sequências de Repetição em Tandem/genética , Quimeras de Transplante/genética , Separação Celular/instrumentação , Separação Celular/métodos , Dissecação/instrumentação , Dissecação/métodos , Endotélio Vascular/patologia , Transplante de Coração/patologia , Hepatócitos/química , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Fígado/patologia , Transplante de Fígado/patologia , Miocárdio/patologia , Sensibilidade e Especificidade
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