RESUMO
Cytokeratin K19 (CK19) expression was evaluated by a reverse transcription PCR method (RT-PCR) in the RNA obtained from peripheral blood stem cell collections (PBSC) from four patients with breast cancers (BC) and 34 mononucleated blood cell (MBC) negative controls (17 PBMC from normal subjects 12 PBSC from different types of leukaemias--M3, M4Eo, M2, etc.--and two from patients with Hodgkin's lymphoma; and three bone marrow (BM) collections). Two BC tissues were taken as positive controls. The method studied (Datta YH, Paul T, Adams PT, Drobyski WR. Sensitive detection of occult breast cancer by reverse transcription polymerase chain reaction. J Oncol 1994;12:475-8) is sensitive enough to allow the detection of CK19 transcripts in a 10(-6) dilution of cDNA reverse transcribed from 1 microgram of BC RNA, but CK19 transcripts were also detected in 64% of the RNA obtained from the MBC controls. However, the amplified product detected in the control samples represents the transcript of the CK19 gene as confirmed by the results of Mae III digestion. It should be pointed out that although the CK19 expression was detected, the levels of expression in PBMC were almost negligible for they disappeared at 1:5 cDNA dilution. Moreover, a direct relationship between the number of BC cells added to PBMC and the increasing dilution levels of the cDNA necessary to prevent CK19 expression was observed. This allows us to conclude that the cDNA dilutions make it possible to distinguish the false from the true positive samples and that, in addition, the cDNA dilutions inform about the degree of BC cell contamination.
Assuntos
Expressão Gênica , Queratinas/genética , Leucócitos Mononucleares/metabolismo , Reação em Cadeia da Polimerase/métodos , Neoplasias da Mama/genética , Estudos de Casos e Controles , DNA Complementar/sangue , DNA Complementar/genética , DNA de Neoplasias/sangue , DNA de Neoplasias/genética , Estudos de Avaliação como Assunto , Feminino , Células-Tronco Hematopoéticas/metabolismo , Doença de Hodgkin/genética , Humanos , Leucemia/genética , Reação em Cadeia da Polimerase/estatística & dados numéricos , Sensibilidade e EspecificidadeAssuntos
Vírus da Mieloblastose Aviária/enzimologia , Vírus da Leucemia Murina de Moloney/enzimologia , Reação em Cadeia da Polimerase/métodos , DNA Polimerase Dirigida por RNA/metabolismo , Rearranjo Gênico , Humanos , Queratinas/genética , Leucemia Promielocítica Aguda/genética , Proteínas de Fusão Oncogênica/genética , Receptores do Ácido Retinoico/genética , Receptor alfa de Ácido Retinoico , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: To present the results of the analysis of DNA content in renal adenocarcinoma and its correlation with histopathological findings as a first step to conduct predictive studies. MATERIAL AND METHOD: DNA analysis was carried out by flow cytometry in deparaffined and fresh samples (1-4 per tumour) from 192 tumours. Correlation to stage, size, growth pattern and grade was studied using the squared chi test. RESULTS: The percentage of non-diploid tumours increased with the number of samples analyzed. Use of multiple sampling allowed to classify as non-diploid an additional 19% tumours. 57% tumours were homogeneously diploid, while the rest were non-diploid showing a wide variety of patterns: homogeneous, heterogeneous, and even tumours with more than one different non-diploid population. A positive correlation was seen between DNA content pattern, pathological stage and grade. CONCLUSIONS: Multiple sampling is essential to obtain representative information on DNA content. Prior to conduct predictive studies, the correlation between DNA content, stage and grade should be studied to preclude addition of non-independent information.
