RESUMEN
Sequential treatment with all-trans retinoic acid followed by chemotherapy significantly improves the long-term survival of patients who have acute promyelocytic leukemia (APL). Consequently, a simple and accurate test is needed to establish the diagnosis of APL and to identify those patients having a relapse of the disease. We describe an accurate, 2-hour indirect immunofluorescent assay for identifying APL cells in bone marrow specimens. The assay uses the PML (PG-M3) murine monoclonal antibody that is directed against the amino-terminal portion of the PML gene product. We observed a distinctive, finely speckled pattern of fluorescence in the NB4 cell line (a positive control), as well as in 15 clinical specimens that were confirmed to have APL by cytogenetic, cytochemical, and immunophenotypic studies, including four cases of microgranular variant of APL. By contrast, a coarse globular pattern of fluorescence was observed in 53 other clinical specimens that did not contain APL. When we performed dilution studies using artificial mixtures of APL cells with normal bone marrow cells, we detected as few as 5% APL cells in the mixture. Finally, there was complete concordance between the immunofluorescent assay and a polymerase chain reaction-based assay for the PML-retinoic acid receptor alpha chimeric gene in 12 other clinical specimens. We conclude that the immunofluorescent assay for PML protein is a rapid, sensitive, and accurate method for determining the presence of APL cells in clinical specimens. This assay therefore should be considered as a cost-effective alternative to other diagnostic tests, such as karyotyping or polymerase chain reaction, for the diagnostic evaluation of APL.
Asunto(s)
Técnica del Anticuerpo Fluorescente Indirecta , Leucemia Promielocítica Aguda/diagnóstico , Proteínas Nucleares , Anticuerpos Monoclonales , Médula Ósea/patología , Estudios de Evaluación como Asunto , Humanos , Proteínas de Neoplasias/inmunología , Reacción en Cadena de la Polimerasa , Proteína de la Leucemia Promielocítica , Factores de Transcripción/inmunología , Células Tumorales Cultivadas , Proteínas Supresoras de TumorRESUMEN
Tumor heterogeneity may adversely affect the flow cytometric measurement of S-phase fraction (SPF) in breast cancer specimens, and 10-20% of breast cancer specimens are not evaluable by flow cytometry due to technical factors such as debris, high coefficients of variation, poor specimen quality, or small sample size. Therefore, we performed this study on 207 specimens of breast cancer in order to determine if the apoptotic rate (AR) could serve as a useful adjunct to flow cytometric SPF measurements in breast cancers. The average AR in each specimen was determined by microscopic examination of tumor tissue that was specifically stained for apoptotic bodies by a commercially available TUNEL (Tdt-mediated dUTP digoxigenin nick end labelling) assay kit. The mean AR (4.5 +/- 3.0, n = 37) in the high SPF (> 10%) group was significantly (P < 0.01) higher than the mean AR (1.3 +/- 1.2, n = 72) in the low SPF (< 6%) group. Although the distributions of AR values in the two groups had substantial overlap, AR values greater than 5.5 per high power field (h.p.f.) were not observed in the low SPF cases but were present in 13 out of 37 cases with a high SPF. Simple linear regression analyses relating SPF to the mean AR in 57 DNA diploid cases and 41 DNA aneuploid cases yielded a minimal correlation (r2 = 0.21) between the two parameters only in the DNA aneuploid group. We conclude that an elevated AR has an association with high SPF in breast cancers, but the association is too weak to permit the general use of AR as a predictor of SPF. Our study also identified a subset of breast cancers with both a high SPF (> 10%) and a high AR (> 5.5/h.p.f.) that may warrant further investigation to determine its clinical significance.