Cytometry

Quantification and characterization of total cellular p53 protein in colorectal cancer.

Tominaga O. Hammel P. Hamelin R. Nagawa H. Muto T. Remvikos Y.
UMR 147 CNRS, Institut Curie, Paris, France.
Immunochemical methods were developed for the optimal detection and characterization of total cellular p53 protein expression, both in the nuclear-attached and soluble fractions of colorectal cancers, in order to improve the correlation between protein deregulation and gene status. Seventy colorectal carcinomas were studied using 3 monoclonal antibodies in a sensitive analyzing system combining flow cytometry (nuclear-bound fraction) and enzyme-linked immunosorbent assay (ELISA; soluble fraction). DNA indices were calculated on the DNA histograms and mutations of the p53 gene were searched for in a subset of 41 cases. Three p53 expression patterns were found: 35 tumors were classified as pattern "A," characterized by high p53 expression including "mutant" conformation and missense mutations of the gene (16/17 cases tested), pattern "B" consisted of 15 tumors with total absence of p53 expression corresponding to nonsense mutations of the gene (8/9 cases tested), and pattern "C" of 20 tumors presenting low or undetectable nuclear-bound p53 but intermediate p53 protein content (pAb (1801+) in the soluble fraction. The latter pattern was associated with wild-type genes (14/15 cases tested), and with tumors that were often localized in the right colon compared to pattern "A" and "B" tumors (45% versus 8%, P < 0.009) and were frequently near-diploid (80% versus 29%, P < 0.0002). No correlation was found between tumor stage and the patterns of p53 expression. The results indicate that both flow cytometry (FCM) and ELISA seem necessary for the proper characterization of the p53 expression pattern, thus achieving a high degree of concordance with molecular analysis of gene mutations.

CD26 expression and dipeptidyl peptidase IV activity in an aggressive hepatosplenic T-cell lymphoma.

Year 1998
Ruiz P. Mailhot S. Delgado P. Amador A. Viciana AL. Ferrer L. Zacharievich N.
Department of Pathology, University of Miami School of Medicine, Florida 33101, USA. pruiz@mednet.med.miami.edu
The transmembrane serine aminopeptidase dipeptidyl peptidase IV (DPP IV) (also known as CD26) participates in several immunological functions and has a binding affinity for several molecules, including collagen, which may be an integral mechanism for T cells to traverse endothelial barriers. Since CD26 is phenotypically expressed in certain T-cell malignancies, this study utilized a novel four-color cytofluorographic procedure to measure DPP IV enzymatic activity concurrently with the expression of other surface markers in an aggressive hepatosplenic T-cell lymphoma. Immunophenotypic analysis by flow cytometry revealed the tumor to be CD2+, CD3+, CD5-, CD7+, TcR-gamma/delta+, CD4-, CD8+/-, CD56+, and CD11c+. The CD26 molecule was also expressed, and DPP IV activity was present, with the maximal activity detectable after 10 min of incubation. These results represent the initial description of enzymatically active CD26 in a T-cell malignancy, and raise the possibility that this molecule may be a participant in the pathogenetic mechanisms utilized by the neoplastic cells.

Flow cytometry description of a novel CD3-/CD7+ intraepithelial lymphocyte subset in human duodenal biopsies: potential diagnostic value in coeliac disease.

Year 1998
Eiras P. Roldan E. Camarero C. Olivares F. Bootello A. Roy G.
Servicio de Inmunologia, Hospital Ramon y Cajal, Madrid, Spain.
Intraepithelial lymphocytes (IEL) represent a heterogeneous cellular compartment of unknown functions and controversial ontogeny. Previous observations in humans indicate that the majority of IEL subsets express the CD3 complex associated with either the alphabeta or the gammadelta T-cell receptor components, and describe the characteristic increase of CD3+TCRgammadelta+ IEL in coeliac disease. In the present work, we analyze the surface antigen expression of intraepithelial lymphocytes isolated from duodenal biopsies of control subjects and coeliac disease patients. We describe a CD3-CD7 + IEL subset frequently found in control subjects (41.41+/-21.8), with the following features: 1) most of these cells are CD45R0+ CD103+ and CD44- CD28- CD5-; 2) a significant percentage express CD56 (44.7%+/-21.3), CD2 (55.1%+/-16.2), and CD94 (16.2%+/-7.3). Furthermore, they are CD122+ and CD25-; 3) this CD3- IEL subset exhibit an activated phenotype expressing higher levels of CD69, CD103, and CD38 than the CD3+ subset. Interestingly, this CD3- subset is drastically reduced in CD patients (2.2+/-2.9 in active disease, 6.3+/-4.6 in treated patients versus 41.4+/-21.8 in control subjects). The imbalanced ratio "increased TCRgammadelta versus decreased CD3- CD7+" is a permanent finding in CD patients following clinical and histological remission. This parameter might provide helpful diagnostic information (easily obtained by 3-color FCM from diagnostic biopsies), and suggest a potential implication in the pathogenesis of coeliac disease.