7 research outputs found
Prognostic impact of ZAP-70 expression in chronic lymphocytic leukemia: mean fluorescence intensity T/B ratio versus percentage of positive cells
<p>Abstract</p> <p>Background</p> <p>ZAP-70 is an independent negative prognostic marker in chronic lymphocytic leukemia (CLL). Usually, its expression is investigated by flow cytometric protocols in which the percentage of ZAP-70 positive CLL cells is determined in respect to isotypic control (ISO-method) or residual ZAP-70 positive T cells (T-method). These methods, however, beside suffering of an inherent subjectivity in their application, may give discordant results in some cases. The aim of this study was to assess the prognostic significance of these methods in comparison with another in which ZAP-70 expression was evaluated as a Mean-Fluorescence-Intensity Ratio between gated T and CLL cells (T/B Ratio-method).</p> <p>Methods</p> <p>Cytometric files relative to ZAP-70 determination according to the three readouts were retrospectively reviewed on a cohort of 173 patients (test set), all with complete clinical and biological prognostic assessment and time-to-treatment (TTT) available. Findings were then validated in an independent cohort of 341 cases from a different institution (validation set).</p> <p>Results</p> <p>The optimal prognostic cut-offs for ZAP-70 expression were selected at 11% (ISO-method) or 20% of positive cells (T-method), as well as at 3.0 (T/B Ratio-method) in the test set; these cut-offs yielded 66, 60 and 73 ZAP-70<sup>+ </sup>cases, respectively. Univariate analyses resulted in a better separation of ZAP-70<sup>+ </sup>vs. ZAP-70<sup>- </sup>CLL patients utilizing the T/B Ratio, compared to T- or ISO-methods. In multivariate analyses which included the major clinical and biological prognostic markers for CLL, the prognostic impact of ZAP-70 appeared stronger when the T/B-Ratio method was applied. These findings were confirmed in the validation set, in which ZAP-70 expression, evaluated by the T- (cut-off = 20%) or T/B Ratio- (cut-off = 3.0) methods, yielded 180 or 127 ZAP-70<sup>+ </sup>cases, respectively. ZAP-70<sup>+ </sup>patients according to the T/B Ratio-method had shorter TTT, both if compared to ZAP-70<sup>- </sup>CLL, and to cases classified ZAP-70<sup>+ </sup>by the T-method only.</p> <p>Conclusions</p> <p>We suggest to evaluate ZAP-70 expression in routine settings using the T/B Ratio-method, given the operator and laboratory independent feature of this approach. We propose the 3.0 T/B Ratio value as optimal cut-off to discriminate ZAP-70<sup>+ </sup>(T/B Ratio less than 3.0) from ZAP-70<sup>- </sup>(T/B Ratio more/equal than 3.0) cases.</p
Applicability and reproducibility of acute myeloid leukaemia stem cell assessment in a multi-centre setting
Leukaemic stem cells (LSC) have been experimentally defined as the leukaemia-propagating population and are thought to be the cellular reservoir of relapse in acute myeloid leukaemia (AML). Therefore, LSC measurements are warranted to facilitate accurate risk stratification. Previously, we published the composition of a one-tube flow cytometric assay, characterised by the presence of 13 important membrane markers for LSC detection
A multicancer-like syndrome in a dog characterized by p53 and cell cycle-checkpoint kinase 2 (CHK2) mutations and Sirtuin gene (SIRT1) down-regulation.
INTRODUCTION: We have investigated SIRT1, p53 and cell cycle-checkpoint kinase 2 (CHK2) gene dysfunction in a dog with a multicancer syndrome-like in order to evaluate their potential role in the determinism of the disease and to establish a possible correlation between SIRT1 transcript level and p53 expression status. MATERIAL AND METHODS: Blood sample and tumour samples from a pure breed English Setter dog with different tumours were used for this study. Nucleotide sequence analysis was performed with a DNA autosequencer in order to examine p53 and CHK2 mutations. In addition, the expression level of SIRT1 was quantified by Southern Blot analysis of Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). RESULTS: Cytological examination revealed five different tumours: a cutaneous sebaceous epithelioma, a cutaneous mast cell tumour, a testicular Sertoli cell tumour, an oral malignant melanoma, and a cutaneous squamous cell carcinoma. Sequencing analysis revealed the presence of a nucleotide substitution, (CGG>CAG) exon 7 of the p53 gene in DNA from peripheral blood mononuclear cells (PBMCs) as well as in the melanoma; whereas the other four cancers showed the loss of the wild-type allele. Furthermore, CHK2 mutation at codon 311 has been identified in the melanoma and sebaceous epithelioma. In addition, SIRT1 cDNA expression decreased in all tumour samples compared to cDNA SIRT1expression level in peripheral blood mononuclear cells (PBMCs) in the same dog. CONCLUSIONS: These results suggest that the germ line mutation of the p53 gene at codon 248 might be, at least, one cause of the multicancer syndrome-like in our dog; furthermore, we show a possible correlation between SIRT1 transcript level and p53 mutations status. The regulatory role of SIRT1 in tumour suppressor pathways suggests that the net effect seen may represent both direct and indirect downstream regulation and it is likely to depend on the presence or absence of functional p53
A monoclonal antibody against mutated nucleophosmin1 for the molecular diagnosis of acute myeloid leukemias
Mutations in the nucleophosmin 1 (NPM1) gene are the most frequent genetic
aberrations of acute myeloid leukemia (AML), and define a clinically distinct subset
of AML. A monoclonal antibody (T26) was raised against a 19-aminoacid
polypeptide containing the unique C-terminus of the type A NPM1 mutant protein.
T26 recognized 10 of the 21 known NPM1 mutants, including the A, B and D types,
which cover ~95% of all cases, and did not cross-react with wild type NPM1 or
unrelated cellular proteins. It performed efficiently with different detection
technologies, including immunofluorescence, immunohistochemistry and flow
cytometry. Within a series of consecutive de novo AML patients, 44/110 (40%) and
15/39 (38%) cases scored positive using the T26 antibody in immunofluorescence and
flow cytometry assays, respectively. T26-positive cases were found to be all carrying
mutations of NPM1 exclusively, as determined by molecular analysis. T26 is the first
antibody that specifically recognizes a leukemia-associated mutant protein.
Immunofluorescence or flow cytometry using T26 may thus become a new tool for a
rapid, simple and cost-effective molecular diagnosis of AML