Expressió de ZAP-70 en linfòcits B normals i síndromes limfoproliferatives B

La Leucemia Limfatica Cronica (LLC), la mes frequent de les leucemies en el mon occidental, es una malaltia produida per la proliferacio i cumul de limfocits B madurs, actualment incurable. Aproximadament el 50 de les LLCs presenten hipermutacions al gen de les immunoglobulines (IgVH), el que indica que podrien representar un subgrup diferent de cellules que passen pel centre germinal, el lloc fisiologic de les hipermutacions.6. A mes a mes, els pacients sense hipermutacions tenen un pitjor pronostic. L'analisi de les immunoglobulines es de gran interes pel pronostic dels malalts dLLC. Malauradament aquest es costos i llarg. Es per aixo que una de les prioritats a la recerca en LLC es identificar marcadors relacionats amb les hipermutacions de les immunoglobulines. Recentment sha descrit la presencia de la proteina ZAP-70 en cellules de LLC sense mutacions a les immunoglobulines. ZAP-70 es una kinasa de la familia de tirosines kinases Syk/ZAP-70 que sexpressa normalment en limfocits T i NK, on participa en la senyalitzacio a traves del TCR. Tambe es troba en limfocits pro/pre-B de ratoli, pero no s'ha descrit en limfocits B humans. La hipotesi de treball del present projecte consisteix en que la expressio de ZAP-70 podria estar relacionada amb lestat mutacional de les immunoglobulines i, per tant, amb la progressio de la LLC cap a estadis mes avancats. Aquesta proteina podria trobar-se en limfocits pro/pre-B humans i en les neoplasies que en deriven, les leucemies agudes limfoblastiques B. RESULTATS I CONCLUSIONS: - El 57 % dels casos de LLC tenen una elevada expressio de ZAP-70. La determinacio de ZAP-70 per citometria es un factor pronostic rapid i fiable i de gran utilitat clinica en la LLC. - L'expressio de ZAP-70 es troba en limfocits pro/pre-B normals i desapareix en estadis maduratius posteriors. - El 56% de les leucemies agudes limfoblastiques B expressen ZAP-70, probablement com a reflex del seu origen cellular - La proteina ZAP-70 es troba expressada de manera aberrant en aproximadament un trenta per cent de les leucemies/limfomes de Burkitt, ja que no es troba el limfocits B de fenotip madur - la proteina ZAP-70 i daltres elements senyalitzadors del pre-TCR/TCR es troben expressats i fosforilats en les cellules de leucemia aguda limfoblastica B, possiblement collaborant amb altres proteines senyalitzadores en garantir la senyalitzacio a traves del pre-BCR. " SUMMARY: BACKGROUND: The mutational status of immunoglobulin variable region genes (IgVH) is an important prognostic parameter in chronic lymphocytic leukemia (CLL). Unfortunately, no good surrogates for IgVH mutations have as yet been identified. The ZAP-70 gene is overexpressed in a subset of CLL cases, this change correlating with the lack of mutations of IgVH. METHODS: To probe the suggestive link between ZAP-70 expression and IgVH mutations, ZAP-70 was analyzed in different cell lines and in 56 CLL pacients using flow cytometry, Western Blot, and/or immunohistochemistry methods. The results were correlated with the IgVH mutational status and patients outcome. RESULTS: Using flow cytometry ZAP-70 expression was higher in IgVH unmutated CLL (n=35) (48 percent of cells ± 21 percent) than in mutated cells (n=21) (5.7 percent of cells ± 3.8 percent). All CLL patients with ZAP-70 expression > 20 percent (n=32) lacked IgVH mutations, whereas 21/24 CLL with ZAP-70 < 20 percent exhibited IgVH mutations. Finally, ZAP-70 expression had prognostic significance for progression and survival in Binet A cases. CONCLUSIONS: ZAP-70 expression by flow cytometry correlated with the IgVH mutational status and had prognostic significance in CLL patients. Therefore, ZAP-70 analysis can be considered a reliable surrogate for the IgVH mutational status in CLL and a useful tool for assessing prognosis in this disease. PURPOSE: ZAP-70 gene is normally expressed in T and NK cells, where is required for the T-cell receptor signaling. It has been described that ZAP-70 contributes to the B-cell development at early stages of B-cell differentiation in mice. The purpose was to investigate the presence of ZAP-70 in normal pro/pre B-cells and mature B-cells, and in tumoral cells from B-acute lymphoblastic leukemias (B-ALLs). EXPERIMENTAL DESIGN: ZAP-70 expression was ascertained by flow cytometry, immunofluorescence, Western blot, and quantitative RT-PCR. Analysis of ZAP-70 and other signalling proteins of the pre-TCR/TCR was performed by Western Blot. RESULTS: ZAP-70 was expressed in pro/pre B-cells, but not in normal mature B-cells derived from bone marrow, peripheral blood or tonsil. Among tumoral cells, ZAP-70 was expressed in 56% of B-ALLs with pro/pre B-cell phenotype and in 4/6 Burkitt/ALL lymphomas. Moreover, other elements of the pre-TCR/TCR signaling pathway, like LAT and Lck, were also found in B-ALL cells. CONCLUSIONS: Amongst normal B-cell subsets, ZAP-70 was found expressed in normal pro/pre B-cells, but not in a significant proportion of normal B-cells with mature phenotype. Moreover, the presence of ZAP-70 in B-ALLs probably reflects their cellular origin. The lack of ZAP-70 expression in normal mature B-cells suggests that its expression in mature-derived neoplasms with different cellular origin, such as Burkitts lymphoma and chronic lymphocytic leukemia, might be due to an aberrant phenomenon. "