ZAP-70 changes

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ZAP-70 levels do change in CLL but not very often.Terry J Hamblin

Abstract
Evidence is accumulating that in some patients with CLL the expression of ZAP-70 changes over time. This diminishes its role as a prognostic marker but increases our understanding of the pathophysiology of the disease.

Commentary
Although the mutational status of the immunoglobulin variable region (IgVH) genes remains the gold standard for prognostic markers in chronic lymphocytic leukaemia (CLL), it is a complicated assay unsuited to routine laboratories. Unfortunately, attempts to find a surrogate have run into several obstacles. CD38 expression measured by flow cytometry was an early contender and initially seemed to have as much predictive value IgVH mutations. Unfortunately, it was subsequently found to give disparate results in 28% and even worse was described as changing over time in 24% (1).

More recently, ZAP-70 expression has attracted great interest. Although transiently expressed during certain stages of B-cell development, most mature peripheral blood B cells do not express ZAP-70, a cytoplasmic tyrosine kinase which is the key signalling molecule for T-lymphocytes and NK-cells. In CLL it acts as an auxiliary molecule for B-cell signalling in the more aggressive cases, probably acting to block inhibitors of signalling through syk. It’s presence in aggressive CLL appears to be due the chaperoning function of heat shock protein 90 (Hsp90). After it was originally identified in gene expression microarrays as a gene differentially expressed in CLL cells with unmutated IgVH genes, several laboratories developed flow cytometric assays that potentially could be used by routine laboratories. Initially, there was 95% concordance with IgVH mutational status, but a later assay using the much more convenient direct immunofluorescence, showed concordance of only 77%, hardly better than CD38, although with this assay ZAP-70 appeared to perform better than IgVH mutations as a prognostic factor (reviewed in 2).

The disparity betweens these assays has prompted a search by several laboratories for an assay that would be reliable, reproducible and of prognostic value. This quest is currently unresolved. The type of anticoagulant to use, allowable storage and transit time, which gating strategy, which monoclonal antibody and which fluorescent dye all remain in dispute and how to express the results is still a contentious issue (3). Nevertheless, several publications have attested to the fact that like IgVH mutations and unlike CD38, ZAP-70 is stable over time.

In today’s issue of Leukemia and Lymphoma, Poulain et al (4) dispute this. Four out of 33 ZAP-70 negative cases subsequently became ZAP-70 positive and this was associated with clinical progression. Two out of 27 ZAP-70 positive patients became negative. In one case this was associated with corticosteroid treatment, a phenomenon that has been observed previously. Although previous publications have reported on the stability of ZAP-70 over time, close scrutiny of these publications confirms that in a small number of patients (around 10%) ZAP-70 expression does change as time passes, although until Poulain et al there has been no indication as to why.

Recently Boelens et al (5) reported that ZAP-70 expression in CLL is higher in lymph node cells than in peripheral blood cells and Cutrona et al (6) demonstrated that within normal germinal centres there is a population of ZAP-70 positive, CD38 positive B cells representing in vivo activated cells.

These observations add to our understanding of ZAP-70 in CLL. It seems likely that both ZAP-70 and CD38 are normal features of activated B cells, rather than markers of neoplastic aberration. In CLL activation events and cell division both take place in proliferation centres and here upregulation of both markers occurs. There seems to be a free exchange of cells between tissue and blood making the presence of both CD38 and ZAP-70 surrogates for the rapidity of cell turnover.

The mutational status of the IgVH genes is fixed in CLL making it unique as a prognostic marker; it assigns cases into one immutable subgroup or the other. Other markers, whether CD38 or ZAP-70 expression or chromosomal abnormalities, can be acquired during the course of the disease. Early stage patients can gain little comfort from knowing that they are not present at diagnosis.


References

1. Hamblin TJ, Orchard JA, Ibbotson RE, Davies Z, Thomas PW, Stevenson FK, Oscier DG. CD38 expression and immunoglobulin variable region mutations are independent prognostic variables in chronic lymphocytic leukemia, but CD38 expression may vary during the course of the disease. Blood 2002, 99:1023-1029.
2. Hamblin AD, Hamblin TJ. Functional and prognostic role of ZAP-70 in CLL. Expert Opinion on Therapeutic Targets 2005; 9:1165-1178.
3. Marti G, Orfao A, Goolsby C. ZAP-70 in CLL: Towards standardization of a biomarker for patient management: History of Clinical Cytometry Special Issue. Cytometry Part B Cin Cytometry 2006; 70B:197-200.
4. Poulain S, Benard C, Daudignon A, Le Baron F, Morel P, Duthilleul P. Is ZAP-70 expression stable over time in B chronic lymphocytic leukaemia? Leukemia and Lymphoma, 2007; 48: 1219-21.
5. Boelens J, Philippe J, Offner F. B-CLL cells from lymph nodes express higher ZAP-70 levels than B cells from peripheral blood. Leuk Res 2007; 31:719-20.
6. Cutrona G, Colombo M, Matis S, Reverberi D, Dono M, Tarantino V, Chiorazzi N, Ferrarini M. B lymphocytes in humans express ZAP-70 when activated in vivo.
Eur J Immunol. 2006; 36:558-69.