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Predicting Progression — ZAP-70 in CLL
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     The harder we look, the more we find. Diagnoses of chronic lymphocytic leukemia (CLL) are becoming more common because we are looking harder. With sensitive techniques, a monoclonal population of B lymphocytes that is indistinguishable from CLL cells may be found in the blood of 3.5 percent of persons older than 40 years of age. Even without such sophisticated methods, simple immunophenotyping, which is available to all, easily singles out CLL from the many other causes of slight lymphocytosis. Such a finding will never have clinical effects in most people — they will die with it rather than from it — but in some people in this group, we really are looking at an early stage of a killer disease.

    The traditional staging systems for CLL, Rai in the United States and Binet in Europe, involve looking for anemia or thrombocytopenia and feeling for lymph nodes, liver, and spleen. These staging systems are very good at telling us whom to treat at the time of diagnosis but not at telling us who will later need treatment. Clinical trials have shown that advanced-stage disease requires treatment, whereas early-stage disease does not. To be precise, treating early-stage CLL with chlorambucil offers no benefit over delaying treatment until the disease progresses. For patients with early-stage CLL, the advice has been "watch and wait," which is often interpreted by the patient as "wait and worry." And because we are looking harder, most patients have early-stage CLL.

    CLL is a tumor of B lymphocytes, and the purpose of B lymphocytes is to make antibodies. During an immune response, a series of point mutations occur in the immunoglobulin genes of a B cell — a process that changes the binding site of the antibody in a way that makes it fit its target antigen as snugly as possible. These somatic mutations occur in the germinal centers of the peripheral lymphoid tissue. The presence of somatic mutations thus acts as a sort of "visa stamp," certifying that a B cell has passed through a germinal center. By sequencing the immunoglobulin heavy-chain variable-region (IgVH) genes, researchers in England and New York were able to divide CLL into two types.1,2 In slightly more than half the cases, the leukemic cells had mutated IgVH genes. These patients had a relatively benign condition that was stable or only slowly progressive, with an average survival of 25 years. By contrast, patients with unmutated CLL cells had aggressive disease and an average survival of eight years.

    Unfortunately, sequencing genes is expensive and time-consuming, and only a few research laboratories have adopted the technique. An easily measured surrogate marker is required. Initially, CD38, a cell-surface enzyme involved in regulating B-cell activation, was thought to be that surrogate, but its measurement gives discordant results in about 30 percent of cases, and its levels may vary during the course of the disease. A comparison of the gene-expression profiles of the two subtypes of CLL identified about 240 genes that are expressed differently in the two types. Of these, the 70-kD zeta-associated protein (ZAP-70) is the most promising surrogate for gene sequencing.

    ZAP-70 is a key signaling molecule for T lymphocytes and natural killer cells. Although it does not normally function in B cells, it is anomalously expressed in CLL cells with unmutated IgVH genes and may enhance the signaling process when the B-cell receptor is engaged (see Figure). It has been technically difficult to develop assays for the measurement of ZAP-70, since T cells, which contain much more ZAP-70 than B cells, must either be separated or visualized separately. However, researchers in Barcelona, Spain, and Bournemouth, United Kingdom, have independently reported effective flow-cytometry assays in which the intracellular content of ZAP-70 closely correlates with the IgVH mutational status.3,4

    Figure. The Role of ZAP-70 in T-Cell and B-Cell Signaling.

    In T-cell signaling, collaboration between ZAP-70 and the other Src kinases, Lck and Fyn, leads to the activation of downstream signaling pathways, including nuclear factor B and mitogen-associated protein kinase, leading to cellular proliferation. In B cells, ZAP-70 is not normally involved in signaling. The engagement of the surface immunoglobulin by antigen alerts the immunoglobulin-associated molecules, CD79a and CD79b, which associate with and phosphorylate Syk, leading to downstream signaling. In CLL with unmutated IgVH genes (U-CLL), ZAP-70 is recruited to the B-cell–receptor complex and, in some as-yet-undiscovered way, enhances signaling. TCR denotes T-cell receptor, and BCR B-cell receptor.

    In this issue of the Journal, Rassenti and colleagues in the United States (pages 893–901) report a comparison between a flow-cytometry assay for ZAP-70 and the mutational status of IgVH genes in predicting the time to first treatment in a large series of patients with CLL. Although the results of the two assays were similar, 23 percent of the results of the two methods were discordant. This discordance rate is greater than the 5 to 6 percent reported by the two European groups. There may be several reasons for this discrepancy. The U.S. study was 50 percent larger than the two European studies combined. The anti–ZAP-70 antibodies used by the three groups differed, and different antibodies may give different results. And the population in the U.S. study was younger (median age, 55 years) than that in the Barcelona study (median age, 60 years) or the Bournemouth study (median age, 67 years).

    More encouragingly, the ZAP-70 level as measured by flow cytometry was apparently better than the mutational status at predicting the need for treatment, and in contrast to the CD38 level, the ZAP-70 level did not change over time. Nevertheless, it seems that knowing both the ZAP-70 level and the IgVH mutational status provides more useful prognostic information than knowing only one. It is too soon to abandon the sequencing of immunoglobulin genes.

    In the meantime, it is necessary to consolidate (through the exchanging of samples) the three methods for measuring ZAP-70 by flow cytometry so that everyone is singing from the same hymn sheet. It is also necessary to prepare for randomized, controlled trials, involving patients who have early-stage CLL and risk factors for a poor prognosis, in which the "watch and wait" approach is compared with the best available treatment (probably a combination of agents that includes fludarabine and a monoclonal antibody). With such a structured approach, CLL may become a paradigm for more common asymptomatic cancers, such as well-differentiated prostate cancer detected by means of serum prostate-specific–antigen levels and ductal carcinoma in situ detected by means of mammography.

    Source Information

    From the Department of Immunohaematology, University of Southampton, Southampton, United Kingdom.

    References

    Damle RN, Wasil T, Fais F, et al. Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood 1999;94:1840-1847.

    Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK. Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 1999;94:1848-1854.

    Crespo M, Bosch F, Villamor N, et al. ZAP-70 expression as a surrogate for immunoglobulin-variable-region mutations in chronic lymphocytic leukemia. N Engl J Med 2003;348:1764-1775.

    Orchard JA, Ibbotson RE, Davis Z, et al. ZAP-70 expression and prognosis in chronic lymphocytic leukaemia. Lancet 2004;363:105-111.(Terry J. Hamblin, M.D.)