Zap-70 and IgV_H Mutation Status in Combination with p53 Functional Analysis Identifies the Response of Poor Prognosis B-CLL Patients to Conventional Cytotoxic Agents.

B-Chronic lymphocytic leukemia (B-CLL) patients whose malignant cells harbour unmutated immunoglobulin heavy chain variable region (IgV_H) genes or express the zeta-associated protein tyrosine kinase ZAP-70 show a worse prognosis than do patients with mutated IgV_H genes or ZAP-70^−ve expression. The inability of malignant cells to activate the pro-apoptotic p53 pathway in response to ionizing radiation (IR) also correlates with a poor prognosis. We studied ZAP-70 expression and IgV_H mutation status in 161 patients with B-CLL in order to determine the degree of concordance between these two prognostic criteria (M104/F57, wbc 2.44–576x10⁹/l lymphocytes 0.56–287x10⁹/l). We also studied the functional status of the p53 pathway and the apoptotic response to ionizing radiation in cells from a subset of patients from both prognostic categories. A human ZAP-70 antibody (clone 2F3-2) was conjugated to the Alexa Fluor 488 dye using a zenon mouse IgG labelling kit and used for a FACS based assay. FACS results were expressed as a ratio of B-cell mean cell fluorescence to T-cell mean cell fluorescence with a cut off at > 0.75 identifying a ZAP-70^+ve sub-group. IgV_H mutational status was studied by sequence analysis of FR1/J_H polymerase chain reaction products. The ability of 5Gy ionizing radiation to augment levels of p53 and its transcriptional target p21^CIP1 was quantified by western blot analysis. Cleavage of the caspase 3 target poly(ADP ribose) polymerase (PARP) was used as a measure of apoptosis induction. ZAP-70^+ve expression was observed in 25% (41/161) of the samples with a median ratio of 0.85 (range 0.76–1.46) while the remaining 120 samples were ZAP-70^−ve, with a median ratio of 0.56 (range 0.19–0.73). IgV_H mutation status was analysed in 92 of these patients. Assignment of prognostic category by both criteria was concordant in 72/92 (78.2%) of the cases of which 54/92 (58.6%) were ZAP^−ve/IgV_H mutated (good prognosis) and 18/92 (19.5%) were ZAP^+ve/IgV_H unmutated (poor prognosis) patients. The remaining 21.7% were discordant, ie., either ZAP^+ve/IgV_H mutated (5.4%) or ZAP^−ve/IgV_H unmutated (16.3%). Isolates from 5/6 ZAP^+ve/IgV_H unmutated patients upregulated p53 in response to IR but nevertheless failed to initiate PARP cleavage, suggestive of a block in the apoptotic pathway distal to p53 induction. In 9 ZAP^−ve/IgV_H mutated isolates studied, 7 induced p53, p21 and PARP cleavage following IR. In conclusion, this large cohort of CLL patients demonstrated a good correlation between ZAP-70 expression and IgV_H mutational status in identifying a poor prognosis sub-group. However, this prognostic category, as defined by both IgV_H mutation status and ZAP-70 expression failed in some cases to predict the ability of B-CLL cells to induce an apoptotic response to DNA damage in vitro. Induction of the p53 pathway was not always sufficient to secure an apoptotic response, especially in the poor prognosis group. A combination of ZAP-70 and IgV_H analysis with a functional assay for DNA damage-induced apoptosis will identify individuals in either prognostic category who are unlikely to respond to conventional cytotoxic drugs. Alternative therapeutic strategies independent of DNA damage-inducing agents may be of value in the treatment of these patients.