ZAP-70 Expression Assessed by Immunohistochemistry Correlates with Time to First Treatment in Patients with Chronic Lymphocytic Leukemia.

ZAP-70 expression is a prognostic indicator in chronic lymphocytic leukemia (CLL). Initial studies measured intracellular ZAP-70 expression by flow cytometry and defined a cut-off of 20% as predictive of time from diagnosis to initial therapy and overall survival. However, due to technical challenges, standardization of methods for evaluation of ZAP-70 expression has been difficult. This has resulted in a limitation of applicability for a potentially useful clinical test. Immunohistochemistry (IHC) is easy to perform and interpret, and therefore may be a more reliable and accurate means for assessing ZAP-70 expression. We investigated the potential prognostic implications of ZAP-70 expression assessed by IHC.

We searched our CLL database for all patients seen at Weill Cornell Medical College between 2007 and 2009 with an identifiable date of diagnosis, date of first therapy, IgVH mutation status, and ZAP-70 status. All patients gave informed consent to participate in the research database. Paraffin-embedded cell blocks were prepared from formalin-fixed peripheral blood mononuclear cells and evaluated by IHC for PAX5, CD3, and ZAP-70. The entire cell clot was reviewed. If more than 20% of cells faintly expressed ZAP-70, the ZAP-70 status was considered to be positive. Patients had IgVH mutations status determined by a commercial laboratory with <98% sequence homology used as a cut-off. Time from diagnosis to first treatment (TT) was estimated by the Kaplan-Meier method; comparisons were performed by log-rank test. Pearson's pairwise correlation coefficient was used to evaluate the relationship between ZAP-70 and IgVH.

One-hundred thirty-seven patients with date of diagnosis and date of first treatment were identified in the database. Sixty-six patients have been treated to date. ZAP-70 status was available for 106 patients, 62 were positive, 44 were negative. In a subset of 20 patients, two blinded pathologists reviewed IHC and found perfect agreement on ZAP-70 status. IgVH mutation status was available for 86 patients, 34 were unmutated, and 52 were mutated. Eighty-two patients had both ZAP-70 status and IgVH mutation status available. There was good correlation between ZAP-70 and IgVH status (r=0.36, p=0.0008). Seventy-five percent of those with unmutated IgVH were ZAP-70 positive. Sixty-one percent of patients with mutated IgVH were ZAP-70 negative. However, only 57% of ZAP-70 positive patients were IgVH unmutated. The predicted median TT was 54 months for the whole cohort (range 0-376+ months, 95% CI 45-96 months). The median TT was 27 months (range 0-112 months) for ZAP-70-positive patients compared to 96 months (range 0-376+ months) for ZAP-70-negative patients (p=0.002). The median TT was 32 months (range 0-112 months) for IgVH unmutated patients compared to 88 months (range 0-376+ months) for IgVH mutated patients (p=0.01).

ZAP-70 as measured by IHC was feasible in a large cohort of patients and ZAP-70 status correlated with time to first treatment. These data suggest that ZAP-70 assessment by IHC may be a reliable prognostic tool in CLL and should be evaluated in larger, multicenter clinical studies.

No relevant conflicts of interest to declare.