Prognostic information and biological insights in chronic lymphocytic leukemia by high-resolution immunophenotypic analysis of ZAP70

Abstract

The E2997 Phase III trial included preservation of valuable chronic lymphocytic leukemia (CLL) patient specimens and relevant clinical outcome data. Using a novel high-resolution technology on a flow cytometry platform, we assessed 79 E2997 samples for the expression of 27 analytes that reflected the activity of signaling pathways and apoptosis. We found that the expression levels of ZAP70 segregated the samples into two subpopulations with the distribution showing a peak-trough-peak configuration. Although prior assessment of ZAP70 by standard procedures did not reveal any prognostic information, we found by using the trough in the distribution as a cutpoint that ZAP70 expression levels were significantly correlated with both progression-free survival and overall survival. Additionally, the cells expressing high versus low levels of ZAP70 demonstrated distinct molecular organization as indicated by the other analytes assessed. Our analysis demonstrates the value of ZAP70 expression as a prognostic indicator and suggests that the different clinical results may be due to the distinct molecular biology of the ZAP70-low versus the ZAP70-high CLL samples.

Keywords: ZAP70; chronic lymphocytic leukemia; phospho-ZAP70; prognosis.

© 2014 International Society for Advancement of Cytometry.

Figures

Figure 1. Representative flow cytometric results

Frozen CLL from the E2997 clinical trial were thawed, stained for CD19 expression, stained for the expression of various analytes, processed by EAS™, and analyzed on a flow cytometer. The gating strategy and flow cytometric histograms are shown. The histograms of the various analytes are shown after gating on the CD19+ events. The dark outlined histogram represents specific stain and the light outlined histogram represents the negative control.

Figure 2. ZAP70 Expression Distribution in CLL Cells

a) ZAP70 expression levels for 77 CLL samples from the E2997 clinical trial were classified into bins of fluorescence intensity and graphed as a distribution. ZAP70 staining was processed by EAS™. The superimposed curve was drawn for visualization of the distribution. The arrow shows the trough of the distribution which was used to stratify the samples. b) The distribution of ZAP70 expression levels in CD19+ B lymphocytes in 42 samples from healthy adult volunteers. The superimposed curve was drawn for visualization of the distribution. c) The distribution of ZAP70 expression levels for the 77 CLL samples analyzed in part a; however, the samples were processed by standard staining methods. ZAP-70 expression was measured as a proportion of the level seen in T lymphocytes from the same sample.

Figure 3. Comparison of ZAP70 Expression by a Standard Method and EAS™

The same 77 CLL samples were assessed for ZAP70 expression by direct staining and reporting by the ratio of fluorescence in the CLL cells compared to the T lymphocytes in each sample and by EAStm and reporting by the ratio of fluorescence in the cells stained with a specific antibody compared to control staining. The correlation coefficient r is 0.60 and the p value is less than 0.001.

Figure 4. Survival Stratified by ZAP70 Expression

CLL samples were stratified based on the level of ZAP70 expression, and progression-free survival (top panel) and overall survival (OS; bottom panel) plots are shown for the stratified groups separately. The p values obtained by the log rank test are shown in the upper right-hand corner of each panel.

Figure 5. Bivariate Correlations

Expression levels of Musashi-2 and PUMA (upper left panel), Bcl-2 and Mcl-1 (lower left panel), phospho-Akt(thr308) and Bcl-2 (upper right panel), and Bak and Bax (lower right panel) are shown for all 79 CLL samples. Each symbol represents an individual sample. Linear regressions are shown. Pearson product moment correlation coefficients are shown in each panel.

Figure 6. Bivariate Correlations Stratified by ZAP70 Expression

a) Expression levels of ZAP70 versus Bcl2L12 (top row), Mcl-1 (middle row), and SHP-1 (bottom row) are shown in samples stratified for ZAP70 expression. Samples with high levels of ZAP70 expression are shown in the left column, and samples with low levels of ZAP70 expression are shown in the right column. Each symbol represents an individual sample. Linear regressions are shown. The Pearson product moment correlation coefficients with the associated p values are shown in each panel. b) Correlations of phospho-ZAP70 and ZAP70 with 23 other analytes with the samples stratified into high ZAP70 expression and low ZAP70 expression. The analytes with correlations greater than 0.5 are shown within a box and by a thick black connector. Analytes in light gray are significantly correlated with phospho-ZAP70 in the samples with low ZAP70 expression and also with ZAP70 in the samples with high ZAP70 expression. Analytes in dark gray are uniquely correlated with phospho-ZAP70 in samples with low ZAP70 expression and with ZAP70 in samples with high ZAP70 expression. There are no correlations with r greater than 0.5 with ZAP70 in the samples with low ZAP70 expression and with phospho-ZAP70 in the samples with high ZAP70 expression.

Figure 6. Bivariate Correlations Stratified by ZAP70 Expression

a) Expression levels of ZAP70 versus Bcl2L12 (top row), Mcl-1 (middle row), and SHP-1 (bottom row) are shown in samples stratified for ZAP70 expression. Samples with high levels of ZAP70 expression are shown in the left column, and samples with low levels of ZAP70 expression are shown in the right column. Each symbol represents an individual sample. Linear regressions are shown. The Pearson product moment correlation coefficients with the associated p values are shown in each panel. b) Correlations of phospho-ZAP70 and ZAP70 with 23 other analytes with the samples stratified into high ZAP70 expression and low ZAP70 expression. The analytes with correlations greater than 0.5 are shown within a box and by a thick black connector. Analytes in light gray are significantly correlated with phospho-ZAP70 in the samples with low ZAP70 expression and also with ZAP70 in the samples with high ZAP70 expression. Analytes in dark gray are uniquely correlated with phospho-ZAP70 in samples with low ZAP70 expression and with ZAP70 in samples with high ZAP70 expression. There are no correlations with r greater than 0.5 with ZAP70 in the samples with low ZAP70 expression and with phospho-ZAP70 in the samples with high ZAP70 expression.