Ibrutinib-induced lymphocytosis in patients with chronic lymphocytic leukemia: correlative analyses from a phase II study

Abstract

Ibrutinib and other targeted inhibitors of B-cell receptor signaling achieve impressive clinical results for patients with chronic lymphocytic leukemia (CLL). A treatment-induced rise in absolute lymphocyte count (ALC) has emerged as a class effect of kinase inhibitors in CLL and warrants further investigation. Here we report correlative studies in 64 patients with CLL treated with ibrutinib. We quantified tumor burden in blood, lymph nodes (LNs), spleen and bone marrow, assessed phenotypic changes of circulating cells and measured whole-blood viscosity. With just one dose of ibrutinib, the average increase in ALC was 66%, and in>40% of patients the ALC peaked within 24 h of initiating treatment. Circulating CLL cells on day 2 showed increased Ki67 and CD38 expression, indicating an efflux of tumor cells from the tissue compartments into the blood. The kinetics and degree of the treatment-induced lymphocytosis was highly variable; interestingly, in patients with a high baseline ALC the relative increase was mild and resolution rapid. After two cycles of treatment the disease burden in the LN, bone marrow and spleen decreased irrespective of the relative change in ALC. Whole-blood viscosity was dependent on both ALC and hemoglobin. No adverse events were attributed to the lymphocytosis.

Trial registration: ClinicalTrials.gov NCT01500733.

Figure 1

Ibrutinib-induced lymphocytosis develops rapidly, peaks within days, and is highly variable between patients. (a–d) Data on 41 patients with complete counts for the first six months on ibrutinib are depicted. (a) Change in mean ALC over time; vertical lines indicate SEM. (b) Mean relative change of ALC on treatment over baseline; vertical lines indicate SEM. (c) Cumulative proportion of patients reaching their peak ALC at the indicated time point. Note >40% of patients reached the peak ALC on day 2. (d) Box and whisker plots demonstrating distinct inter-patient variability in ALCs.

Figure 2

Variability in development and resolution of ibrutinib-induced lymphocytosis. (a) Hierarchical clustering of change in ALC normalized to baseline (day 1) in individual patients (n=41) reveals three distinct patterns. The degree of change is color coded according to the legend shown. (b–c) The mean for each of the three patient clusters is shown as (b) ALC or (c) relative change of ALC compared to pre-treatment. (d) The pre-treatment ALC for the three patient clusters are significantly different by Student’s T-test.

Figure 3

Ibrutinib rapidly decreased total tumor burden and increases the rate of cell death (a) A graphic representation of the estimated tumor burden in different anatomic compartments pre-treatment is shown for four representative patients (identified by study code). (b–d) Change in tumor burden from pre-treatment (Pre) to 2 and 6 months (MO) on treatment in different tissues. (b) Graphic representation of total lymph node volume computed from whole body CT scans. (c) Graphic representation of spleen volume computed from CT scan. (d) CLL cell infiltration of bone marrow visualized by CD79a staining. (e) Changes in disease burden on treatment is shown for the four representative patients. Also see Supplementary table S1. (f) The viability of circulating CLL cells was measured in fresh whole blood samples using the LIVE/DEAD stain (n=12, each symbol represents a different patient). Ibrutinib doubles the rate of cell death (P=0.03 by paired Student’s T-test.)

Figure 4

Ibrutinib induced lymphocytosis is driven by the release of cells from the lymph node. (a) More CLL cells in the lymph node (LN) express Ki67 (proliferation marker) and CD38 (activation marker) compared to CLL cells in the peripheral blood (PB). All data are from untreated patients, red symbols represent ibrutinib study patients (n=5). (b) Shown is the relative change in the frequency of Ki67 positive CLL cells in the peripheral blood on day 2 (after one dose of ibrutinib) compared to pre-treatment (n=28). (c) Change in the frequency of CD38 positive CLL cells on day 2 compared to pre-treatment (n=11, CD38+ patients only). (d) Efflux of CLL cells from the lymph node increases the fraction of Ki67 positive cells in the blood. Shown is the data for one representative patient. The frequency of Ki67 expressing CLL cells pre-treatment in lymph node (measured in single cell suspension by flow cytometry) and in the the peripheral blood is shown. On day 2, 30% of the ALC consists of “additional cells” and the frequency of Ki67 positive CLL cells has increased to 8.2%. If all the additional cells derive from the lymph node (with Ki67 expression in 12.8%) the predicted frequency of Ki67 positive cells is 8.3%. (e–f) Regression analysis of predicted vs. actual percent Ki67 expression (e) and ALC values (f). Dashed lines indicate 95% confidence intervals (n=8).

Figure 5

Whole blood viscosity is rarely elevated in CLL and is influenced by ALC and hemoglobin. (a) Correlation of whole blood viscosity (normal range 3.6–6 cP, shaded) with ALC (n= 105; R=0.41; P<0.001). (b) Correlation of whole blood viscosity with hemoglobin concentration (n= 105; R=0.59; P<0.001). (c) Whole blood viscosity pre-treatment (pre) and on treatment days 2 and 28. (d) Change in hemoglobin in patients with an initial value ≥ 10 g/dL (n=26). (e) Contour plot of whole blood viscosity as a function of both ALC and hemoglobin. Whole blood viscosity measurements are color coded from low (white) to high (red). Correlations by Pearson’s test.