Abstract 3531: Changes in immune cell populations in relapsed/refractory CLL patients treated with a Bruton's Tyrosine Kinase (BTK) Inhibitor, Ibrutinib (PCI-32765), in combination with Bendamustine and Rituximab (BR)

Although chemoimmunotherapy has led to higher response rates in chronic lymphocytic leukemia (CLL), most patients relapse due to emergence of resistant subclones. Here, we analyzed changes in immune cell (IC) populations of 29 subjects with relapsed/refractory CLL enrolled in PCYC-1108, a Phase 1b, open-label, parallel-group, nonrandomized, multicenter study of ibrutinib (PCI-32765) in combination with BR (O'Brien et al; ASCO 2012). Mononuclear cells were collected and cryopreserved at 4 time points: 1: just prior to therapy, 2: cycle 1 day 15 (d15), 3: at start of cycle 3 (d 60) and; 4: at the end of study (d 180). Based on a decrease in absolute lymphocyte count (ALC), 27/29 patients responded to this combination therapy. We analyzed residual B, T and monocyte populations and prognostic markers, CD38 and CD49d in each patient and grouped the cases based on percent reduction of CD19+CD5+ (“CLL B cells”) at all time points while on therapy. 10/29 cases (∼34%) showed > 50% reduction (Grp1) whereas 41% (12/29) showed greater retention of CLL cells (Grp2), and 7/29 cases (∼24%) showed variable changes (Grp 3). Median reduction in ALC in Grp1 was 97.8% (range: 68.3-99.75) vs 87.7% (range: 77.3- 99.6) for Grp2. Grp 1 cases showed a decrease in CD23+ and CD38+ CLL B cells at d15; although CD23+ cells decreased further CD38+ cells increased in 8/10 Grp1 cases. CD49d+ CLL cells remained stable in 6/10 Grp 1 and 11/12 Grp 2 cases but increased in 4/10 Grp1 cases. A marked increase in CD3+ and CD14+ cells accompanied the decrease in CLL B cells in cases in Grp1. Grp2 followed a different trend: CD3+ cells decreased until d60 and eventually increased, albeit mildly whereas their percentages of CD14+ cells did not change throughout therapy. Such dynamic changes in IC populations prompted us to assess the presence of dividing CLL cells, in the face of reduced ALC. Surprisingly, although Grp1 showed decreased percentages of CLL cells, 3/10 cases developed increased numbers of Ki-67+ CLL cells, achieving 19-43% at d180. In Grp2, only 0.1-4.0% Ki-67+ CLL B cells were found. BCR-induced phosphorylation of BTK was analyzed by phosphoflow. Both Grp1 and Grp2 exhibited increased BTK phosphorylation at d15 which decreased precipitously at subsequent time points. Together, these studies indicate that combining ibrutinib with BR effectively reduced numbers of circulating CLL cells in >93% of cases, but relative proportions of CLL cells only in ∼1/3 relapse/refractory CLL cases.