ALBERT

Description: Key PointsActivation of BCR and canonical NF-κB signaling in the lymph node correlates with survival in MCL. Mutations and polymorphisms in BCR and NF-κB pathways may confer cell autonomous signaling and affect response to ibrutinib.

Description: Chronic lymphocytic leukemia (CLL) cells depend on microenvironmental factors for proliferation and survival. In particular, tissue resident CLL cells show an increase in both B-cell receptor (BCR) and NF-κB signaling; pathways known to regulate survival, proliferation and migration of CLL cells. One key signaling molecule in this pathway is Bruton’s tyrosine kinase (BTK) that is activated directly downstream of the BCR and known to be up-regulated in CLL cells (Herman et al., Blood 2011). Ibrutinib, a covalent BTK inhibitor currently in clinical trials for CLL, has been shown to induce apoptosis and inhibit proliferation and tumor burden both in vitro and in mouse models of CLL (Herman et al., Blood 2011; Ponader et al., Blood 2012, Herman et al., Leukemia 2013). Recently, in a multicenter study, ibrutinib has been shown to induce objective clinical responses and reduce lymphadenopathy in the majority of patients, regardless of the presence of adverse prognostic markers (Byrd et al., NEJM 2013). It has been shown that CLL cells in the lymph node and bone marrow microenvironments demonstrate higher levels of BCR and NF-κB signaling as well as increased cell activation and proliferation (Herishanu et al., Blood 2011). We therefore sought to determine the in vivo effect of ibrutinib on tumor cell activation and proliferation in these microenvironmental niches. We have previously demonstrated that ibrutinib inhibits BCR and NF-κB in the lymph node microenvironment within the first 24 hours after initiation of therapy (Herman et al., ASH 2012). We here expand our previous results by evaluating the long term effects of ibrutinib in the tissue compartment. Because repeat sampling of lymph node tissue on therapy is not practical, we assessed changes in the bone marrow compartment. We obtained bone marrow aspirates pre-treatment and after two cycles of ibrutinib (Day 56). We first evaluated the BCR gene signature using a previously validated set of BCR regulated genes (Herishanu et al., 2011). We found that BCR signaling was significantly inhibited in CLL cells sampled from the bone marrow in 8/8 patients evaluated (P = 0.01). Similarly we also found inhibition of the NF-κB gene signature in all patients evaluated (P = 0.01). In fact, every patient evaluated demonstrated a reduction in both signatures, but there was substantial variation among patients in the extent to which these pathways were inhibited. This variability did not appear to correlate with clinical and prognostic factors, such as IGHV mutation status, deletion of chromosome 17p, or prior treatment status. However, the degree of inhibition of NF-κB signaling was strongly correlated with the degree of inhibition of BCR signaling (r = 0.96, P 〈 0.001), suggesting that the BCR (and/or a second equally BTK dependent pathway) plays a major role in activating NF-κB also in bone marrow resident CLL cells. To confirm our results we next evaluated the phosphorylation of proteins activated downstream of the BCR. We found that CLL cells showed a significant reduction in both PLCγ2 and ERK phosphorylation (mean reduction 52.9% and 71.2%, respectively; P 〈 0.01). Next, using the proliferation marker Ki67 we found a significant reduction in tumor proliferation in the bone marrow on ibrutinib (from a median of 6.6% KI67+ CLL cells pre-treatment to 1.1% on Day 56, P = 0.003). Lastly, we also found a significant reduction in the cell surface expression of the activation markers CD69 and CD86 (mean reduction of 57% (P = 0.001) and 82% (P = 0.03), respectively). In conclusion, our data demonstrate that ibrutinib effectively inhibits BCR, NF-κB, and ERK signaling. This occurs very quickly as demonstrated in the lymph node and is sustained on treatment as shown in the bone marrow. The strong and sustained reduction in proliferation and activation of CLL cells in the tissue microenvironment suggests that BTK is indeed a central hub mediating the nourishing and protective effects of the tumor microenvironment. This work was supported by the Intramural Research Program of NHLBI, NIH. We thank our patients for donating blood and tissue samples to make this research possible. We acknowledge Pharmacyclics for providing study drug. Disclosures: Off Label Use: Ibrutinib in chronic lymphocytic leukemia.

Description: Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of mature, monoclonal B-cells in the peripheral blood (PB), bone marrow, and secondary lymphoid organs. Ibrutinib targets Bruton’s tyrosine kinase (BTK); which is activated downstream of the B-cell receptor (BCR), a pathway known to regulate the survival, proliferation, and migration of B-cells. In vitro, ibrutinib has been shown to induce apoptosis and inhibit proliferation (Herman et al., Blood 2011). Recently, clinical trials demonstrated that ibrutinib is well tolerated and can induce objective clinical responses in CLL (Advani et al., JCO 2013; Byrd et al., NEJM 2013). Interestingly, ibrutinib leads to a transient lymphocytosis that resolves over the first 2-3 months on treatment. This lymphocytosis is thought to arise because of a redistribution of CLL cells due to the release of tumor cells from the tissue compartment into the peripheral blood and/or due to inhibition of re-entry of circulating cells into the tissue sites. In our single agent phase II trial of ibrutinib in CLL (NCT01500733) over 40% of patients reached their peak absolute lymphocyte count (ALC) already within 24 hours of starting treatment. We therefore hypothesized that the increase in ALC may be primarily due to efflux of cells from the lymph node, and that this would result in an increased number of cells in the blood having the immunophenotypic characteristics of tissue resident cells. Indeed, compared to the matching pre-treatment samples, circulating CLL cells on day 2 showed increased expression of the activation marker CD38 and of the proliferation marker Ki67 (P 〈 0.01). Consistent, with a treatment-induced release of already activated cells from tissue sites, by day 14 the circulating cells have lower expression of CD38 and Ki67 than at baseline. Next we sought to determine whether ibrutinib decreases the expression of the adhesion molecules CD62L and CD49d. By day 28 of treatment, we indeed found a statistically significant decrease in the expression of CD62L (P= .02; median reduction 38%) but only a minimal reduction in CD49d (median reduction 13%; P 〉 0.05). To evaluate the in vivo effect of ibrutinib on cell adhesion we chose a functional assay that models tumor-stromal interactions in the tissue microenvironment. We collected PB tumor cells from patients pre-treatment and on day 28 of treatment. In vitro, these cells were allowed to adhere to fibronectin-coated plates for 1 hour. After rigorous washing, we quantified the number of adherent cells by fluorescence microscopy. CLL cells from ibrutinib treated patients showed a dramatic reduction in their ability to adhere to extracellular matrix component fibronectin compared to matching pre-treatment cells (median inhibition 96%, range 75%-99%; P〈 0.001). This reduction in adhesion was demonstrable both within 24 hours and after 4 weeks on therapy. Interestingly, the inhibitory effect of ibrutinib on cell adhesion was independent of IGHV mutational status. In conclusion, we present direct in vivoevidence that the ibrutinib induced lymphocytosis develops within hours and is, in significant part, due to the release of previously activated cells from the tissue microenvironment. The rapid onset of the lymphocytosis and the dramatic defect in adhesion, in the absence of major changes in the expression of adhesion molecules, suggests that ibrutinib directly interferes with an intracellular signaling network required for cell adhesion. This work was supported by the Intramural Research Program of NHLBI, NIH. We thank our patients for donating blood samples to make this research possible. Also, we acknowledge Pharmacyclics for providing study drug. Disclosures: Off Label Use: Ibrutinib in chronic lymphocytic leukemia.

Description: Key Points Ibrutinib inhibits both BCR and NF-κB signaling in lymph node and bone marrow resident CLL cells. Rapid and sustained reduction of cellular activation and tumor proliferation was achieved in all anatomic compartments.