Abstract 1789
Ibrutinib (PCI 32765) is an orally administered covalent inhibitor of Bruton's Tyrosine Kinase (BTK). Ibrutinib has significant activity in chronic lymphocytic leukemia/small ...lymphocytic lymphoma (CLL/SLL) and is typically well tolerated (Byrd ASCO 2011, O'Brien ASH 2011). Rarely serious bleeding in patients concurrently on oral anticoagulation has been reported but was not related to thrombocytopenia (O'Brien ASH 2011). However, grade 1 or 2 ecchymosis/contusion is a frequent adverse event in patients on ibrutinib. In addition to being essential for B cell receptor signaling BTK is also involved in the signaling of the glycoprotein (GP)VI and GPIV von Willebrand (vW) receptors (Liu, Blood 2006). Thus, it is possible that ibrutinib could increase the bleeding risk by interfering with thrombus formation. In addition, lymphoproliferative disorders and some drugs have been associated with acquired vW-disease (AvWD).
In an ongoing single center, open label phase II trial we treat CLL/SLL patients with ibrutinib 420 mg daily on 28 day cycles (NCT01500733). We measured platelet (PLT) function on the PFA-100 instrument, vW-factor (vWF) antigen levels and activity (vWF-Ag/vWF-Act), and factor VIII (FVIII) on baseline, days 2 and 28. Here we report on effects of ibrutinib on platelet counts and function in 25 patients who completed >2 cycles.
PLT counts prior to treatment ranged from 36 k/μl to 256 k/μl with a median of 102 k/μl. Twelve (48%) patients had a pre-treatment PLT count <100 k/μl. Median PLT counts for days 14, 28, and 56 increased to 140, 137, and 135 k/μl, respectively (P<.01). 76% of patients showed an increase after only 2 weeks on drug (median increase 25 k/μl (range 4–183 k/μl) that was sustained at subsequent timepoints. On day 14, 6 patients (24%) had a decrease in PLT count by a median of 13 k/μl from baseline; of these, 3 had a pre-treatment PLT count of <100 k/ul and 1 developed grade III thrombocytopenia (42 k/μl) that resolved to >100 k/μl by day 56. 20% (5 of 25) of patients reported grade 1 spontaneous ecchymosis with no correlation to platelet count, PFA testing, or vWF measurements. Of note we performed lymph node core biopsies in 35 patients taking ibrutinib with minimal bruising. Only 2 patients had more extensive local bruising/ecchymosis at the biopsy site.Test (normal range)baselineday 2day 28PLT count (161-347 k/μl)102140137PFA-100 EPI (86-154 sec)109110110PFA-100 ADP (73-129 sec)747979vWF-ACT (52-156 IU/dL)182nd99vWF-AG (50-197 IU/dL)158nd101FVIII (41-184 IU/dL)181nd157All values are median, nd = not done
In 19 patients PFA-100 measurements of epinephrine (EPI) and adenosine diphosphate (ADP) stimulated platelet aggregation times were available (test requires PLT count >100 k/μl). Median changes in closure times with EPI and ADP on treatment were not significantly different from baseline (See table). Four (21%) patients started with abnormally prolonged EPI closure times (one on aspirin, one on ibuprofen; discontinued with the start of ibrutinib) which resolved by day 28 in 3 and decreased in 1. Three (16%) patients had a prolongation of EPI closure times on day 2 that resolved by day 28 in 2 and decreased in 1. All closure times on ADP were low or normal. No patients with abnormal PFA testing demonstrated spontaneous ecchymosis. From baseline to day 28 vWF-Act, vWF-Ag and FVIII decreased (P<0.05; n=24). All 3 values were high normal to elevated prior to treatment and decreased to normal on treatment.
This preliminary report does not identify any significant ibrutinib effect on platelet function. PLT counts improved rapidly in the majority of patients and when seen transient decreases have been minimal. Three patients (16%) developed an abnormal reading in PLT function tests on treatment but none developed spontaneous echymosis or bleeding. The observed normalization of mildly elevated baseline levels of vWF and FVIII seems most consistent with a reduction in acute phase reactants and there was no evidence for AvWD on ibrutinib. The apparent functional tolerance of BTK inhibition in platelets is likely attributable to redundancy in the affected signaling pathways.
This work was supported by the Intramural Research Program of NHLBI, NIH. We thank our patients for participating in these research studies.
No relevant conflicts of interest to declare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract 184
Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of mature B cells in peripheral blood (PB), bone marrow (BM) and lymph node (LN). Recent studies identified the LN ...as an important site of tumor cell activation and proliferation (Herishanu et al 2011). Using in vivo labeling of “newly-born” cells with deuterated water (2H2O; heavy water), the proliferation rate of CLL cells was estimated to range between 0.1 to >1% of the clone per day (Messmer et al 2005). Furthermore, a CXCR4dimCD5bright population of CLL cells in the PB contained more deuterium (2H)-labeled DNA and hence “recently-born cells than the CXCR4brightCD5dim population (Calissano et al 2011). Possible explanations for this observation include that CXCR4dimCD5bright cells proliferate more rapidly in the PB or that these cells are recent emigrants from tissues where proliferation occurs. As prior studies were performed on PB cells, the growth rates and characterization of the proliferative fraction of CLL cells in the LN remain unknown. Here we used 2H-labelling to directly compare cellular growth rates in PB, BM, and LN.
Patients drank 2H2O for 28 days; on day 13 an excisional LN biopsy and a BM aspirate were obtained. PB samples were obtained at baseline and on days 13 and 28. CLL cells were isolated using positive selection, or sorted based on reciprocal differences in CXCR4 and CD5 density for isolation of “proliferative” (CXCR4dimCD5bright), “intermediate” (CXCR4intCD5int), and “resting” (CXCR4brightCD5dim) fractions of CLL clones. 2H incorporation into the DNA of newly divided cells was measured by mass spectrometry. Raw values were normalized to the 2H2O content in total body water. Cellular growth rates were calculated by dividing the fraction of 2H-labeled cells by the number of days from the start of the labeling period.
To date, samples from 6 treatment-naïve CLL patients have been analyzed. On day 13, up to 16%, 9%, and 24% of the CLL cells sampled from PB, BM, and LN, respectively were 2H-labeled. The resulting mean estimated growth rate in % of the clone per day was for PB 0.41 (0.09 – 1.13), for BM 0.41 (0.28 – 0.68), and for LN 0.83 (0.31 – 1.84). The difference in growth rates between PB and LN was statistically significant (P<.02) and on average 2.5 times higher in the LN than in the PB. On day 28, the total fraction of 2H-labeled PB cells had further increased with the calculated growth rate in agreement with the growth rate in PB on day 13. CLL cells in the BM had a mean growth rate of 0.41% (0.28 – 0.68) of the clone per day, which was not significantly different than the growth rate in the PB. In fact in 2 patients the growth rate in the BM was lower than in the PB. The growth rates determined in LN and PB on day 13 were inversely correlated to the lymphocyte doubling time (r=-0.65 by Pearson correlation) and tended to be higher in patients with ZAP70 positive CLL. In keeping with the growth rates measured by 2H labeling, the fraction of Ki67-expressing CLL cells was higher in the LN than in the PB % cells mean ± SD (PB = 3.8 ± 1.6, LN = 18.4 ± 3.1; P=.005). Interestingly, while the average CLL growth rate in LNs by 2H-labeling was 2.5-times the rate in PB, the Ki-67 positive fraction in LNs was 5-times the fraction in the PB, further supporting the view that active clonal proliferation occurs predominantly in the LN, from which recently-born cells enter the PB. Consistent with a prior study of PB CLL cells (Calissano et al 2011), the CXCR4dimCD5bright subset in LN exhibited higher 2H-labeling than the bulk of the clonal cells (intermediate fraction) and the resting fraction. These studies indicate that a CXCR4dimCD5bright subset exists in LN-resident CLL cells and has higher 2H-labeling than the rest of the clone. Specifically, the calculated growth rate of the CXCR4dimCD5bright subset was on average 3.2-times the growth rate of all CLL cells in the LN. Moreover, the data suggest that sampling the PB for newly-born cells is a reliable measure of the degree of proliferation occurring in LNs.
Taken together our data show that the proliferation rate of CLL cells is higher in the LN than in the BM and PB and suggest that some of the newborn cells exit the LN within days. A clonal subset of CXCR4dimCD5bright cells is present in both the LN and PB and might harbor the proliferative core of the disease.
Gatmaitan:KineMed: Employment. Emson:KineMed: Employment. Chiorazzi:KineMed: Dr. Chiorazzi holds stock options in KineMed, Inc. Other.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract 865
Chronic lymphocytic leukemia (CLL), remains incurable, except by allogeneic stem cell transplantation. While multiple chemotherapeutic options are available, relapse and eventually ...development of chemotherapy resistance are common. Drug resistance of CLL cells is mediated by genetic alterations including deletion of chromosomes 17p (p53 locus) and 11q (ATM locus), and by pro-survival signals emanating from the tissue microenvironment. Thus new therapies are needed that are active against tumor cells in protective tissue sites and those with adverse cytogenetic features. To identify possible new therapies for CLL, we conducted a high throughput screening assay of primary CLL cells exposed to 7 step-wise dilutions of each member of a 2,816 compound library (including FDA-approved drugs and known bio-actives from commercial suppliers). As a control for “unspecific” cytotoxicity, we used PBMCs from normal donors. Of 102 compounds efficacious against CLL cells of all 6 patients tested, 5 were not or minimally toxic in normal lymphocytes. One of these 5 compounds is Auranofin (AF), an FDA-approved oral disease-modifying anti-rheumatic drug.
Next, we investigated the efficacy of AF against an expanded cohort of CLL samples (n=50) and investigated its mechanism of action. Samples were characterized for IGHV mutational status, ZAP70 expression, CD38 expression, and FISH cytogenetics. PBMCs were tested against serial dilutions of AF (final concentrations: 0.125–4μM) for 24 hours in flat bottom 96-well plates. MTS assay was performed to quantify cell viability. A dose-dependent response was observed in all samples. The inhibitory concentration at 50% (IC50) was calculated using GraphPad Prism Software. For a majority of samples, the IC50 was <1μM, a concentration easily achieved in vivo. AF cytotoxicity was independent of the IGHV mutational status, ZAP70 and CD38 expression, and cytogenetic subgroups (including 17p and 11q deletion). To assess whether AF is selectively cytotoxic for CLL cells, we determined drug-induced apoptosis of PBMCs of CLL patients for CLL cells (identified as CD19+/CD3-; >95% of those are CLL cells) and T-cells (CD19-/CD3+) separately using Annexin V staining. AF reduced viability of T-cells on average by 10% and of CLL cells by 50% (n=8; P<.05 for comparison between T and CLL cells). To determine the effect of the microenvironment on AF toxicity, we cultured primary CLL cells in the presence or absence of Nurse-Like Cells (NLC), a model of the protective effect of the microenvironment. NCLs enhance CLL cell viability and confer resistance to chemotherapeutic agents such as fludarabine. However, co-culture on NLCs did not protect CLL cells from the cytotoxic effect of AF. To investigate the effect of AF on tumor biology, total RNA (2.5 μg) from CLL PBMCs treated for 4 and 10 hours with AF in vitro was profiled on Human Genome U133 Plus 2.0 arrays (Affymetrix) and compared to untreated controls. There were 81 genes whose expression changed >2-fold at P<.01, most prominently several genes encoding heat-shock proteins and antioxidant enzymes such as heme oxygenase and thiroredoxin reductase. In Ingenuity Pathway analysis, the most significantly upregulated pathway in response to AF was the NRF2-mediated Oxidative Stress Response (P<.001). Combined, this suggests that AF induced oxidative stress resulting in the upregulation of a detoxifying response. To study the effect of AF on redox balance we used dihydroethidium (DHE) and concomitantly measured cell viability using 3,3′-dihexyloxacarbocyanine iodide (DiOC6) by flow cytometry. AF induced a time- and dose-dependent increase in ROS production in CLL cells that correlated with the onset of apoptosis. In conclusion, AF is selectively cytotoxic for CLL cells and its activity is independent of classic mechanisms of chemotherapy resistance. Based on these observations AF is now being studied in a clinical proof of concept phase IIa clinical trial for patients with relapsed/refractory CLL (NCT01419691).
No relevant conflicts of interest to declare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract 2899
Bruton's tyrosine kinase (BTK) is essential for B cell receptor (BCR) signaling. Ibrutinib (PCI 32765) is a covalent inhibitor of BTK that has demonstrated durable antitumor activity in ...chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) (Byrd ASCO 2011, O'Brien ASH 2011). Ibrutinib induces a transient increase in the absolute lymphocyte count (ALC) occurring during a period of rapid reduction in lymphadenopathy. This is thought to be due to inhibition of CLL cell adhesion and migration leading to a release of CLL cells from tissue sites.
This phase II trial (NCT01500733) using ibrutinib 420 mg daily on 28 day cycles enrolled two cohorts of CLL/SLL patients; 1.) >65 years old; and 2.) having deletion of chromosome 17p). Treatment naive and previously treated patients in need of treatment were eligible. Response was assessed after 2 cycles using CT scans. Bone marrow (BM) biopsies in a subset of patients were obtained at baseline and either on day 28 or 56. Whole blood viscosity was measured. Here we report on effects of ibrutinib in 26 patients (n=11, >65 yo; n=15, del 17p) who completed >2 cycles.
Fourteen patients (54%) had a partial response (PR) by IWCLL criteria, and 62% achieved a >50% reduction in lymphadenopathy measured as the sum of the products of the greatest diameter (SPD) of the 4 largest lymph nodes as assessed by CT, irrespective of ALC. The effects of ibrutinib on total tumor burden were measured by concomitantly assessing lymph node (LN), spleen, blood, and BM disease. The median reduction in lymphadenopathy was 52% (range 22–72%) on day 56. Despite at times massive reduction in tumor burden, there was no evidence of tumor lysis. The rates of nodal responses in subgroups of patients defined by deletion 17p, non deletion 17p, treatment naive and relapsed/refractory patients was comparable. There was an overall reduction in splenic disease with a median decrease in spleen size (measured as longest cranio-caudal extension) of 11% (0.5–40%; P<0.01). A trend towards more rapid resolution of splenomegaly in treatment naïve patients was seen. BM evaluations are complete in 10 patients: cellularity decreased in 3 of 7 and remained unchanged in 4 on day 28; on day 56 cellularity was decreased in 3 of 3 patients. Using immunohistochemistry for CD79 we confirmed that the reduction in BM infiltration was due to reduced tumor infiltration. As seen in other studies, the increase in ALC peaked within the first 2 months followed by a slow decline. The median ALC increased from 75 k/μl (3.5 – 308) to 102 k/μl on day 28 (P<.01). However, only 62% of patients had an increased ALC on treatment days 14 and 28 compared to baseline and the change in ALC at the end of the first cycle (day 28) was very heterogeneous (>95% reduction to ∼700% increase). Treatment naïve patients showed more pronounced relative increases in ALC and the most dramatic increase occurred in patients with a lower starting ALC. The rise in ALC correlated with an increase in whole blood viscosity (r=0.52; P<0.05, n=18). However, all viscosity measurements, both at baseline and on treatment, were within the normal range (3.6–6.0 cP) and there were no clinical signs or symptoms of a hyperviscosity syndrome.
Reduction in lymphadenopathy with a transient increase in ALC has been reported in other studies and is thought to reflect a redistribution of tumor burden into the blood. While a treatment induced rise in ALC was common in our patients, up to a third had resolution of blood disease within a few weeks. This and the often dramatic reduction in nodal disease with or without an increase in ALC, and the concomitant reduction in tumor infiltration of BM and spleen indicate a significant contribution of cell death, the mechanism of which remains to be defined. Interestingly resolution of disease in the BM appears to be slower than in the LN. This may reflect more active BCR signaling in the LN and consequently more pronounced changes in tumor biology on ibrutinib in this site. In addition, treatment naïve patients appear to respond more rapidly raising the question whether untreated CLL is more dependent on BTK-dependent signaling pathways in the tissue microenvironment.
This work was supported by the Intramural Research Program of NHLBI, NIH. We thank our patients for participating in these research studies.
No relevant conflicts of interest to declare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
Background: B-cell receptor (BCR) activation plays a pivotal role in the pathogenesis and for progression of CLL. Ibrutinib (PCI-32765), an irreversible inhibitor of Bruton's tyrosine kinase ...(BTK), can disrupt BCR signaling and has durable antitumor activity in CLL. We initiated a phase II single center trial to extend this experience and assess the impact of ibrutinib on tumor cells in vivo. Methods: Treatment naïve (TN) and relapsed/refractory (R/R) pts with CLL are treated with 420 mg ibrutinib daily. Pts are enrolled in 2 cohorts: 1) deletion (del) of chromosome 17p and 2) >65yo and treated until disease progression. Response is evaluated at 6 months (mo) and every 6 mo thereafter. 17p del was assessed by FISH cytogenetics. Spleen volume on computed tomography (CT) scans was computed using a General Electric Advanced Workstation Server. Results: With a median follow-up of 9 mo we report on the first 53 patients: for del 17p n=29; 15 TN, 14 R/R; and for >65yo n=24; 8 TN, 16 R/R. Estimated event free survival at 12 mo is 94%. Most adverse events were mild with non hematologic toxicities (regardless of causality) grade ≥3 in <13% of pts. Two deaths on study were not treatment related. At 6 mo 95% of pts (n=44 evaluable) had a nodal response (median reduction in lymph node size of 73%) and 52% of pts had a partial response (PR) by IWCLL criteria. One pt had progressive disease (presumed Richter's transformation). All pts had a decrease in splenomegaly. The median reduction of splenic volume computed from CTs was 55% (30-1716 ml). Tumor infiltration in bone marrow biopsies (n=26), decreased by a median 82% as assessed by immunohistochemistry for CD79a and the median ALC decreased from 79 k/µl (0.5-402) to 32 k/µl (0.8-167) at 6 mo for a median reduction of 62%. We evaluated the in vivo effects of ibrutinib using blood and tissue samples collected pre and during ibrutinib treatment. On treatment expression of genes known to be induced by B-cell receptor (BCR) activation (Herishanu et al 2011) was significantly reduced in CLL cells in the peripheral blood (PB) in all pts (n=15; median reduction 57%; P<.001). Consistent with inhibition of BCR signaling, we found concurrent reduction in PLCγ2 phosphorylation; a direct target of BTK. Patients also donated matched lymph node (LN) core biopsies pre-treatment and within the first 4 days on treatment. Ibrutinib inhibited BCR signaling in the LN (n=8, P=.004) to a similar degree as observed in the PB. Similarly, ibrutinib decreased expression of NF-κB target genes in PB and LN. In keeping with inhibition of BCR and NF-κB signaling surface markers of cellular activation such as CD38, CD69, and CD86 were significantly reduced by ibrutinib as measured by flow cytometry (P<.02). Finally, ibrutinib significantly reduced tumor proliferation as determined by the percentage of PB CLL cells expressing Ki67 (median reduction of 80%; P<.001). Conclusions: Ibrutinib as a single agent is well tolerated and highly effective in both TN and R/R pts achieving rapid disease control in blood, lymph nodes, spleen and bone marrow that is durable. Correlative studies on patient samples during ibrutinib treatment provide direct evidence for on target effects of ibrutinib in vivo demonstrating effective and sustained inhibition of BCR and NF-κB signaling in both PB and LNs. Ibrutinib deserves further investigation as targeted therapy for CLL.
We thank our patients for participation and donation of blood and tissue samples. Supported by the Intramural Research Program of NIH.
Citation Format: Adrian Wiestner, Sarah Herman, Rashida Mustafa, Janet Valdez, Jade Jones, Nakhle Saba, Andrew Lipsky, Diane C. Arthur, Gerald Marti, Francine Thomas, Irina Maric, Stefania Pittaluga, Xin Tian, Susan Soto, Georg Aue, Mohammed Z. Farooqui. Potent single agent activity of Ibrutinib (PCI-32765) in patients with chronic lymphocytic leukemia (CLL): clinical and translational results from an ongoing phase II study. abstract. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-141. doi:10.1158/1538-7445.AM2013-LB-141
Chronic lymphocytic leukemia (CLL) is an adult lymphoid malignancy with a variable clinical course. There is considerable interest in the identification of new treatments, as most current approaches ...are not curative. While most patients respond to initial chemotherapy, relapsed disease is often resistant to the drugs commonly used in CLL and patients are left with limited therapeutic options. In this study, we used a luminescent cell viability assay based on ATP levels to find compounds that were potent and efficacious in killing CLL cells. We employed an in-house process of quantitative high throughput screening (qHTS) to assess 8 concentrations of each member of a 2,816 compound library (including FDA-approved drugs and those known to be bio-active from commercial suppliers). Using qHTS we generated potency values on each compound in lymphocytes donated from each of six individuals with CLL and five unaffected individuals. We found 102 compounds efficacious against cells from all six individuals with CLL ("consensus" drugs) with five of these showing low or no activity on lymphocytes from a majority of normal donors, suggesting some degree of specificity for the leukemic cells. To our knowledge, this is the first study to screen a drug library against primary CLL cells to identify candidate agents for anti-cancer therapy. The results presented here offer possibilities for the development of novel drug candidates for therapeutic uses to treat CLL and other diseases.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Newer biological treatment strategies have been developed in the last decade with some promising outcomes. Their safety, however, has been questioned lately with multiple reports of increased risk ...for malignancies and infectious complications. These reports render their use suboptimal. We report a 44-year-old woman receiving adalimumab (Humira®) for advanced juvenile rheumatoid arthritis who then developed acute myelogenic leukemia.
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