ALBERT

Description: Introduction The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib induces objective clinical responses in the majority of CLL patients (Byrd et al., NEJM 2013). Ibrutinib covalently binds to BTK and with once daily dosing (420 mg, PO) results in 〉 90% inhibition of kinase activity. Germline inactivating mutations in BTK lead to an immunodeficiency syndrome first described by the pediatrician Dr. Bruton in boys suffering from recurrent bacterial infections. These kids, diagnosed with what is now known as Bruton’s agammaglobulinemia, have a severe defect in B cell maturation resulting in the virtual absence of immunoglobulins. Hypogammaglobulinemia is a common complication of CLL and likely is a significant contributor to the increased rate of infections that are a leading cause of death in CLL. Thus, to what degree ibrutinib affects normal B cell function and immunoglobulin levels may in part determine the safety profile of continuous treatment with this agent. Patients and Methods Here we present data from a phase II trial (NCT01500733) of ibrutinib 420 mg daily on 28 day cycles for relapsed/refractory (RR) and treatment naïve (TN) CLL/SLL patients (pts). Serum immune globulins (IgG, IgM, IgA), serum free light chains, and immunofixation electrophoresis were obtained at baseline, and every 6 months thereafter. For statistical analysis of pre-treatment to on-treatment measurements the paired Student t-test was used. Results Here we report on 25 patients (10 TN, 15 RR) who completed 〉12 months on ibrutinib and never received immunoglobulin replacement therapy. By 6 and 12 months, there was a non-statistically significant trend toward decreased IgG levels (ref. range 642-1730) from a pre-treatment median of 601 to 587 mg/dL (at 6 months) and 495 mg/dL (at 12 months; P = 0.14). In contrast, median serum IgA (ref. range 91-499) rose from 42 (baseline) to 58 (at 6 mo) to 61 mg/dL by 12 months (P〈 0.005). Three patients had a clonal IgM on electrophoresis, which decreased with treatment. In the remaining 22 patients IgM (ref. range 34-342) rose from 16 (baseline) to 25 (6 months) to 23 mg/dL by 12 months (P upper limit of normal (median 5.7 mg/dl). At 6 and 12 months there was a 76% and 72% reduction of the KSFLC (P〈 0.01), and in 7 pts the level normalized by 6 months. In contrast, prior to therapy the lambda serum free light chains (LSFLC, ref. range 0.66-2.32 mg/dL) were low (median 0.62 mg/dL) in these patients and increased by 68% (P upper limit of normal (median 8.4 mg/dL), which decreased on ibrutinib by 〉 80% (P〈 0.03) and normalized in 88% of pts by 12 months. The KSFLC in most of these patients was in the low normal range and only increased by 19% from baseline by 12 months. Thus, ibrutinib effectively reduces the clonal light chain, a correlate of tumor control, while the non-clonal light chains, presumably in part reflecting normal B-cells, are low pre-treatment and increase during treatment. Conclusion Consistent with other reports we see little change in IgG levels in the first 12 months. Importantly, ibrutinib leads to a significant increase in both IgA and IgM serum levels, suggesting a beginning recovery of humoral immunity. The reduction of clonal light chains, a tumor marker, correlates with clinical response. In contrast, the increasing levels of the non-clonal light chain may herald a recovery of the normal B-cell (and possibly plasma cell compartment) raising the possibility that ibrutinib may selectively target CLL cells while allowing the re-growth of normal B-cells. We are currently investigating this further. Supported by the Intramural Research Program of NHLBI. We thank our patients for participating and acknowledge Pharmacyclics for providing study drug. Disclosures: Off Label Use: Ibrutinib not FDA approved for CLL.

Description: Abstract 54 Severe aplastic anemia (SAA) is characterized by trilineage marrow hypoplasia and a paucity of hematopoietic stem cell (HSC) progenitors. SAA is treated with immunosuppressive therapy (IST) or allogeneic HSC transplantation (HSCT), with a successful outcome after either treatment in a majority of patients. However, 20–40% of patients without a suitable donor for HSCT and a suboptimal response to IST may have persistent severe thrombocytopenia. Thrombopoietin (TPO) is the principal regulator of platelet production, and it exerts its effects through binding the megakaryocyte progenitor TPO receptor mpl, which stimulates production of mature megakaryocytes and platelets. Several lines of evidence support the concept that signaling through mpl also influences expansion and maintenance of primitive HSCs and multi-potent progenitor cells. Eltrombopag, is a small molecule TPO receptor agonist that stimulates mpl, and increases platelet counts in patients with chronic immune thrombocytopenic purpura (ITP). It is approved for the treatment of chronic ITP (Promacta®). We conducted a non-randomized, pilot phase II study of eltrombopag in SAA patients who remained severely thrombocytopenic at least six months after one or more rounds of IST (clinical trials.gov identifier NCT00922883). Consecutive patients fulfilling the inclusion criteria received eltrombopag 50mg daily with dose escalation every two weeks to a maximum dose of 150mg daily. Primary end points assessed after three months of treatment were changes in peripheral blood counts (platelets, hemoglobin, absolute neutrophil counts), with hematologic response criteria defined a priori for each lineage. Secondary endpoints included the incidence of bleeding events, and health related quality of life. Patients who achieved hematologic responses were maintained on eltrombopag through an extended access protocol. We completed our planned accrual of twenty five patients, and 22 are evaluable for response to date. Median age was 45 years old (range 18–77 years), and the median time from the last course of IST was 13 months (range 6–54 months). Median follow up time was 9 months (range 1–24 months). Nine of twenty two patients (41%) achieved hematologic responses: seven of twenty-two patients (32%) achieved platelet responses with transfusion independence for eight weeks or greater; six patients had improved hemoglobin levels after starting treatment (mean hemoglobin increase of 3.8 g/dL) and 4 patients who were previously dependent on red blood cell transfusions have achieved transfusion-independence. Five neutropenic patients had increased neutrophil counts after treatment with eltrombopag (mean increase 660 cells/uL). Plasma TPO levels did not predict for hematologic response to eltrombopag. Serial bone marrow biopsies performed on patients with hematologic responses demonstrated normalization of trilineage hematopoiesis and cellularity in three of four responders receiving a year or more of therapy, with no increase in reticulin fibrosis (Figure 1). These results represent the first evidence that TPO stimulation can expand the HSC pool in humans, with clinically meaningful trilineage hematologic improvements in patients with SAA, resulting in transfusion-independence and improved quality of life with a simple daily oral regimen. Updated response data on the full 25 patients will be presented at the Society's meeting. Disclosures: Off Label Use: Eltrombopag for severe aplastic anemia.

Description: Lenalidomide is active in lymphoid malignancies, but its mechanism of action remains ill defined. One possible mechanism is immune activation due to increased expression of costimulatory molecules on tumor cells. In CLL lenalidomide treatment has been uniquely complicated by tumor flare reactions (TFR: pain and lymph node swelling) resulting in treatment related mortality. To investigate effects of lenalidomide on CLL cells we exposed PBMC from 17 CLL patients enrolled in a phase II clinical trial of single agent lenalidomide and normal donors (n=10) in-vitro to 2μM lenalidomide for 48 hours and measured costimulatory molecules CD80 and CD86 on B-cells and activation marker CD69 on T-cells by flow cytometry. CD80 expression increased on average 2-fold on CLL cells but remained unchanged on normal B-cells (p=0.01 for log2 MFI CLL vs normal). CD69 expression on T-cells followed a similar pattern, albeit with more interindividual variability among CLL samples (p=0.03 for log2 MFI CLL vs. normal). Next we wished to correlate the degree of in-vitro activation with the clinical effects of lenalidomide treatment in the same patient. Our observations in several patients suggested that the dominant feature of lenalidomide treatment is a cytokine release syndrome (CRS). Indeed, on day 8 of treatment we detected increased serum levels of TNFa, IL-1ra, CCL2, CCL3, CCL4 and IL-8. To correlate the CRS with in-vitro measurements, we applied uniform criteria for diagnosis. Patients who experienced at least 2 of the following symptoms (% of patients with the symptom, n=18) were considered to have a CRS: increase in lymph node size and or ALC by 〉25% (50%), fever 〉38C (44%), pain (61%), fatigue (72%), chills (33%), hypotension/dehydration (39%), and rise of creatinine (33%). Onset of symptoms was within 8 to 72 hours (average 38 hours) after initiation of therapy (20mg patients 1–10, 10mg patients 11–18). The CRS score, summarizing number and severity of symptoms in each patient, averaged 3.14 (range 0–10) with no difference between the 20mg and10mg cohort. The CRS score correlated (Pearson r-value, p50% in CD3+ cells in the lymph node and the average pre/post ratio of T-cells was 1.14 (p=0.37). In summary, lenalidomide upregulated expression of CD80 on B-cells and of CD69 on T-cells from CLL patients but not on normal B and T-cells. The in-vitro response correlated with the clinical onset and severity of a CRS in-vivo. However, the degree of T-cell activation or of the CRS did not predict the effect on the leukemic cell count. Our data suggest the possibility that immune activation in CLL may be primarily responsible for side effects but not be required for disease control. This interpretation is consistent with the good clinical activity of lenalidomide in several lymphoid malignancies in the absence of notable immune effects and if confirmed, has important implications for future use of lenalidomide and the formulation of combination regimens.

Description: Introduction: Pre-treatment cytogenetics and post-treatment minimal residual disease (MRD) status are predictive biomarkers in CLL. We hypothesized the two markers can guide a risk-adapted treatment strategy to achieve deep and durable responses in previously untreated CLL/SLL. Methods and Patients: 31 subjects were enrolled. Risk assessment was performed before treatment and after induction. Based on pre-treatment FISH, patients were categorized into either high-risk (with deletion 17p or 11q) or standard-risk (no deletion 17p or 11q) and treated with up to 6 cycles of OFC (Ofatumumab 300mg i.v. on cycle 1 day 1, 1000mg i.v. on cycle 1 day 8 and on day 1 of subsequent cycles; Fludarabine 25mg/m2 i.v. on day 1-3; Cyclophosphamide 250mg/m2 i.v. on day 1-3;) or OF (Ofatumumab given as described for the high-risk group; Fludarabine 25mg/m2 /d i.v. on day 1-5), respectively. Three months after completion of chemoimmunotherapy, a second risk stratification was performed based on presence or absence of MRD. MRD negative (MRDneg) state was defined as

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: Abstract 4427 Severe aplastic anemia (SAA) is characterized by trilineage marrow hypoplasia and a paucity of hematopoietic stem cell progenitors. SAA is treated with immunosuppression or allogeneic stem cell transplantation (SCT), with a successful outcome in a majority. However, 20–40% of patients without a suitable donor for SCT do not respond to immunosuppression and may have persistent severe thrombocytopenia. Thrombopoietin (TPO) is the principal regulator of platelet production, and it exerts its effects through binding the megakaryocyte progenitor TPO receptor mpl, which stimulates production of mature megakaryocytes and platelets. Eltrombopag, a small molecule TPO mimetic that binds to mpl, increases platelet counts in healthy subjects, and in patients with chronic immune thrombocytopenic purpura. Both TPO and eltrombopag stimulate more primitive multilineage progenitors and stem cells in vitro. Patients with SAA and thrombocytopenia have very elevated TPO levels; nevertheless, we asked whether pharmacologic doses of eltrombopag could stimulate hematopoiesis in these patients without other options. We are conducting a pilot phase II study of eltrombopag in SAA patients with severe thrombocytopenia refractory to immunosuppressive therapy. Consecutive eligible adult patients were treated with oral eltrombopag at an initial dose of 50 mg daily, with escalation to a maximum dose 150 mg daily, with the goal of maintaining a platelet count of 〉20,000/uL above baseline. Treatment response was measured after three months and was defined as platelet count increases to 20,000/uL above baseline, or stable platelet counts with transfusion-independence for a minimum of 8 weeks. Nine patients have been enrolled and six are evaluable for response to date. Two patients did not respond to treatment. Three patients achieved platelet responses by 12 weeks of treatment, and all have sustained their responses (median follow up 10 months). Four patients exhibited improved hemoglobin levels 12 weeks after starting treatment (median hemoglobin increase of 2.1 g/dL) and two patients who were previously dependent on packed red blood cell transfusions have achieved transfusion-independence. Three neutropenic patients exhibited increased neutrophil counts after treatment with eltrombopag (median increase 0.46K cells/uL). These results provide evidence that eltrombopag can improve platelet counts in patients with severe refractory thrombocytopenia, and perhaps more surprisingly, have a clinically relevant impact on erythropoiesis and myelopoiesis. Updated data will be presented at the Society's meeting. Disclosures: Off Label Use: Eltrombopag for thrombocytopenia in refractory severe aplastic anemia patients.

Description: Introduction Immune dysregulation is a hallmark of CLL, making these patients particularly vulnerable to infectious complications. Patients with CLL are at increased risk of developing varicella zoster virus (VZV) reactivation (shingles) due to their advanced age and immunocompromised status. A new recombinant (non-live) adjuvanted shingles vaccine (SHINGRIX; RZV) has the potential to reduce VZV reactivation, without the risk of giving a live vaccine to immunocompromised individuals. RZV is proven to reduce the risks of herpes zoster and postherpetic neuralgia in healthy adults ≥ 50 years of age, however its efficacy in immunocompromised individuals, including CLL, remains unknown. We report preliminary safety and efficacy of RZV in treated and untreated CLL patients. Methods In this phase II open-label study (NCT03702231), patients with CLL who were either treatment naïve or receiving treatment with a Bruton's tyrosine kinase inhibitor (BTK-I) (ibrutinib or acalabrutinib) received 2 doses of RZV via intramuscular injection at 0- and 3-months. Subjects were followed for 6 months and received assessment of serologic response at 3- and 6-months. Based on results of prior published studies, serologic response was defined as a ≥ four-fold rise in VZV anti-glycoprotein E (anti-gE) blood IgG serum titer after completing the RZV vaccine series. Additionally, serologic response was analyzed 3 months following the first vaccine administration to study the kinetics of the humoral vaccine response. All subjects completed an adverse event (AE) diary documenting any local (injection site) or systemic AE that started within 7 days after receiving the first and second vaccine dose. The data reported herein are as of July 12th 2019; updated results will be presented at the meeting. Results Safety data are available on 57 subjects who received at least one vaccine dose. The most frequent local and systemic AEs were injection site pain (67%), injection site reaction (32%) and generalized myalgias (25%) (Table 1). All adverse reactions were grade 1-2, except for 2 (4%) grade 3 reactions. No serious AEs were reported. All AEs resolved or returned to baseline within 7 days of vaccine administration. Seven subjects have completed the primary endpoint and have had serologic response assessment at 6-months. Serologic responses were observed in 3 (43%) patients. All patients (n = 7) achieved ≥ 2.2-fold rise in VZV anti-gE titers at 6-months. Preliminary analysis of 3-month samples (n = 43) shows early evidence of increasing titers after the first dose of RZV. Conclusions RZV administration appears to be safe in CLL patients that are treatment naïve or receiving treatment with a BTK-I. Compared to previously reported toxicities in the healthy population, no increase in frequency or severity of AEs were observed. Preliminary data suggests that RZV induces humoral immune responses in patients with CLL. Disclosures Wiestner: Pharmayclics: Research Funding; Acerta: Research Funding; Merck: Research Funding; Nurix: Research Funding.

Description: Abstract 979 Lenalidomide's mechanism of action in chronic lymphocytic leukemia (CLL) is not well understood. In vitro data suggest that anti-leukemic immune responses are important. Tumor flare reactions during treatment have been associated with response in some but not other studies. In vivo data that mechanistically link immune stimulation to clinical responses are lacking. We designed an independent, single center, phase II trial of lenalidomide in relapsed/refractory CLL (clinicaltrials.gov: NCT00465127). Here we report final clinical data and results of multiple translational analyses that indicate that an IFNy centered immune response is critical for response. A 3 week on, 3 weeks off treatment scheme (42 day cycles) was chosen to pulse immune stimulation while trying to minimize myelosuppression. The starting dose was 20 mg daily for the first 10 patients and 10 mg for the subsequent 23. Response was measured at 24 weeks. 5 patients, 4 with del 17p, achieved a PR by IWCLL criteria (16%) and were eligible to continue drug for 4 more cycles; the PFS in these patients was 16 months compared to 7 months for all other (p

Description: Key Points Most cases of ibrutinib-resistant CLL were due to mutations in BTK and/or PLCG2 and often composed of multiple independent subclones. High sensitivity testing identified resistance mutations up to 15 months before manifestation of clinical progression.

Description: Abstract 1803 Background: Chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and related B-cell malignancies are incurable diseases that universally relapse after initial therapy. Resultant cytopenias in refractory patients are a common barrier to salvage therapy. Innovative targeted agents with favorable tolerability profiles that can overcome acquired mechanisms of resistance are urgently needed. ON 01910.Na (rigosertib) is a selective non-ATP competitive multikinase inhibitor that potently inhibits PI3 kinase and induces reactive oxygen species and NOXA-dependent apoptosis in vitro. Pre-clinical testing of rigosertib demonstrated selective cytotoxicity against CLL and MCL cells with minimal effects on normal B and T cells (Chapman et al 2012). Extensive clinical testing of rigosertib in patients with solid tumors or myelodysplastic syndromes (MDS) has indicated lack of myelosuppression and overall good tolerability (Raza et al ASH 2011 #3822). Here, we present the results from the phase I study assessing the safety and maximum tolerated dose (MTD) of intravenous rigosertib in patients with relapsed CLL, MCL, and related B-cell malignancies. Materials and Methods: phase I dose-escalation clinical trial was conducted to evaluate the safety and efficacy of rigosertib in patients with CLL, MCL, MM, and HCL who were refractory or relapsed after ≥1 lines of therapy. Baseline cytopenias were permitted unless ANC 〈 500 or platelets were 〈 10K and unable to be supported with transfusion. Pts with GFR 〈 40ml/min, serum sodium 〈 134meq/L, and active ascites were excluded. Dose escalation followed a traditional 3+3 design and dosing cohorts were 1200mg/m2, 1500mg/m2 and 1800mg/m2 over 48 hours and 1800mg (flat dose), and 2100mg (flat dose) over 72 hours. Infusions were delivered via an ambulatory infusion pump and repeated in 14 day cycles for up to 4 cycles. Response was determined in patients who completed 4 cycles. Pts who demonstrated a biologic response without DLT were allowed to continue infusions until disease progression. Primary endpoint was toxicity after 2 cycles. Secondary endpoints included the toxicity with extended dosing and measures of biologic activity after 4 cycles. Results: Increasing doses of rigosertib were evaluated in 16 pts with relapsed CLL (10), MCL (2), MM (2), and HCL (2). All patients were evaluated for toxicity, while 10 patients completed 4 cycles of therapy and were evaluable for secondary endpoints. Median age was 61 yrs [range 52–65]. Drug-related adverse events (AEs) were reported in 15 pts (94%) and were almost exclusively grade ≤2. Most frequent drug-related AEs were fatigue 31%, musculoskeletal pain 31%, nausea 19%, constipation 19%, and diarrhea 12%. Grade 3/4 drug-related AEs included 2 cases of G4 neutropenia (both patients had neutropenia at baseline) and 1 case of syncope; there was 1 cardiac death in a patient with pre-existing heart disease that was classified as unrelated. No dose-limiting toxicities (DLTs) were observed. Analysis of blood samples collected for pharmacokinetics is planned. Response data in the 13 patients evaluable for response indicated that 7 had stable disease and 6 had disease progression. No clinical responses or evidence of biologic activity was observed. Conclusions: Escalating doses of rigosertib were well tolerated in patients with relapsed/refractory B-cell malignancies with rare G3/G4 toxicities. Of note, most patients with baseline cytopenias tolerated the therapy well. The highest dose level studied in this study is one step up from the dose level of the ongoing pivotal trial of rigosertib in MDS. However, as a single agent no clinical responses were observed with rigosertib in B-cell malignancies. Further development of rigosertib in lymphoid malignancies will require either combination therapy or alternative dosing schedules. Disclosures: Wilhelm: Onconova: Employment, Equity Ownership.