Goldman, Stanton

Abstract BACKGROUND: Burkitt Lymphoma (BL) represents approximately 40% of all childhood and adolescent non-Hodgkin lymphoma (Miles/Cairo, BJH 2012). Children with relapsed or progressive BL develop chemoimmunotherapy-resistant disease and can rarely be cured with salvage therapy (Cairo et al, JCO 2012). Bruton's tyrosine kinase (BTK) is a regulator of normal B-cell development and is activated upon B-cell receptor (BCR) stimulation. Chronic active BCR signaling through BTK activation can be inhibited by the selective and covalent BTK inhibitor, ibrutinib (Young/Staudt et al, Nat Rev Drug Dicov 2013). Preclinical studies of ibrutinib in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) suggest that its inhibitory effect on cell proliferation is in the range of 1.0uM to 25.0uM (Herman et al, Blood 2011; Cinar et al, Leuk Res 2013), which is higher than the typical plasma concentration range in patients at the recommended dose of 560mg (<0.6uM) (Advani et al, JCO 2013). Despite these relatively high IC50 values in vitro, ibrutinib has been highly effective in the treatment of patients with refractory CLL and MCL (Byrd et al, NEJM 2013 and Wang et al, NEJM 2013). Ibrutinib was initially approved by the FDA (IMBRUVICA, USPI) for patients with MCL in November 2013 and CLL in February 2014, who have received at least one prior therapy. BL, however, is associated with tonic vs chronic active BCR signaling; while, both CLL and MCL have chronic active BCR signaling. These findings suggest that the antitumor activity of ibrutinib in BL, which is associated with tonic BCR signaling, may be less pronounced than in Activated B cell-like diffuse large B-cell lymphoma (ABC-DLBCL), CLL or MCL. OBJECTIVES: We hypothesize that ibrutinib may be a potential adjuvant agent in the treatment of BL. Therefore, we investigated the in vivo anti-tumor activity of ibrutinib in BL xenografted NSG mice. METHODS: Raji Burkitt lymphoma (BL) cells (ATCC) were exposed to ibrutinib (0-10uM, generously provided by Janssen R&D LLC) alone and in combination with dexamethasone (1uM) for 5 days and then MTS cell proliferation assay (Promega) was performed. The IC50 values were determined with CompuSynTM software (Chou and Martin, ComboSyn, 2005) based on data derived from MTS assay. Raji BL cells were stably transfected with a firefly luciferase expression plasmid (ffluc-zeo), kindly provided by Laurence Cooper MD, PhD. NSG mice (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ, 6-8 weeks old) from Jackson laboratory were irradiated (2.5 Gy) and then mice were subcutaneously injected with one million ffluc-zeo tumor cells. Tumor progression and tumor burden were monitored by Bioluminescent Imaging system using the Xenogen IVIS-200 (Caliper Life Sciences). Mice were orally gavaged with either vehicle or ibrutinib (12.5mg/kg) for 10 days (once a day) at post-tumor cells injection. Analysis of survival rates were determined by the Kaplan-Meier method and differences evaluated by log-rank test using the Prism Version 6.0 software. RESULTS: Ibrutinib compared to control induced a significant dose-dependent decrease in cell proliferation in Raji BL cells (5uM, 0.561 ± 0.170, p<0.03, IC50=5.20uM) following 120 hours (5 days) of ibrutinib treatment. Interestingly, we observed a significant decrease in cell proliferation in Raji BL cells (5uM, 0.213 ± 0.078, p<0.002, IC=0.50 ± 0.374uM) following ibrutinib and 1uM dexamethasone combination treatment following 120 hours (5 days) of treatment. We observed a significant decrease of tumor progression by tumor luminescence signal intensity following ibrutinib treated Raji BL xenografted NSG mice at day 20 (12.5mg/kg, p<0.001 and 25mg/kg, p<0.05) and day 25 (12.5mg/kg, p<0.05 and 25mg/kg, p<0.05) compared to control (Figure 1A).Ibrutinib (12.5 mg/kg) treated mice (n=39) significantly prolonged survival compared to vehicle controls (n=28) (p<0.0001) (Figure 1B). CONCLUSIONS: Ibrutinib (12.5mg/kg) significantly decreased tumor progression and significantly increased survival in Raji BL xenografted NSG mice, suggesting that ibrutinib may be a potential adjuvant agent for therapy in the treatment of BL. Future studies will investigate the efficacy of combination therapy with ibrutinib on survival in BL xenografted NOD/SCID mice. Disclosures Galardy: Mission Therapeutics: Research Funding.

Abstract Abstract 2702 Introduction/Background Tumor Lysis Syndrome (TLS) is a potentially life-threatening metabolic syndrome that frequently complicates the management of newly diagnosed mature B-NHL in children and adults (Cairo et al., BJH 2010). We have demonstrated rasburicase to be superior to allopurinol in the rapid reduction of uric acid (UA). (Goldman/Cairo et al, Blood 2001) In the previous FAB/LMB/96 international mature B-NHL trial, variables associated with a significant inferior EFS included advanced stage, elevated LDH, primary mediastinal involvement (PMBL) and combined BM/CNS disease (Cairo et al, JCO, 2011). We have previously reported the pharmacokinetics and safety of the addition of rituximab to a modified FAB/LMB/96 backbone in children and adolescents with advanced stage mature de-novo B-NHL. (Cairo et al., ASCO, 2010) Here we focus on outcome in those specific high-risk sub-categories of advanced childhood mature B-NHL following the incorporation of a recombinant urate oxidase (Rasburicase) and targeted immunotherapy (Rituximab) to the FAB/LMB/96 chemotherapy backbones. Material and Methods Children with newly diagnosed stage III/IV or high-risk (BM+±CNS+) de-novo mature B-NHL were prospectively treated with cyclophosphamide, vincristine, and prednisone (COP) reduction therapy preceded by 4–24 hours of non-alkaline hydration and rasburicase (0.2mg/kg) (generously supplied by Sanofi-Aventis). Laboratory and clinical TLS (LTLS and CTLS) was prospectively monitored as previously defined (Cairo & Bishop, BJH, 2004). Immunochemotherapy consisted of FAB B4 or C1 therapy (adriamycin 1 hr infusion) as we have previously described (Patte et al and Cairo et al, Blood, 2007) with the addition of rituximab 375 mg/m2/dose (generously supplied by Genentech) with 2 doses administered in each induction cycle and 1 dose in each consolidation cycle. Results With respect to LTLS and CTLS, 78 patients were evaluable: group B (stage III/IV) with NL-LDH (<2X ULN, n=23), Group B with HI-LDH (≥ 2X ULN, n=18), and Group C (n=37). The mean initial UA levels (mg/dl +/− SD) were 4.7±2, 8.7±4.6, and 7.1±4.6, respectively. At presentation, 31% of patients (all groups) had an elevated (> 8mg/dl) UA, and 47% received elective doses of rasburicase in the days prior to the first day of chemotherapy. The incidence of LTLS prior to rasburicase in Group B NL-LDH, Group B HI-LDH, and Group C, was 4.3%, 5.5%, and 16.6%, respectively. The incidence of CTLS prior to rasburicase was 0%, 11%, and 8.6%, respectively. Following the administration of rasburicase, the incidence of new onset LTLS and CTLS for the entire cohort was 13% (2.6% with hyperuricemia) and 4% (1% with hyperuricemia), respectively. Furthermore, there was a significant improvement in the estimated GFR in those with renal impairment from day -1 prior to rasbursicase to day+7 post R-COP (55 ± 29 ml/min1.73 m2 to 136 ± 29 respectively; p<0.0007; Figure 1). Following combined FAB 96 chemotherapy (with 1 hour Adriamycin) and rituximab, the 3- year EFS (95% CI) for pilot patients (6 doses of rituximab) with Stage III/IV and Group C BM/CNS patients were 95% (80–99%) (Figure 2) and 89% (73–98%), respectively. Interestingly, two of four patients with centrally reviewed mediastinal primaries developed recurrent disease, while none of the 19 stage III/IV patients with LDH < 2X ULN developed recurrent disease or toxic deaths. Conclusions Rasburicase and non-alkaline hydration largely prevented (94% of the time) new onset TLS with hyperuricemia safely during COP reduction therapy in children with advanced stages of de-novo mature B-NHL. For patients with mediastinal primaries, the high failure rate with group B4 therapy + rituximab suggests it would be appropriate to investigate new treatment strategies. Excluding this histology, combined modified FAB/LMB 96 chemotherapy and rituximab resulted in only 5 deaths (2 toxic deaths, 3 disease related deaths) among our cohort of 62 with advanced mature B-NHL. In comparing these current results from the previous FAB/LMB 96 trial without rituximab, the stage III/IV patients had a 95% vs. 84% and patients with CNS+ disease a 93% vs. 72% 3 year EFS. (Patte et al., Cairo et al. Blood, 2007) Future efforts to intensify chemotherapy in patients with CNS involvement should proceed only with careful toxicity monitoring, as preliminarily these patients seem to have excellent outcome with this immunochemotherapy approach. Disclosures: Gross: Pfizer Oncology: Consultancy; Roche/Genentech: Consultancy; Eli Lilly: Consultancy.

Abstract BACKGROUND: Primary Mediastinal B-Cell Lymphoma (PMBL) is a rare form of B-cell non-Hodgkin lymphoma (Lones/Cairo et al, JCO 2000). We have previously reported a significant decrease in EFS among pediatric and adolescent patients with PMBL compared with other stage III non-PMBL diffuse large B-cell lymphoma (DLBCL) patients following FAB/LMB 96 therapy, suggesting that new therapeutic targeted agents are needed (Gerrard/Cairo et al, Blood 2013). Activation of the B-cell receptor (BCR) signaling pathway has now emerged as a central oncogenic pathway that promotes growth and survival in both normal and malignant B-cells. The cascade of signaling pathways downstream of the BCR includes BTK, MAPK, NF-κB and AKT. PMBL has a constitutively activated NF-kB pathway (Rosenwald et al, JEM 2003). Ibrutinib is a selective and covalent BTK inhibitor that inhibits chronic active BCR signaling and downstream signaling including the NF-kB pathway (Herman et al, Blood 2014) and is an active agent in ABC-DLBCL, which also has an activated NF-kB pathway (Griner et al, PNAS 2014). Preclinical studies of ibrutinib in CLL and MCL suggested that the inhibitory effects on cell proliferation were seen in the range of 1.0uM to 25.0uM (Herman et al, Blood 2011; Cinar et al, Leuk. Res 2013). Despite these relatively high IC50 values in vitro, ibrutinib has been highly effective in the treatment of patients with refractory CLL and MCL (Byrd et al, NEJM 2013 and Wang et al, NEJM 2013). Ibrutinib was approved by the FDA (IMBRUVICA, USPI) for patients with MCL in November 2013 or CLL in February 2014, who have received at least one prior therapy. However, the antitumor activity against PMBL of ibrutinib alone and in combination with other agents is currently unknown. OBJECTIVES: We hypothesize that ibrutinib in combination with other agents may be a future targeted agent in the treatment of PMBL. Therefore, we investigated the effect of ibrutinib alone and in combination with dexamethasone, rituximab and carfilzomib on cell proliferation and apoptosis in a PMBL cell line. METHODS: The Karpas-1106P PMBL cell line (DSMZ, Germany) was treated with ibrutinib alone (0-10uM, generously provided by Janssen R&D LLC) and in combination with dexamethasone (1uM), rituximab (20ug/ml) and carfilzomib (5nM) for 5 days treatment. The MTS cell proliferation assay (Promega) and western blot analysis were performed. Significant differences were determined by Student's t -test.The IC50 values were determined with CompuSyn software (Chou and Martin, ComboSyn, 2005). RESULTS: We observed a significant down-regulation of phosphorylated BTK after 5 days with ibrutinib treatment in Karpas-1106P cell line with 1uM ibrutinib (0.178 ± 1.63, p<0.0001) and the expression of total BTK protein was also significantly decreased with 1uM ibrutinib following 5 days of treatment (0.803±0.190, p<0.005) compared to control (1.000±0.00). We also observed a significant dose-dependent decrease in cell proliferation (0-10uM, p<0.0001) in ibrutinib-treated Karpas-1106P PMBL cell line. In combination with dexamethasone, the ibrutinib IC50 values for inhibition of cell proliferation were significantly decreased from 0.71uM ± 0.172 (ibrutinib alone) to 0.04uM ± 0.007 (p<0.005) with 1.0uM dexamethasone combination following 5 days of treatment. Second, in combination with rituximab experiment, the ibrutinib IC50 values was significantly decreased from 0.845uM ± 0.289 (ibrutinib alone) to 0.37uM ± 0.056 (p<0.05) with 20ug/ml rituximab following 5 days of treatment. Lastly, the ibrutinib IC50 values were significantly decreased from 0.72uM ± 0.185 (ibrutinib alone) to 0.002uM ± 0.001 (p<0.005) with 5nM carfilzomib combination following 5 days of treatment. CONCLUSIONS: Ibrutinib alone and in combination with dexamethasone, rituximab and carfilzomib significantly inhibited cell proliferation in the Karpas-1106P PMBL cell line. Ibrutinib's potential as an adjuvant agent in combination treatment in patients with PMBL is supported by these experiments. Future studies will focus on the in vivo effects of ibrutinib alone and in combination with dexamethasone and carfilzomib in a NOD/SCID PMBL xenograft mouse model. Disclosures Galardy: Mission Therapeutics: Research Funding.

Abstract BACKGROUND: Burkitt lymphoma (BL) represents approximately 40% of all childhood and adolescent non-Hodgkin lymphoma (Miles/Cairo, BJH, 2012). Children with relapsed or progressive BL develop chemotherapy-resistant disease and can rarely be cured with salvage therapy (Cairo et al, JCO, 2012). Bruton's tyrosine kinase (BTK) is a regulator of normal B-cell development and is activated upon B-cell receptor (BCR) stimulation. Chronic active BCR signaling through BTK activation can be inhibited by the selective and covalent BTK inhibitor, ibrutinib (Young et al, Nat Rev, 2013). Ibrutinib has been highly effective in the treatment of refractory patients with CLL and MCL and has been approved by the FDA for patients with CLL or MCL who have received at least one prior therapy (USPI) (Byrd et al, NEJM, 2013 and Wang et al, NEJM, 2013). BL, however, is associated with tonic or possibly chronic active BCR signaling; while, both CLL and MCL have chronic active BCR signaling. We have previously demonstrated ibrutinib significantly decrease BL proliferation and viability in vitro (Lee/Cairo et al, ASH, 2014) OBJECTIVE: We hypothesize that ibrutinib may be a potential adjuvant agent in the treatment of BL. Therefore, we investigated the in vivo anti-tumor activity of ibrutinib in BL xenografted NOD/SCID (NSG) mice. METHODS: The luciferase expression plasmid (ffluc-Zeo), kindly provided by L. Cooper, MD) was transfected into Raji cells. Cells were subcutaneously injected into NSG mice (6-8wks old, NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, Jackson lab). Tumor cells engraftment and progression were examined by Bioluminescent Imaging using the Xenogen IVIS-200 (Caliper Life Sciences). Mice were orally gavaged with vehicle control or Ibrutinib (1.25, 12.5 and 25mg/kg/day, generously provided by Janssen R&D, LLC) for 10 days. Statistical probability of survival and curve were determined by Kaplan Meier method. RESULTS: We observed a significant decrease in tumor luminescence intensity following ibrutinib treated BL xenografted NSG mice at day 20 (12.5mg/kg, p&lt;0.001 and 25mg/kg, p&lt;0.05) and at day 25 (12.5mg/kg, p&lt;0.05 and 25mg/kg, p&lt;0.05) compared to vehicle control. Furthermore, ibrutinib (12.5 mg/kg) treated mice significantly prolonged survival compared to vehicle control mice (p&lt;0.05). The median survival of mice following ibrutinib treatment (1.25, 12.5 and 25 mg/kg) were 31.5, 37.5 and 22.5 days, respectively, compared to control (24 days). CONCLUSIONS: Ibrutinib significantly decreased tumor progression and significantly increased the survival in BL xenografted NSG mice following 12.5mg/kg ibrutinib treatment. These results indicate that ibrutinib may be a potential adjuvant agent therapy in the treatment of BL. Future directions will be investigated the efficacy of ibrutinib in combination with dexamethasone in BL xenografted NSG mice. Citation Format: Sanghoon Lee, Changhong Yin, Timmy O'Connell, Matthew Barth, Janet Ayello, Lauren Harrison, Carmella van de Ven, Rodney Miles, Paul Galardy, Stanton C. Goldman, Megan Lim, Michelle Hermiston, Linda McAllister-Lucas, Lisa G. Roth, Sherrie L. Perkins, Mitchell S. Cairo. Ibrutinib significantly improves survival in a human Burkitt lymphoma (BL) xenograft NSG mouse model: Ibrutinib may be a potential adjuvant agent in the treatment of BL. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2608. doi:10.1158/1538-7445.AM2015-2608

SummaryPatient‐specific primers from 10 children/adolescents with Burkitt leukaemia (BL) ± central nervous system disease who were treated with French‐British‐American/Lymphome Malins de Burkitt 96 C1 plus rituximab were developed from diagnostic blood/bone marrow. Minimal residual disease (MRD) was assessed by real‐time polymerase chain reaction at the end of induction (EOI) and consolidation (EOC). Seventy per cent (7/10) and 71% (5/7) were MRD‐positive at EOI and EOC, respectively, with no disease recurrences. MRD after induction and consolidation did not predict relapse and subsequent therapy appeared to eliminate MRD. Thus, assessing MRD at a later time point is warranted in future trials to determine its clinical significance.

SummaryChildren and adolescents with Burkitt Lymphoma (BL) and combined central nervous system (CNS) and bone marrow involvement still have a poor prognosis with chemotherapy alone. We therefore investigated in children and adolescents with bone marrow (≥25% blasts) and/or CNS‐positive Burkitt lymphoma the chemoimmunotherapy combination of rituximab (375 mg/m2) and the standard chemotherapy arm of our previously reported French‐American‐British (FAB) Lymphome Malins de Burkitt (LMB) 96 trial. Central pathological and cytogenetic characterization was also performed. There were 40 evaluable patients with Burkitt histology (25 with leukaemia and 15 with CNS disease ± leukaemia). The chemoimmunotherapy regimen was well tolerated. The incidence of grade III/IV mucositis during induction cycles with combined chemotherapy and rituximab was 31% and 26%, respectively. The 3‐year event‐free survival (EFS)/overall survival (OS) was 90% (95% confidence interval [CI], 76–96%) in the entire cohort and 93% (95% CI, 61–99%) in patients with CNS disease. Based on the results of this trial, an international randomized study of FAB/LMB 96 chemotherapy ± rituximab for high‐risk patients is currently under investigation.

SummaryLaboratory (LTLS) and clinical (CTLS) tumour lysis syndrome (TLS) are frequent complications in newly diagnosed children with advanced mature B cell non‐Hodgkin lymphoma (B‐NHL). Rasburicase, compared to allopurinol, results in more rapid reduction of uric acid in paediatric patients at risk for TLS. However, the safety and efficacy of rasburicase for the treatment or or prevention of TLS has not been prospectively evaluated. Children with newly diagnosed stage III‐IV, bone marrow+ and/or central nervous system+ mature B‐NHL received hydration and rasburicase prior to cytoreductive therapy. Rasburicase was safe and well‐tolerated and there were no grade III–IV toxicities probably or directly related to rasburicase. Patients with an initial lactate dehydrogenase ≥2× upper limit of normal had a significantly elevated uric acid level (P = 0·005), increased incidence of TLS (P−0·005) and lower glomerular filtration rate (GFR; P < 0·001). Following rasburicase, there was only a 9% and 5% incidence of LTLS and CTLS, respectively. Furthermore, there was a significant improvement in estimated GFR from Day 0 to Day 7 following rasburicase (P = 0·0007) and only 1·3% of patients required new onset renal assisted support after rasburicase administration. A TLS strategy incorporating rasburicase prior to cytoreductive chemotherapy proved safe and effective in preventing new onset renal failure and was associated with a significant improvement in GFR.