ALBERT

Description: Background: Autologous stem cell transplantation (ASCT) has been used as consolidation following induction therapy to reduce relapse (Le Gouill S et al et al, ASH 2014) in mantle cell lymphoma (MCL). Rituximab maintenance therapy post induction has been shown to improve overall survival (Kluin-Nelemans et al. NEJM 2012). Bortezomib has been shown to be effective against patients with relapsed/refractory MCL (Fisher et al, JCO 2006). However, the efficacy of the combination of bortezomib and rituximab as maintenance therapy post ASCT has not been established. Although CCND1 overexpression is pathognomonic in MCL, quantitative monitoring of CCND1 mRNA has not been established as a method of MRD monitoring. We aim to increase the 2 year disease free survival (DFS) for patients with MCL post ASCT with maintenance bortezomib and rituximab, and to explore the use of quantitative CCND1 mRNA expression as minimal residual disease (MRD) monitoring in this study. We report our interim analysis of DFS, toxicity, and quantitative CCND1 mRNA monitoring here. Methods: This is a multicenter phase II study in patients with MCL post ASCT. All patients were in complete remission (CT scan and bone marrow biopsy) post ASCT prior to entering study. Bortezomib was given at 1.3 mg/m2subcutaneously once a week for 4 weeks every 3 month for 2 years. Rituximabwas given at 375 mg/m2 intravenously once a week for 4 weeks every 6 months for 2 years. CT scanning and/or FDG-PETwere performed at every 6 months intervals to assess for disease status. The primary endpoint was 2 year DFS after start maintenance therapy. Secondary endpoints included toxicity, relapse rate, OS, and CCND1 mRNA as MRD monitoring. Blood was collected at baseline post ASCT and on day 1 of each cycle (3 month interval) at City of Hope. CCND1 mRNA was assessed using droplet digital PCR technology (ddPCR) on RNA obtained from peripheral blood mononuclear cells. JVM2 (MCL cell line), normal healthy patient, and an untreated patient with MCL involvement served as controls. All values were normalized to HPRT1 (housekeeping gene) and then to level of JVM2 (100%). Results: Sixteen patients were accrued, with 15 eligible for analysis. See Table 1 for baseline characteristics. The median follow- up was 19.1 months from time of starting maintenance therapy to time of last contact. The 2 year DFS is 100% (95% CI: N/A) and 2 year OS is 100% (95% CI: N/A). There have been no relapses and no treatment- related deaths on the study. Treatment was well tolerated. Grade 3/4 toxicities possiblyattributable to study drug included neutropenia (n=4), lymphopenia (3), pneumonia (1), and skin infection (1). Grade 2 possibly related toxicities included neutropenia (1), thrombocytopenia (2), anemia (1), arthralgia (1), peripheral sensory neuropathy (1), pruritis (1), and hypertension (1). Peripheral neuropathy toxicities included grade 1 sensory neuropathy (4), grade 1 motor neuropathy (1), and grade 2 sensory neuropathy (1). For MRD analysis, 12/15 patients had up to 12 months time point and 8/12 patients had up to 24 months time point. Out of 105 samples collected, ddPCR yield quantifiable CCND1 mRNA results in all time points tested except 3 samples. High CCND1 mRNA was detected in untreated patient with MCL involvement and JVM2. Low CCND1 mRNA was detected in normal healthy control and all samples on maintenance therapy. In all 12 patients analyzed, the relative CCND1 mRNA level was never above 12% of control (JVM2), and majority of the samples were below 5% (Figure 1). There were also very little intra-patient fluctuations. Conclusions: The combination of bortezomib with rituximab for treating MCL is well tolerated post ASCT and has encouraging results with no relapses seen to date. Quantitative CCND1 mRNA monitoring by ddPCR is feasible and correlates with clinical outcome. Table 1. Characteristics N (%) or Median (range) Median age 62 (45-66) Gender Male Female 14 1 Stage II Stage III Stage IV 1 4 10 Months from Transplant to Initial Maintenance 3.1 (2.3-5.8) Extra Nodal Disease No Yes 4 11 MIPI (At initial diagnosis) Low INT High 8 6 1 ASCT in 1st CR(n=13) -R-bendamustine -R-HCVAD/MTX/ARA-C -NORDIC -RCHOP ASCT in 2nd CR (n=2 ) -RCHOP followed by R-HCVAD/MTX/ARA-C -RCHOP followed by R-bendamustine 3 4 4 2 1 1 Median number of cycles 6 (2-8) Figure 1. Quantitative CCND1 mRNA monitoring by ddPCR Figure 1. Quantitative CCND1 mRNA monitoring by ddPCR Disclosures Chen: Seattle Genetics, Inc.: Consultancy, Other: Travel expenses, Research Funding, Speakers Bureau; Genentech: Consultancy, Speakers Bureau; Millennium: Consultancy, Research Funding, Speakers Bureau. Off Label Use: 90-Y ibritumomab tiuxetan is a radiotherapeutic antibody targeting CD20. It is part of a regimen indicated for treating patients with relapsed/refractory, low-grade or follicular B cell non-Hodgkin lymphoma (NHL), or patients with previously untreated follicular NHL who achieved a complete or partial response to first-line chemotherapy.. Holmberg:Up to date: Patents & Royalties; Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Membership on an entity's Board of Directors or advisory committees, Research Funding. Siddiqi:Seattle Genetics: Speakers Bureau; Kite pharma: Other: attended advisory board meeting; Pharmacyclics/Jannsen: Speakers Bureau. Nademanee:Gilead: Consultancy; Seattle Genetics Inc.: Research Funding; Spectrum: Research Funding; Celgene: Consultancy. Forman:Amgen: Consultancy; Mustang: Research Funding.

Description: Abstract 65 Background Specific oncogenes when overexpressed in lymphoma are associated with poor prognosis. Small interfering ribonucleic acids (siRNAs) can be custom designed to inhibit target oncogenes, but delivery of siRNA into lymphoma cells is a difficult task. Aptamers are synthetic in vitro selected nucleic acids that bind with high specificty to their target molecules. B cell activating factor (BAFF) enhances the maturation and survival of B cells via binding to its receptor, BAFF-R, which is expressed on the surface of B cell lymphoid malignancies. We have designed an aptamer against BAFF-R (R1) as a delivery vehicle for siRNA by covalently linking it to an siRNA molecule that inhibits oncogene target STAT3 (si-STAT3). We aim to downregulate STAT3 mRNA both in vitro (primary lymphoma cells) and in vivo (mouse xenograft models) with our aptamer/siRNA construct (R1-STAT3). Methods In vitro experiments used 2 mantle cell lymphoma cell (MCL) lines (Jeko-1, Z138), one diffuse large B cell lymphoma (DLBCL) cell line (Daudi), one negative control cell line (CEM, no BAFF-R expression), and primary lymphoid tumors. Primary lymphoid tumors were obtained from patients with at least 15% peripheral blood lymphoma involvement (chronic lymphoid leukemia-CLL, marginal zone lymphoma-MZL, and MCL). The selection and synthesis of aptamers used SELEX, gel shift, and filter binding assays. The visualization of aptamers utilized Z-axis confocal microscopy and Cy3 labeled aptamers. siRNA against STAT3 was generated using standard algorithms. Conjugation of aptamer to siRNA was done by two separate methods (chimera and stick, Zhou 2009). Downregulation of STAT3 was accessed by qRT-PCR and western blot analysis. We used NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice and MCL cell lines for in vivo mouse xenograft experiments. One million Z138 cells transduced with lentiviral vectors expressing luciferase were injected subcutaneously into the flanks of mice. Tumor engraftment was visualized by palpation and Xenogen imaging. Mice were then treated with R1 alone, R1-STAT3, and a negative control via intratumoral injection. Dosage and schedule of administration were consistent for all experimental groups. Mice were then sacrificed and subcutaneous tumors analyzed for STAT3 expression. We also injected one million Z138 cells transduced with a lentiviral vector expressing luciferase intravenously via tail and visualized engraftment by Xenogen imaging. Results Confocal microscopy showed easy entry of Cy3 labeled R1 aptamers and aptamer/STAT3 siRNA (R1-STAT3) conjugate into lymphoma cell lines (MCL and DLBCL), and primary lymphoma cells (CLL and MCL), but not into CEM cells. Intravenous injection of R1 showed biodistribution of R1 in lung/liver/spleen/BM and tumor site. qRT-PCR for R1 showed the highest concentration of R1 in tumor sites as compared to spleen/liver (4 log increase). These results show R1 aptamer can selectively target BAFF-R expressing lymphomas in vitro and in vivo. Table 1 shows R1-STAT3 conjugates downregulate STAT3 mRNA and STAT3 protein in MCL cells lines, but has no effect on CEM cells. Subcutaneous injection of MCL cells caused subcutaneous tumor formation which was easily palpable. We then performed Intratumoral injection of R1 and R1-STAT3 into subcutaneous tumors and analyzed STAT3 mRNA expression. Table 1 shows downregulation of STAT3 mRNA with R1-STAT3 as compared to R1 injection alone (83% downregulation). These results shows R1-STAT3 can efficiently downregulate STAT3 mRNA in vivo as well as in vitro. Conclusions BAFF-R functions as an efficient delivery vehicle for siRNA therapy in BAFF-R expressing B cell lymphomas. The aptamer/siRNA construct selectively and efficiently downregulates target oncogenes in primary lymphoma cells and in mouse xenograft models. Disclosures: No relevant conflicts of interest to declare.