Ritchie, David

PURPOSE Allogeneic hematopoietic cell transplantation (HCT) improves outcomes for patients with AML harboring an internal tandem duplication mutation of FLT3 ( FLT3-ITD) AML. These patients are routinely treated with a FLT3 inhibitor after HCT, but there is limited evidence to support this. Accordingly, we conducted a randomized trial of post-HCT maintenance with the FLT3 inhibitor gilteritinib (ClinicalTrials.gov identifier: NCT02997202 ) to determine if all such patients benefit or if detection of measurable residual disease (MRD) could identify those who might benefit. METHODS Adults with FLT3-ITD AML in first remission underwent HCT and were randomly assigned to placebo or 120 mg once daily gilteritinib for 24 months after HCT. The primary end point was relapse-free survival (RFS). Secondary end points included overall survival (OS) and the effect of MRD pre- and post-HCT on RFS and OS. RESULTS Three hundred fifty-six participants were randomly assigned post-HCT to receive gilteritinib or placebo. Although RFS was higher in the gilteritinib arm, the difference was not statistically significant (hazard ratio [HR], 0.679 [95% CI, 0.459 to 1.005]; two-sided P = .0518). However, 50.5% of participants had MRD detectable pre- or post-HCT, and, in a prespecified subgroup analysis, gilteritinib was beneficial in this population (HR, 0.515 [95% CI, 0.316 to 0.838]; P = .0065). Those without detectable MRD showed no benefit (HR, 1.213 [95% CI, 0.616 to 2.387]; P = .575). CONCLUSION Although the overall improvement in RFS was not statistically significant, RFS was higher for participants with detectable FLT3-ITD MRD pre- or post-HCT who received gilteritinib treatment. To our knowledge, these data are among the first to support the effectiveness of MRD-based post-HCT therapy.

Abstract Abstract 4082 N-methyl-2-pyrrolidone (NMP) is considered an inert drug delivery vehicle that is widely used as an industrial organic solvent. NMP is found in a range of pharmaceutical preparations and bio-prosthetic cements. In particular, NMP is used to solubilize kinase inhibitors for pre-clinical testing in experimental mice. The Vk*MYC transgenic mouse is a model of multiple myeloma (MM) that recapitulates the human disease. We have adapted Vk*MYC MM using a syngeneic transplant approach to shorten latency and facilitate testing of novel therapeutics. In this model, we noted delayed disease progression and improved survival in NMP treated mice compared to alternative vehicle controls. To confirm this observation, matched cohorts of mice bearing monoclonal kappa-secreting MM were treated with 20% NMP/80% polyethylene glycol (PEG) (n = 13), PEG alone (n = 10) or PBS (n = 7) at 100uL per 20g mouse daily p.o. Mice were monitored by serial serum protein electrophoresis and quantitative kappa immunoglobulin (IgK) estimation. At the commencement of therapy, cohorts were well matched for disease burden; mean M-spikes 12%, 14% and 14% of total serum protein for NMP, PBS and PEG respectively (p=0.58 NMP vs. PBS, p=0.68 NMP vs. PEG). After 28d treatment, NMP-treated mice had lower paraproteinemia compared to PBS and PEG controls (mean M-spike NMP 31%, PBS 42% and PEG 40%; p < 0.05 NMP vs. PBS or PEG), with concordant suppression of IgK (NMP 18g/L vs. PBS 39g/L, p<0.05). Survival in NMP-treated mice was also significantly longer than that in PBS-treated controls (median survival 50d and 40d respectively; p<0.05 log rank). Survival of PEG-only treated mice did not differ from PBS controls (median survival 39.5d; p=0.49). We compared the structure of NMP to known MM therapeutics, revealing that NMP is a subunit of thalidomide and its analogues. We next tested NMP for cytotoxicity and immune-modulatory drug (IMiD) activity in vitro. NMP (1uM – 10mM) was not directly cytotoxic to healthy donor peripheral blood mononuclear cells (PBMCs) or the U266 myeloma cell line. However, treatment of PBMC with 10uM NMP significantly enhanced NK cell cytotoxicity against the NK cell-sensitive target cell line K562 from 26% to 42% (p < 0.05). Further, NMP favored a Th-1 cytokine profile without directly altering the frequency of CD4, CD8, NK or regulatory T cell subsets in vitro. As compared to the IMiD lenalidomide, NMP inhibited TNF-a production from LPS stimulated monocytes. Given the IMiD-like effects of NMP, we assessed its ability to alter the expression of NK cell ligands on myeloma targets. NMP upregulated both NKG2D and DNAM-1 ligands (MIC-A, MIC-B, ULBP-1, ULBP-2 and CD155) on U266 cells, thereby enhancing the NK cell mediated lysis of the U266 cell line. Our results demonstrate the in vivo anti-myeloma activity and IMiD-like properties of NMP, indicating that it is a putative active thalidomide subunit. The detection of IMiD-like activity was facilitated by a sensitive, immune competent MM model. NMP should no longer be considered inert for the purposes of drug delivery. These findings mandate re-evaluation of drug efficacy data where NMP has been used as the drug delivery vehicle, particularly in immune-competent cancer models. Our findings with NMP may allow the development of new IMiDs. Finally, occupational or iatrogenic exposure to NMP should be further assessed as a potential risk for immunologically mediated adverse effects. Disclosures: No relevant conflicts of interest to declare.

Abstract Multiple myeloma is a blood cancer that arises in the bone marrow from malignant plasma cells. Although treatments have dramatically improved during the last decade, multiple myeloma remains an incurable disease responsible for more than 10 000 deaths per year in the United States. CD137 is a costimulatory molecule expressed on activated T cells and NK cells. We recently established that agonist monoclonal antibodies (mAbs) against CD137 (clone 3H3) significantly reduce tumor burden and improve survival in a mouse model of multiple myeloma1. We found that anti-CD137 mAb therapeutic activity requires the presence of both CD8 T cells and NK cells but is independent of Fc receptors. Moreover, we observed increased numbers of T cells in the bone marrow of anti-CD137 mAb treated mice. CD8 T cells were increased while the percentage of Tregs was reduced. Thus, anti-CD137 mAbs protect mice against multiple myeloma by inducing potent NK cell and CD8 T cell responses. In the present study, we investigate further the therapeutic interest and the potential limitations of anti-CD137 mAbs. To this aim, we used multiple myeloma cell lines derived from Vk*MYC transgenic mice. Wild-type (WT) mice intravenously injected with Vk*MYC cells develop a disease very similar to human multiple myeloma. High percentages of malignant plasma cells are observed in the bone marrow and in the spleen 5 weeks after injection and disease progression can be followed by the level of serum gamma-globulin (M protein). We first observed that, although early anti-CD137 mAb treatment efficiently protected the mice against multiple myeloma, this treatment loses efficacy when given more than 3 weeks after Vk*MYC cell injection. Interestingly, CD4 cell depletion restores the efficacy of late anti-CD137 mAb treatment, indicating that Treg-mediated suppression may limit the therapeutic efficacy of anti-CD137 mAbs. In line with previous studies, we observed that NK cell numbers in the spleen are dramatically reduced 12 days after the initiation of anti-CD137 mAb treatment. NK cell numbers are also decreased in other organs including liver, lung and blood. Importantly, we found that NK cells are still poorly represented in the spleen 3 weeks after the last treatment with anti-CD137 mAbs. Finally, we observed that anti-CD137 mAb treatment induced T cell activation and increases T cell infiltration in the liver. Consistent with the cases of severe hepatitis observed in patients treated with anti-CD137 mAbs, we noted that some of anti-CD137 mAb treated mice that had survived multiple myeloma developed severe liver damage. Such liver damage could be observed more than 100 days after the last therapy with anti-CD137 mAbs. In conclusion, we report several side effects induced by anti-CD137 mAbs treatment that last long after the treatment has been interrupted. These results encourage being particularly careful when administrating this drug to patients. Our study demonstrates the utility of mouse models to investigate the anti-tumor benefits versus toxicity of immunotherapies. We are planning to use this approach to assess the therapeutic efficacy of anti-CD137 mAbs in combination with other treatments. We speculate that the prior reduction of tumor burden by chemotherapy or stem cell transplantation will allow better efficacy of anti-CD137 mAbs at lower doses. The use of mice injected with multiple myeloma cell lines will help determining which combination allows efficient anti-tumor responses at late multiple myeloma stages with limited toxicity. 1 Guillerey C*, De Andrade LF*, Vuckovic S, Miles K, Ngiow SF, Yong MCR, Teng MWL, Colonna M, Ritchie DS, Chesi M, Bergsagel PL, Hill GR, Smyth MJ*, Martinet L* Immunosurveillance and therapy of multiple myeloma is CD226-dependent; Journal of Clinical Investigation, May 1;125(5):2077-89. Citation Format: Camille Guillerey, Lucas Ferrari de Andrade, Slavica Vuckovic, David Ritchie, Marta Chesi, Leif Bergsagel, Geoffrey R. Hill, Ludovic Martinet, Mark J. Smyth. Anti-CD137 mAb therapy of multiple myeloma. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B155.