Citations

7525 Background: New prognostic markers in chronic lymphocytic leukemia (CLL) are in demand. Different groups have developed models which combine multiple prognostic markers into a single index to classify CLL patients (pts). The CLL-International Prognostic Index (CLL-IPI) combines five parameters: age, clinical stage, TP53 status, IGHV mutational status, and serum β2 microglobulin levels. B-cell maturation antigen (BCMA) is a cell membrane receptor expressed exclusively on late stage B-cells and plasma cells with elevated serum (s) levels found in B-cell malignancies, such as multiple myeloma (MM). In MM, sBCMA levels can be used to monitor disease status and predict overall survival (OS). To further evaluate this biomarker in other hematologic malignancies, we studied it in CLL. Methods: Untreated (UNTX) CLL pts seen and consented at Mayo Clinic were identified. sBCMA levels were measured on stored sera of 331 UNTX CLL pts using an ELISA-based assay with a polyclonal anti-BCMA antibody from R&D Systems (Minneapolis, MN). The Mann-Whitney analysis was used to assess differences between CLL pts and healthy controls. The relationships between sBCMA and both time to first treatment (TFT) and OS were also assessed using Cox Regression models with an optimal sBCMA cutoff of 40.9 ng/mL. Results: The median age of pts was 61 years, and 71% were male. The distribution of CLL-IPI risk groups was as follows: 135 (41%) Low; 114 (34%) Intermediate; 67 (20%) High; 15 (5%) Very High. The median level of sBCMA in CLL pts (48.6 ng/mL) was higher (P <0.0001) than those of healthy controls (n = 104; 36.03 ng/mL). In CLL pts, sBCMA is significant in univariable analyses of TFT (HR 2.9 (95%CI, 2.0-4.2); P < 0.0001) and OS (HR 2.5 (95%CI, 1.5-4.0); P < 0.0003), and remains significant when adjusting for sex and CLL-IPI factors (HR 2.3 (95%CI, 1.6-3.3), P < 0.0001; HR 1.9 (95%CI 1.1-3.1), P = 0.01, respectively). Conclusions: sBCMA is elevated in CLL pts compared to healthy controls. After adjusting for CLL-IPI and sex, sBCMA levels provided independent prognostic value in predicting TFT and OS in this cohort. Measuring sBCMA with a readily accessible ELISA-based test, provides incremental value over the current CLL-IPI model in predicting prognosis of CLL.

7522 Background: Ibrutinib therapy is associated with an increased risk (~15%) of AF. An accurate risk assessment model for the development of AF in pts starting ibrutinib is not established, and outcomes after AF are not well described. Methods: An IRB approved retrospective review of CLL pts treated with ibrutinib at Mayo Clinic between Oct 2012 and Nov 2018 was conducted. Results: 299 pts were identified with a total of 565 years of ibrutinib exposure (Table). After a median follow-up of 24 months (range 0–70 months), 51 pts developed treatment-emergent AF (13 [25%] CTCAE 3 or higher). Our study assessed 3 clinical prediction models, the Framingham (Schanbel:Lancet 2009), Italian (Visentin:Blood 2018;132:3118), and Shanafelt (Shanafelt:Leuk Lymp; 2017) risk scores. Based on a lower Akaike information criteria, the Italian score was best able to predict risk of treatment emergent AF (2-year risk of AF with score 0 6%; 1-2 8%; 3-4 26%; 5+ 47%). Thirty (61%) pts were treated with medical therapy for AF (27 rate control; 2 rhythm control; 1 both). 16 (31%) pts underwent interventional therapy (3 AV node ablation; 11 cardioversion; and 2 pacemaker). Twelve (23%) pts temporarily held ibrutinib and resumed their original dose, 22 (43%) pts continued reduced dose ibrutinib and 11 (22%) continued their initial ibrutinib dose. Six (12%) pts permanently discontinued ibrutinib. Of 51 pts with treatment-emergent AF, 41 (80%) had a CHA2DS2-VASc score of ≥2 (41% received anticoagulation alone; 12% received antiplatelet therapy alone; 10% received both). No pt developed a thrombotic stroke after treatment-emergent AF. Two major bleeds, (1 GI and 1 intracranial) occurred, one in a pt on concomitant antiplatelet and anticoagulation therapy, and one in a pt receiving neither. The development of AF was associated with shorter event-free survival (HR 2.5, 95%CI 1.5-4.2, p<.001) and shorter overall survival (HR 3.5, 95%CI 2.0-6.3, p<.001). Conclusions: The Italian score was the best predictor of AF development in this cohort of ibrutinib treated pts. Although the vast majority of pts who develop AF after ibrutinib are able to continue therapy, the occurrence of AF appears to be associated with shorter EFS and OS. [Table: see text]

Cellular immunotherapy is a rapidly progressing field in multiple myeloma (MM). Multiple clinical trials have reported impressive efficacy of B cell maturation antigen (BCMA) directed chimeric antigen receptor cell therapy (BCMA CART) in MM. While trials demonstrated an overall response rate of 70-90% in patients with relapsed/refractory MM, the durable response rate is around 30%. Most patients lose their CART cells and the disease relapses within the first year, suggesting an inhibition by the MM tumor microenvironment (TME). Therefore strategies to overcome this inhibition would represent a major advance in CART cell therapy for MM. Cancer associated fibroblasts (CAFs) within the TME play a critical role in promoting tumor growth and in the generation of an immunosuppressive microenvironment. We hypothesized that CAFs from bone marrows of patients with MM (MM-CAFs) inhibit BCMA CART cells and contribute to their failure and that targeting both the malignant plasma cells and CAFs can overcome this resistance. To test this hypothesis, we isolated MM-CAFs and studied their interaction with BCMA CART cells generated from normal donors (41BB costimulated, lentivirally transduced). Our initial findings suggest that MM-CAFs inhibit BCMA CART cell antigen specific proliferation in the presence of the BCMA+ MM cell line OPM2, and this inhibition is predominantly mediated through the secretion of TGF-β (Fig A). MM-CAFs also promoted MM tumor growth in an MM-TME xenograft model established in the laboratory (Fig B). Here, immunocompromised NOD-SCID-γ-/- (NSG) mice were engrafted with 1x106 luciferase+ BCMA+ OPM2, in combination with either 1x106 CAFs or vehicle control intraveneously (IV). Subsequent tumor burden was monitored by bioluminescent imaging of these mice. The presence of CAFs in this model significantly accelerated MM progression (Fig B). Based on these findings, we aimed to develop CART cell therapy targeting both malignant MM cells and their CAFs and to determine whether this strategy can reverse MM-CAF induced CART cell inhibition. To identify targets for these CART cells, we first verified the expression of Fibroblast Associated Protein (FAP), an established CAF target, on MM-CAFs. Flow cytometric analysis of MM-CAFs showed significantly higher expression of FAP, compared to fibroblasts derived from normal bone marrow (Fig C). In addition, our screening flow cytometric analysis identified CS1 as another protein overexpressed by MM-CAFs (Fig C). We therefore designed and generated FAP CART cells (41BB costimulated, lentivirally transduced) and CS1 CART cells (CD28 costimulated, lentivirally transduced). We also generated dual CART cells for both BCMA-FAP CART cells and BCMA-CS1 CART cells. These dual CART cells were generated through the dual transduction of two lentiviral vectors during CART manufacturing. Next, we evaluated the impact of CAFs on effector functions of BCMA CART cells compared to dual targeting CART cells. When CART cells were stimulated with the BCMA+ MM cell line MM1S, in the presence of MM-CAFs, the antigen specific proliferation of BCMA CART cells, but not the dual targeting CART cells was significantly inhibited (Fig A). Similarly, in the presence of MM-CAFs, production of key effector cytokines by BCMA CART cells, but not the dual CART cells was reduced (Fig D). Finally, to verify the significance of our laboratory findings, we investigated the impact of CAFs on CART cell functions in vivo. First, using OPM2 xenografts, treatment with BCMA CART cells were able to completely eradicate MM (Fig E). However, to determine the effect of targeting CAFs, we used our MM-TME model. Here, NSG mice were engrafted with the luciferase+ MM cell line OPM2, along with MM-CAFs, as described in Fig 1B. Mice were then imaged for engraftment and randomized to treatment with 1) untransduced control T cells, 2) BCMA CART cells, 3) BCMA-FAP CART cells, or 4) BCMA-CS1 CART cells. A lower dose (1x106 IV) of CART cell was used to induce relapse post BCMA CART cells. Treatment with BCMA CART cells led to a transient antitumor activity in this MM-TME model (mice died within 2 weeks), while dual targeting CART cells resulted in durable remissions and long term survival of these mice (Fig F). In summary, we demonstrate for the first time that dual targeting both malignant plasma cells and the CAFs within the TME is a novel strategy to overcome resistance to CART cell therapy in multiple myeloma. Figure Disclosures Sakemura: Humanigen: Patents & Royalties. Cox:Humanigen: Patents & Royalties. Parikh:Janssen: Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria; AstraZeneca: Honoraria, Research Funding. Kay:Celgene: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; MorphoSys: Other: Data Safety Monitoring Board; Agios: Other: DSMB. Kenderian:Lentigen: Research Funding; Kite/Gilead: Research Funding; Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Tolero: Research Funding; Novartis: Patents & Royalties, Research Funding; Morphosys: Research Funding.

Background The CLL international prognostic index (CLL-IPI) is a validated staging system for newly diagnosed CLL patients that combines age, clinical stage, serum Beta-2 microglobulin, TP53, and IGHV mutation status into a single score and stratifies CLL patients into four prognostic risk groups with a c-statistic=0.72. Next-generation sequencing identified ~60 putative driver genes recurrently mutated in CLL, and genome-wide association studies (GWAS) identified 41 single nucleotide polymorphisms (SNPs) associated with CLL risk. We previously showed that a polygenic risk score (PRS) of the weighted average of the number of risk alleles among these 41 SNPs is associated with risk of both CLL and MBL. We and others have also shown that the tumor mutational load (TML), i.e., the total number of somatically mutated driver genes, is associated with time to first treatment (TTT). Herein, we examine whether TML and PRS add prognostic value beyond CLL-IPI for disease progression in a cohort of CLL and high-count MBLs. Methods We used the Mayo Clinic CLL resource to identify newly diagnosed (&lt;2 years) previously untreated CLL and high-count MBL patients based on the 2008 International Workshop CLL (IWCLL) criteria . We extracted DNA from CD5+/CD19+ clonal B-cells and sequenced the coding regions of 59 recurrently mutated CLL driver genes using a custom SureSelect panel and Illumina HiSeq 4000. The median coverage depth per sample was 2737X. Somatic mutations were called using MuTect2 in tumor-only mode. Germline variants were eliminated based on minor allele frequencies &gt;0.01%, and by those identified in 1000 Genomes Project, ExAC and/or ESP6500 databases, unless present in known mutation hotspots or COSMIC. After filtering high/moderate impact mutations, we calculated the TML. Peripheral blood DNA was genotyped on Illumina Omni Express array; after extensive quality control metrics, we calculated the germline PRS as the weighted average of the number of risk alleles among the CLL susceptibility SNPs. Cox regression was used to estimate hazard ratios (HR) and 95% confidence intervals (CI) to test for associations with TTT. We analyzed the low/intermediate risk groups and the high/very high risk groups in stratified analyses and tested for interactions between CLL-IPI and TML or PRS. Survival curves are displayed using the Kaplan-Meier method using P-values from the log-rank test; the PRS is displayed by tertile categories. Results Of 166 patients (120 CLL, 46 MBL), 67% were male and the median age at diagnosis was 62 years (range: 28-85). The median follow-up was 5.3 years, and 73 were treated for progressive disease as defined by the 2018 IWCLL criteria. Low risk CLL-IPI comprised 51%, intermediate risk 27%, high risk 18%, and very-high risk 4%. The most commonly somatically mutated genes were TP53 (8%), CHD2 (8%), BIRC3 (8%), SF3B1 (7%), and NOTCH1 (7%). We found that 42%, 32%, and 26% of patients had 0, 1, or 2+ genes with somatic mutations, respectively. The median PRS was 8.3 (range: 6.1-11.3) which is consistent with our prior PRS study on CLL/MBL and higher than 7.53 found in 7983 controls from our prior work. When we modeled TML, PRS, sex, CLL/MBL status and CLL-IPI together with the joint effects of CLL-IPI with TML and PRS, both interactions were significant in the model (P=0.008 for TML*CLL-IPI and P=0.014 for PRS*CLL-IPI), with c-statistic=0.81 (CI:0.77-0.85). When we stratified by CLL-IPI, TML was associated with shorter TTT in the low/intermediate risk (N=129, continuous HR=2.36 per TML category, CI: 1.61-3.47, P=1.3x10-5, Figure 1.A), but there was no evidence of an association in the high/very-high risk (N=37, continuous HR=1.12 per TML category, CI: 0.70-1. 78, P=0.64, Figure 1.B), after adjusting for PRS, sex, and CLL/MBL status. However, the PRS was associated with a shorter TTT in the high/very-high risk (continuous HR=1.77 per SD, CI: 1.00-3.12, P=0.048, Figure 1.D), with no evidence of an association in the low/intermediate risk (continuous HR=0.95 per SD, CI: 0.95-1.29, P=0.74, Figure 1.C), after adjusting for TML, sex, and CLL/MBL status. Conclusions In our study, TML was prognostic among the low/intermediate CLL-IPI risk groups while the PRS was prognostic among the high/very-high CLL-IPI risk groups. Sequencing CLL driver genes and genotyping relevant germline SNPs at time of diagnosis may provide incremental value over the CLL-IPI in predicting TTT among patients with CLL/MBL. Disclosures Parikh: MorphoSys: Research Funding; Genentech: Honoraria; AbbVie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; Janssen: Research Funding; Ascentage Pharma: Research Funding; Acerta Pharma: Research Funding. Ding:Merck: Research Funding; DTRM Biopharma: Research Funding. Cerhan:Celgene: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; NanoString: Research Funding. Kay:MorphoSys: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Agios: Other: DSMB.

Treatment with CD19-directed chimeric antigen receptor T cell (CART19) therapy has resulted in unprecedented clinical outcomes and was FDA-approved in acute lymphoblastic leukemia and non-Hodgkin B-cell lymphoma. However, its success in chronic lymphocytic leukemia (CLL) has been modest to date. An increasing body of evidence indicates that impaired CART cell fitness is the predominant mechanism of the relative dysfunction in CLL. The immunosuppressive microenvironment in CLL is well known and in part may be related to the abundance of circulating extracellular vesicles (EVs) bearing immunomodulatory properties. We hypothesized that CLL-derived EVs contribute to CART cell dysfunction. In this study, we aimed to investigate the interaction between circulating EVs isolated from CLL patient plasma (designated as CLL-derived EVs) and CART19 cells. We enumerated and immunophenotyped circulating EVs from platelet free plasma in untreated patients with CLL. We determined their interaction with CART19 cells using second generation, 41BB co-stimulated, lentiviral transduced CART19 cells generated in the laboratory from normal donors (FMC63-41BBζ CART cells). Our findings indicate that CLL-derived EVs impair normal donor CART19 antigen-specific proliferation against the CD19+ mantle cell lymphoma cell line Jeko-1 (Figure 1A). Next, we characterized CLL-derived EVs using nanoscale flow cytometric analysis of surface proteins and compared to healthy controls. Although the total EV particle count was not different between CLL and healthy controls (Figure 1B), there were significantly higher PD-L1+ EVs in patients with CLL (Figure 1C). Based on these results, we sought to assess the physical interaction between CLL-derived EVs and CART cells from normal individuals. When CLL-derived EVs were co-cultured with CART19 and CLL B cells and imaged with super-resolution microscopy, EVs were localized at the T cell-tumor junction (Figure 1D). Furthermore, CLL-derived EVs are captured by T cells as indicated by a significant reduction in the absolute count of EVs when co-cultured with resting T cells (Figure 1E). Having demonstrated that 1) there is an excess of PD-L1+ EVs in patients with CLL (Figure 1C) and 2) CLL-derived EVs physically interact with CART cells (Figures 1D-E), we sought to establish their functional impact on CART19 cells. Here, CART19 cells were stimulated with irradiated CD19+ JeKo-1 cells at a 1:1 ratio in the presence of increasing concentrations of CLL-derived EVs. There was a significant upregulation of inhibitory receptors such as PD-1 and CTLA-4 on the T cells (Figure 1F). This is associated with a reduction in CART effector cytokines (i.e., TNFβ) at higher concentrations of EVs (Figure 1G), suggesting a state of exhaustion in activated CART19 cells in the presence of CLL-derived EVs. This was further supported by transcriptome interrogation of CART19 cells. Here, CART19 cells were stimulated via 24-hour co-culture with the irradiated CD19+ cell line JeKo-1, in the presence of CLL-derived EVs at ratios of 10:1 and 1:1 EV:CART19 and then isolated by magnetic sorting. RNA sequencing of these activated CART19 cells indicated a significant upregulation of AP-1 (FOS-JUN) and YY1 (Figures 1H), known critical pathways in inducing T cell exhaustion. Finally, to confirm the impact of CLL-derived EVs on CART19 functions in vivo, we used our xenograft model for relapsed mantle cell lymphoma. Here, immunocompromised NOD-SCID-ɣ-/- mice were engrafted with the CD19+ luciferase+ cell line JeKo-1 (1x106 cells I.V. via tail vein injection). Engraftment was confirmed through bioluminescent imaging and mice were randomized to treatment with 1) untreated, 2) CART19 cells, or 3) CART19 cells co-cultured ex vivo with CLL-derived EVs for six hours prior to injection. A single low dose of CAR19 (2.5x105) was injected, to induce relapse. Treatment with CART19 cells that were co-cultured ex vivo with CLL-derived EVs resulted in reduced anti-tumor activity compared to treatment with CART19 alone (Figure 1I). Our results indicate that CLL-derived EVs induce significant CART19 cell dysfunction in vitro and in vivo, through a direct interaction with CART cells resulting in a downstream alteration of their exhaustion pathways. These studies illuminate a novel way through which circulating and potentially systemic EVs can lead to CART cell dysfunction in CLL patients. Disclosures Cox: Humanigen: Patents & Royalties. Sakemura:Humanigen: Patents & Royalties. Parikh:Ascentage Pharma: Research Funding; Janssen: Research Funding; AstraZeneca: Honoraria, Research Funding; Genentech: Honoraria; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding. Kay:Agios: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; MorphoSys: Other: Data Safety Monitoring Board. Kenderian:Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Lentigen: Research Funding; Novartis: Patents & Royalties, Research Funding; Tolero: Research Funding; Morphosys: Research Funding; Kite/Gilead: Research Funding.

Despite the success of chimeric antigen receptor T (CART) cell therapy, it is limited by 1) lower rates of durable responses related to inadequate CART cell expansion and trafficking to tumor sites and 2) development of life-threatening complication such as cytokine release syndrome (CRS). Development of a strategy to efficiently and robustly image and track CART cells in the clinic would allow the in vivo characterization of T cell expansion and trafficking to tumor sites as well as the development of strategies to potentially overcome these limitations. The sodium iodide symporter (NIS) is a characterized and sensitive reporter system that has been used for cell imaging in the clinic. We hypothesized that the incorporation of NIS into CART cells would be a sensitive and efficient way to assess CART cell expansion, trafficking, and toxicity. To test our hypothesis, we used two CART cell constructs that are characterized in preclinical models and studied extensively in the clinic: CART19 (41BB costimulated) and BCMA-CART cells (41BB costimulated). First, we generated NIS+CART19 and NIS+BCMA-CART cells through dual transduction of lentiviral vectors (Fig A) and revealed the exclusive 125I uptake by these NIS+CARTs and its inhibition by the NIS inhibitor KClO4in vitro (Fig B). We then analyzed T cell functions of NIS+CART cells. Here, NIS+CART19 or CART19 cells were cultured with the CD19+ acute lymphoblastic leukemia (ALL) cell line NALM6. There was no difference in CART cell cytotoxicity (Fig C), proliferation, or cytokine production (not shown) between NIS+CART19 and CART19. This indicates that the incorporation of NIS into CART cells does not impair their antitumor activity. Next, we evaluated the sensitivity of NIS+CART19 cell detection by TFB-PET in vivo; imaging was performed using an Inveon TFB-PET/CT scanner. Mice received 250 μCi 18F-TFB 45 minutes prior to image acquisition. NIS+CART cells were detectable with TFB-PET when cells were subcutaneously injected at a dose of 1.25x106 cells (Fig D). Having demonstrated that the incorporation of NIS in CART cells provides a sensitive way of their detection by TFB-PET and does not interfere with their effector functions, we tested its efficiency to assess CART cell trafficking in vivo, using multiple myeloma (MM) xenografts. Here, immunocompromised NOD-SCID-ɣ-/- (NSG) mice were engrafted with the BCMA+OPM2 MM cell line (1x106 IV). After engraftment, the tumor burden was assessed by bioluminescence imaging (BLI) and mice were randomized to receive 1) BCMA-CART or 2) NIS+BCMA-CART cells (5x106 IV). Mice were then serially imaged for 1) bioluminescence as a measure of disease burden, and 2) with TFB-PET to assess CART cell expansion and trafficking. As expected, BLI demonstrated that MM predominantly engrafts in bones (Fig E). TFB-PET confirmed trafficking of the NIS+BCMA-CART cells to the bones, corresponding to the areas involved by MM based on BLI (Fig E, right). Both BCMA-CART and NIS+BCMA-CART cells exhibited similarly potent antitumor activity in this model (not shown). Finally, we aimed to explore whether TFB-PET can detect CART massive expansion in vivo and predict the development of CRS. Here, we used an established CRS model in our laboratory. NSG mice were engrafted with patient derived relapsed ALL blasts (5x106 IV). Engraftment was confirmed by peripheral blood sampling. When the leukemic burden is &gt;10 CD19+ cell/µl, mice were treated with either high dose NIS+CART19 cells (5x106 IV) or PBS control. One week after NIS+CART19 cell treatment, mice developed muscle weakness, hunched bodies, and weight loss (Fig F), which correlate with an extreme elevation of cytokines (Sterner et al. Blood 2018). TFB-PET revealed a significant uptake in the bone marrow, spleen, liver, and lungs (Fig G) of the diseased mice but not control mice. Mice were then euthanized, and tissues were harvested. Flow cytometric analysis confirmed an extensive infiltration of CART cells in the liver and spleen. This demonstrates the ability of TFB-PET to detect NIS+CART cell expansion in vivo, correlating with the development of CRS. In summary, our results robustly show that the incorporation of NIS into CART cells provides a sensitive, clinically applicable platform to image CART cells using TFB-PET and to assess their expansion, trafficking to tumor sites, and the development of CRS. These studies illuminate a novel way to noninvasively assess CART cell functions in vivo. Figure Disclosures Sakemura: Humanigen: Patents & Royalties. Suksanpaisan:Imanis: Employment. Cox:Humanigen: Patents & Royalties. Parikh:Janssen: Research Funding; AstraZeneca: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria. Kay:Agios: Other: DSMB; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; MorphoSys: Other: Data Safety Monitoring Board. Peng:Imanis: Equity Ownership. Russell:Imanis: Equity Ownership. Kenderian:Kite/Gilead: Research Funding; Lentigen: Research Funding; Morphosys: Research Funding; Tolero: Research Funding; Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Novartis: Patents & Royalties, Research Funding.