Shao, Zuoyi

Abstract Introduction Chimeric antigen receptor T cell (CAR-T) expansion has been consistently identified as a predictor of clinical response. However, there are no clinically available test to measure CAR-T presence after infusion for the FDA approved CAR-T therapy. We hypothesize that the lymphocyte expansion which can be readily measured as absolute lymphocyte count (ALC) in blood count differential could be a clinically accessible surrogate for CAR-T expansion in patients who receive FDA approved CD19 CAR-T to treat aggressive B-cell non-Hodgkin lymphoma (NHL). We examined the ALC levels in the first 2 weeks after CAR-T infusion and correlation with clinical outcomes in NHL patients who received CAR-T at our institution. Methods Records were reviewed for patients who received CAR-T between 6/2016 and 1/2021 at Mayo Clinic, Rochester. CAR+ T cells were identified by flow cytometry using an anti-FMC63 antibody. Receiver operator curve was generated using nominal logistic regression to predict complete response (CR) as best response. Progression-free survival and overall survival were calculated using Kaplan-Meier method and between-group differences were assessed using log-rank test. Continuous variables were compared using wilcoxon text and categorical variables were compared using chi-square test. Results Among the 87 patients who received CAR-T for NHL, majority of the pts received axicabtagene ciloleucel (86, 99%). The highest ALC level (ALC peak) in the first 15 days were identified for all patients and median ALC peak was 0.44 X10 9/L (range, 0 - 2.55x10 9/L). The median time to ALC peak was 10 ±3 days. Increasing ALC peak levels correlated with increased CAR+ T cells in blood measured by flow cytometry (n=16, R=0.63, p=0.0008). Using ROC analysis, an ALC peak level of 0.67 was identified as the cut point for best association with CR (AUC=0.68, p=0.0004). Baseline demographics were similar between the high ALC peak (N=35) and low ALC peak (N=52) groups, as shown in Table 1. There was no difference in the incidence, high grade, or duration of cytokine release syndrome (CRS) and immune effector associated neurotoxicity syndrome (ICANS) between the two groups (Table 2). Patients who received a higher cumulative dose of steroid for management of CRS and ICANS had lower ALC peak. A higher ALC peak was associated with an increased CR rate and durable CR at month 6. Similarly, a higher ALC peak was associated with increased progression-free survival (PFS, 6.4 months vs. 2.7 months, p=0.004) and overall survival (OS, 31 months vs. 12 months, p=0.04), as shown in figure 1. Conclusion Given the typical CAR-T expansion seen in the first 2 weeks post infusion, ALC peak in the first 15 days is a clinically accessible, reliable surrogate for CAR-T expansion and predicts durable CR and longer PFS, OS. This study was supported in part by the Mayo Clinic Center for Individualized Medicine, Bernard E. and Edith B. Waterman, Henry J. Predolin Foundation and other generous benefactors of Mayo Clinic. Figure 1 Figure 1. Disclosures Bennani: Verastem: Other: Advisory Board; Purdue Pharma: Other: Advisory Board; Daichii Sankyo Inc: Other: Advisory Board; Kyowa Kirin: Other: Advisory Board; Vividion: Other: Advisory Board; Kymera: Other: Advisory Board. Paludo: Karyopharm: Research Funding. Wang: Genentech: Research Funding; Eli Lilly: Membership on an entity's Board of Directors or advisory committees; LOXO Oncology: Membership on an entity's Board of Directors or advisory committees, Research Funding; MorphoSys: Research Funding; InnoCare: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees. Ansell: Bristol Myers Squibb, ADC Therapeutics, Seattle Genetics, Regeneron, Affimed, AI Therapeutics, Pfizer, Trillium and Takeda: Research Funding. Lin: Janssen: Consultancy, Research Funding; Legend: Consultancy; Bluebird Bio: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Vineti: Consultancy; Gamida Cell: Consultancy; Kite, a Gilead Company: Consultancy, Research Funding; Novartis: Consultancy; Takeda: Research Funding; Merck: Research Funding; Sorrento: Consultancy; Juno: Consultancy.

Abstract Background: Chimeric antigen receptor T cell therapy (CAR-T) is a recent therapeutic advance for the treatment of myeloma. Among the FDA approved and investigational CAR-T, primary refractory disease post CAR-T infusion is uncommon, however most patients eventually relapse. To gain insight to the myeloma cell and immune cell characteristics associated with early relapse (PD) versus durable response (DR), we examined myeloma cells and immune cells in both the bone marrow and blood by single cell RNA-seq. Method: Bone marrow aspirate and blood samples were collected prior to BCMA targeted CAR-T therapy. CD138 + and CD138 neg cells from bone marrow (BM) and peripheral blood mononuclear cells (PBMNC) were collected for 5' based scRNA-seq (10X Genomics). CD138 + cells were clustered by Harmony integration. Top expressing genes in unique clusters for PD and DR were analyzed by Ingenuity Pathway Analyses. To profile CD138 neg BM cells and PBMNC from myeloma patients, the scRNA-seq dataset was mapped to the human PBMC reference using the Seurat v4 reference-guiding approach. Differential gene expression between responders and non-responders was performed using the FindMarkers function for each of the immune cell clusters in the BM and PB. Results: BM and PB samples from 15 patients were analyzed. Among these, 5 patients had relapsed disease more than 1-year post CAR-T infusion (Durable Response, DR), and 10 patients had relapse within 1 year (early relapse, PD). Among these, CD138+ cells were available from 3 DR and 4 PD for analysis. Multimodal expression of BCMA were seen among the CD138+ cells across all samples. Using the mode of the highest expression level as a cut-off, patients with DR had a trend toward having higher fraction of BCMA high CD138+ cells compared to pts with PD. Clustering of all CD138+ cells by Harmony integration identified 12 distinct clusters, among which two clusters were unique to pts in DR, and one was unique to patients in PD. Ingenuity pathway analysis identified the top marker genes in these 3 clusters to be enriched in pathways for IL-15 signaling, BCR signaling, and primary immunodeficiency signaling. (Figure 1A-E.) Differential gene expression for immune cell clusters from CD138 neg BM cells and PBMNC were compared between pts in PD and DR. Interestingly, different patterns were seen in immune cells in PB and BM (Figure 1G). For example, compared to pts in PD, those with DR had decreased signaling in CD16 PB monocytes for interferon signaling and differentiation to macrophages, whereas the CD16 monocytes in the BM in pts in DR had increased expression of mitochondrial genes for anti-oxidant stress, polarization to M1 macrophage and anti-apoptosis. Similarly, pts in DR had CD8 Tcm in the BM with increased expression of genes involved in cell adhesion and anti-apoptosis, and CD4 Tcm in the BM with increased expression of genes in protein metabolism. Conclusion: scRNAseq analysis of myeloma cells suggest different tumor transcriptome profile may be identified in myeloma cells that could relapse early after CAR-T therapy. In addition, host immune profile both in the BM microenvironment and in systemic PB circulation could be associated with durable clinical response. Figure 1 Figure 1. Disclosures Kapoor: Pharmacyclics: Consultancy; Glaxo SmithKline: Research Funding; Amgen: Research Funding; Sanofi: Research Funding; AbbVie: Research Funding; Ichnos Sciences: Research Funding; Sanofi: Consultancy; Regeneron Pharmaceuticals: Research Funding; Takeda: Research Funding; Karyopharm: Research Funding; Karyopharm: Consultancy; Cellectar: Consultancy; BeiGene: Consultancy. Dingli: GSK: Consultancy; Janssen: Consultancy; Sanofi: Consultancy; Novartis: Research Funding; Apellis: Consultancy; Alexion: Consultancy. Kumar: Bluebird Bio: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Tenebio: Research Funding; Novartis: Research Funding; BMS: Consultancy, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche-Genentech: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Antengene: Consultancy, Honoraria; Carsgen: Research Funding; Oncopeptides: Consultancy; Beigene: Consultancy; Amgen: Consultancy, Research Funding; Merck: Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding. Lin: Merck: Research Funding; Kite, a Gilead Company: Consultancy, Research Funding; Takeda: Research Funding; Bluebird Bio: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Sorrento: Consultancy; Juno: Consultancy; Legend: Consultancy; Gamida Cell: Consultancy; Vineti: Consultancy; Novartis: Consultancy; Janssen: Consultancy, Research Funding.

7558 Background: A low absolute lymphocyte to monocyte ratio (ALC/AMC) has been found to predict decreased survival in lymphoma patients receiving chemotherapy and stem cell transplant. We report its clinical significance and additional cellular phenotype changes in patients receiving chimeric antigen receptor T-cell (CART) therapy. Methods: Records were reviewed for patients (pts) who received axicabtagene ciloleucel between 6/2016 and 12/2020. Receiver operator curve was generated using nominal logistic regression to predict CR as best response. Survivals were calculated using Kaplan- Meier method. Blood immune phenotype were assayed by multiparametric flow. Principle component analysis (PCA) was performed using ClusterVis. Results: Low ALC/AMC (≤0.8) prior to lymphodepletion (LD) chemotherapy on day -5 was associated with lower CR rate (AUC=0.68, Table). Our cohort of 81 pts had similar baseline characteristics except that noted in Table. Low ALC/AMC ratio is associated with shorter EFS and OS (EFS: 2.6 vs. 6.4 months, P<0.0001; OS: 5.3 months vs. not reached, P=0.0006), respectively. Prognostic association remained significant in multivariate analysis including ASCT, bridging therapy and CRP. Interestingly, compared to the high ALC/AMC group, the low ALC/AMC group had decreased CD8 Tem, increased CD16+CCR2+ monocytes and increased monocytes’ producing IL12, IL-10, and IL-1β (n=26). Unsupervised PCA identified 3 clusters: 1. Low ALC/AMC, all non-CR; 2. High ALC/AMC, some non-CR; 3. High ALC/AMC, all CR. Compared to cluster 1 and 2, cluster 3 had increased CD4 Tnaive, CD8 Tcm and IL-17 producing CD4 T and NK cells. Conclusions: ALC/AMC is a clinically accessible test that is strongly associated with CAR-T response and survival. Immune characterization revealed that the biologic effect is not just associated with cell ratio. Increased inflammation has been found to negatively impact CAR-T response, with some cytokines known to be from the myeloid lineage. We show that CRP is elevated in the low ALC/AMC group with increased cytokine production by monocytes. In addition, presence of T cell subset and IL-17 producing cells, before LD, are associated with clinical response. Further investigation on optimizing host immunity may help improve clinical outcome with CAR-T.[Table: see text]