ALBERT

Description: Abstract 3875 Introduction: Immunologic environment influences progression of lymphoid malignancies. Specifically, shifts in subsets of natural killer (NK) and T cells as well as tumor expression of inhibitory ligands may contribute to ability to evade host detection. Immune dysfunction may be particularly important in CLL/SLL, as prevalent circulating tumor cells engage in persistent, widespread interactions with immune cells; commonly-used mAb therapies (e.g. rituximab, alemtuzumab) rely upon ADCC mediated by NK cells and other innate effectors; and disease course is highly variable and not fully accounted for by tumor-intrinsic prognostic factors. Therefore, to better characterize the immune system in CLL/SLL, we prospectively assessed NK and T cell frequency, phenotype, and function in a series of CLL/SLL patients. Methods: Serial blood samples (up to 3 samples each, 3–6 months apart) were collected from 31 untreated CLL/SLL patients (median age 66) and 15 healthy age-matched controls (HC), and peripheral blood lymphocytes (PBL) analyzed directly ex vivo by multiparameter flow cytometry (160 distinct parameters evaluated, primarily on T and NK cells). NK cell-mediated natural and antibody-dependent cytotoxicity were also assessed by CD107a degranulation assay following PBL co-culture with rituximab, 721.221 EBV-transformed lymphoma cells, or both. Differences in parameters between patients and controls, or between progressors and non-progressors [categorized based on updated NCI-WG criteria (Blood 2008;111:5446)] were analyzed by Wilcoxon rank-sum test. All subjects signed IRB approved informed consent forms. Results: CLL/SLL VS. HC: CLL/SLL samples displayed a marked decrease in the ability of the cytolytic CD56dim NK cells to degranulate in response to tumor, both with or without rituximab (Table 1). CD56dim NK cells from CLL/SLL patients also displayed a more immature phenotype (↓CD57, ↓NKG2D, ↑CD27, ↓KIR) than those from HC, suggesting either a block in differentiation or elimination of the most-differentiated cells. NK cell expression of NKp44, CD69, CD62L, CD137, granzyme B, perforin, or PD-1, as well as tumor-induced NK cell production of IFNγ, did not differ. CLL/SLL patients had increased total T cells with a decreased CD4:CD8 ratio, associated with increased total number of CD8 T cells, greater activation of naive CD4 T cells and transition to a memory phenotype. Treg (CD4+CD25+FoxP3+) frequency was significantly higher in CLL/SLL patients (4.5% vs. 1.8% of CD4 T cells, p=0.005), as was PD-1 expression on both CD4 and CD8 T cells, while CD137 and ICOS expression was similar in both groups. PROGRESSORS VS. NON-PROGRESSORS: With median follow-up of 16.5 months (range 1–37), 7 of 31 patients have met criteria for progression. Compared to non-progressors, progressors showed changes in the CD56bright NK cell compartment suggestive of increased activation and accelerated differentiation, with increased expression of CD69, granzyme B, perforin, CD16, and KIR. However, no significant functional differences in NK cells, or consistent differences in T cell subsets, have been observed to date. Conclusions: CLL/SLL patients have a shift toward less mature NK cells, associated with deficits in NK cell degranulation against tumor targets, compared with healthy donors. Those CLL/SLL patients who progressed had greater CD56 bright NK cell phenotypic aberrancies than non-progressors, though these findings require confirmation with a larger cohort. Taken together, our findings support the hypothesis that immune dysfunction in CLL/SLL may be due in part to a block in NK cell differentiation or loss of more mature cells, and current studies are exploring these possibilities and potential mechanisms. Given these findings, along with the immunosuppressive changes observed in the T cell compartment (↑Tregs, ↑PD-1), these data support therapeutic strategies in CLL/SLL aimed at augmenting NK and/or T cell function. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Monoclonal antibodies (mAbs) are an emerging therapeutic class for MM patients (pts). Elotuzumab, a mAb in late-phase clinical development, targets the SLAMF7 receptor expressed highly on MM cells. While its primary mechanism of action is through CD16-mediated ADCC, elotuzumab can also directly activate SLAMF7-expressing NK cells. Gaining a greater understanding of phenotypic and functional changes in NK cells over the course of the disease, and how these changes impact capacity for ADCC, may help identify profiles that can better select pts likely to benefit from elotuzumab or other mAb therapies. Methods: We prospectively performed a comprehensive flow cytometry-based analysis of lymphocyte subsets, focusing on expression of NK cell activating and inhibitory receptors, activation and maturation markers, and degranulation in 30 MM pts (12 newly-diagnosed (ND), 18 relapsed/refractory (RR)) and 19 aged-matched healthy donors (HD). Over 140 immune parameters were analyzed, with differences in expression between HD and pt subsets compared by Wilcoxon rank-sum test. We analyzed correlations between expression of certain markers with each other, and with elotuzumab-induced NK cell degranulation against MM cell targets (MM1R) in a 2-hour co-culture assay. We also compared NK cell parameters in blood and bone marrow (BM) from pts with matched samples available. Results: Within the blood, there was no difference in relative NK cell frequency between the groups, and little difference phenotypically between HD and ND pt NK cells, except for decreased DNAM1 expression in ND. In contrast, in comparison to HD, CD56dim NK cells in RR pts were less mature with a higher CD56bright to CD56dim NK cell ratio and reduced expression of the terminal differentiation/maturation markers, CD57 and KLRG1. RR pts also showed increased expression of the activation marker CD69 on all NK cells, and their CD56dim NK cells had increased levels of the natural cytotoxicity receptors, NKp30 and NKp46 and decreased expression of activating receptors DNAM1 and NKG2D. SLAMF7 expression was also increased in RR pts, but only on the CD56bright subset. Consistent changes in NK cell expression of checkpoint/co-stimulatory molecules (eg. PD-1, Tim3, LAG3, CD137) were not seen. Despite these phenotypic changes, no significant differences between groups were noted for elotuzumab-induced ADCC against MM1R targets, as measured by CD107a degranulation by CD56dim NK cells, with significant variability noted within groups. Interestingly, the expression levels of SLAMF7 on CD56dim NK cells directly correlated with CD16 levels, particularly within RR pts (Fig.), suggesting cooperative interactions between these receptors that may be beneficial in MM patients treated with elotuzumab. In addition, degranulation toward elotuzumab-treated MM1R targets was significantly associated with surface expression levels of both SLAMF7 and CD16 on the CD56dim NK cells. The status of NK cells was also compared between matching blood and BM samples from ND (n=7) and RR (n=8) pts. NK cell phenotype and degranulation in blood and BM were similar in ND pts, but in RR pts, expression of CD69 and SLAMF7 were higher on BM-derived NK cells, and CD56dim NK cells from BM demonstrated greater degranulation toward elotuzumab-treated MM1R targets. DNAM1 expression was reduced, but NKG2D, NKp30, and NKp46 were upregulated on various NK cell populations in BM from RR pts compared to peripheral blood. Conclusions: Taken together, our data indicate that NK cells in RR MM pts had increased activation, reduced maturation status, and distinct changes in activating receptor expression levels that are often further enhanced in the BM microenvironment. Furthermore, CD56dim NK cells in many RR pts had parallel increased expression levels of CD16 and SLAMF7, which correlated with enhanced degranulation toward elotuzumab-treated MM target cells. The fact that these changes are seen primarily in RR pts rather than untreated ND pts implies a significant impact of disease evolution and prior therapy on the NK cell compartment, and supports further exploration of these parameters as potential biomarkers of activity of elotuzumab and other therapeutic mAbs in myeloma. Figure 1. Figure 1. Disclosures Campbell: Bristol-Meyers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding. Cohen:Bristol-Meyers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees.