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Objectives: This study aims to retrospectively assess C-lectin-like molecule 1 (CLL-1) bimodal expression on CD34+ blasts in acute myeloid leukemia (AML) patients (total N = 306) and explore potential CLL-1 bimodal associations with leukemia and patient-specific characteristics. Methods: Flow cytometry assays were performed to assess the deeper immunophenotyping of CLL-1 bimodality. Cytogenetic analysis was performed to characterize the gene mutation on CLL-1-negative subpopulation of CLL-1 bimodal AML samples. Results: The frequency of a bimodal pattern of CLL-1 expression of CD34+ blasts ranged from 8% to 65% in the different cohorts. Bimodal CLL-1 expression was most prevalent in patients with MDS-related AML (P = .011), ELN adverse risk (P = .002), NPM1 wild type (WT, P = .049), FLT3 WT (P = .035), and relatively low percentages of leukemia-associated immunophenotypes (P = .006). Additional immunophenotyping analysis revealed the CLL-1- subpopulation may consist of pre-B cells, immature myeloblasts, and hematopoietic stem cells. Furthermore, (pre)-leukemic mutations were detected in both CLL-1+ and CLL-1- subfractions of bimodal samples (N = 3). Conclusions: C-lectin-like molecule 1 bimodality occurs in about 25% of AML patients and the CLL-1- cell population still contains malignant cells, hence it may potentially limit the effectiveness of CLL-1-targeted therapies and warrant further investigation. Keywords: CD34+ blasts; CLL-1; acute myeloid leukemia; bimodality; bone marrow aspirates; flow cytometry.

Introduction:Programmed death-ligand 1 (PD-L1) contributes to tumor escape from immune surveillance by binding to programmed death-1 (PD-1), a negative regulator of T-cell responses. PD-L1 is expressed by both tumor cells and immune cells in the tumor microenvironment. In contrast, the role of PD-L2 in tumor immunity is unclear. To better understand the contribution of PD-L1/L2 to immune escape in marrow-based hematologic malignancies, we used multi-color flow cytometry and immunohistochemistry (IHC) to characterize cell-specific PD-L1/PD-L2 expression in patients with multiple myeloma (MM), myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). Results:PD-L1 was detectable (> 2% positive cells) in 100% of patients, with distinct disease-specific patterns. PD-L1 was expressed most widely in MM, predominantly by malignant plasma cells (median, 95% of plasma cells; n = 5) and lymphocytes (median, 12% of marrow lymphocytes; n = 5). In contrast, in MDS and AML, PD-L1 was more commonly expressed by non-tumor hematopoietic cells: MDS median, 36% of CD34− myeloid precursors and 47% of lymphocytes (n = 13) vs 12% of CD34+ myeloid blasts; AML median, 19% of CD34− myeloid precursors and 26% of lymphocytes (n = 7) vs 16% of CD34+ myeloid blasts were PD-L1+. A higher proportion of CD8+ T cells expressed PD-L1 compared with CD4+ T cells in all 3 malignancies: the CD8:CD4 ratio for PD-L1+ T cells was 3.02 in MM (n = 11), 1.92 in MDS (n = 10), and 1.29 in AML (n = 7). PD-L2 expression was largely absent in AML and MDS (< 2% of CD34+ blasts or lymphocytes expressed PD-L2 (n = 13 MDS and n = 7 AML) but was expressed in a subset of patients with MM on plasma cells (median, 18%; n = 5) but not lymphocytes (< 2%). Across all indications on both tumor cells and lymphocytes, PD-L1 was expressed by a larger fraction of cells than PD-L2 (AML: CD34+ blasts, P < .01 and lymphocytes, P = .005 [n = 7]; MDS: CD34+ blasts, P < .01 and lymphocytes, P = .0001 [n = 13]; MM: plasma cells, P = .03 and lymphocytes, P = .04 [n = 5]). These results were confirmed in a distinct set of 16 cases of primary AML analyzed independently, with detectable PD-L1 expression on myeloid blasts in 14 of 16 cases (88%) without co-expression of PD-L2 (0 of 16 cases). Comparisons of flow cytometry and IHC revealed that some IHC methods may underestimate the prevalence of PD-L1/PD-L2 in marrow-based hematologic malignancies, and results comparing different methods for detecting PD-L1/PD-L2 in the bone marrow will be presented. Conclusions:PD-L1 is highly prevalent in MM, MDS and AML, with significant expression by non-tumor hematopoietic cells, particularly CD8+ T cells. PD-L2 expression was largely absent in myeloid diseases but detectable in MM. Interestingly, PD-L1 expression was most common on tumor cells in MM and on non-tumor hematopoietic cells in MDS, whereas expression on non-tumor and tumor cells in AML was comparable. These data support clinical development of anti-PD-L1/PD-1 therapies in MM, MDS and AML. Future analyses will determine whether different patterns of PD-L1 expression are associated with clinical efficacy. Authors M Dail, L Yang, S Rodig, and J Venstrom contributed equally to this work. Disclosures Dail: Genentech, Inc.: Employment. Green:Genentech, Inc.: Employment. Ma:Genentech, Inc.: Employment. Robert:Genentech, Inc.: Employment. Kadel:Genentech, Inc.: Employment. Koeppen:Roche: Employment, Equity Ownership. Adamkewicz:Genentech, Inc.: Employment. Byon:Genentech, Inc.: Employment. Woodard:Genentech, Inc.: Employment. Rodig:Bristol-Myers Squibb: Honoraria, Research Funding; Perkin Elmer: Membership on an entity's Board of Directors or advisory committees. Venstrom:Genentech: Employment.

Introduction:TIGIT (T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif [ITIM] domain) is an inhibitory immunoreceptor expressed by T and natural killer (NK) cells that is an important regulator of anti-tumor and anti-viral immunity. TIGIT shares its high-affinity ligand PVR (CD155) with the activating receptor CD226 (DNAM-1). We have recently shown that TIGIT blockade, together with PD-L1/PD-1 blockade, provides robust efficacy in syngeneic tumor and chronic viral infection models. Importantly, CD226 blockade abrogates the benefit of TIGIT blockade, suggesting additional benefit of TIGIT blockade through elaboration of CD226-mediated anti-tumor immunity, analogous to CTLA-4/CD28 regulation of T-cell immunity. Whether TIGIT and CD226 are expressed in patients with multiple myeloma (MM) and how TIGIT expression relates to PD-L1/PD-1 expression is unknown. Here we evaluate expression of TIGIT, CD226, PD-1 and PD-L1 in patients with MM to inform novel immunotherapy combinations. Methods:We performed multi-color flow cytometry (n = 25 patients), and multiplex qRT-PCR (n = 7) on bone marrow specimens from patients with MM to assess expression of TIGIT, CD226, PD-1, and PD-L1 on tumor and immune cells. Cells were stained with fluorescently conjugated monoclonal antibodies to label T cells (CD3, CD4, CD8), NK cells (CD56, CD3), plasma cells (CD38, CD45, CD319, CD56), inhibitory/activating receptors (PD-1, TIGIT, PD-L1, CD226), and an amine-reactive viability dye (7-AAD). Stained and fixed cells were analyzed by flow cytometry using BD FACSCanto™ and BD LSRFortessa™. Results:TIGIT, CD226 and PD-L1/PD-1 were detectable by flow cytometry in all patients with MM who were tested, with some overlapping and distinct expression patterns. TIGIT was commonly expressed by marrow-infiltrating CD8+ T cells (median, 65% of cells), CD4+ T cells (median, 12%) and NK cells. In contrast, CD226 was more commonly expressed by marrow-infiltrating CD4+ T cells (median, 74%) compared with CD8+ T cells (median, 38%). PD-1 was expressed by marrow-infiltrating CD8+ T cells (median 38%) and CD4+ T cells (median, 16%). TIGIT was co-expressed with PD-1 on CD8+ T cells (67%-97% TIGIT+ among PD-1+), although many PD-1-negative CD8+ T cells also expressed TIGIT (39%-78% of PD-1-negative). PD-L1 was also expressed by CD8+ (median, 23%) and CD4+ (median, 8%) T cells in addition to MM plasma cells (median, 95%), albeit with significantly lower intensity on T cells compared with plasma cells. The expression of TIGIT and PD-L1 mRNA was highly correlated (R2 = 0.80). Analysis of PVR expression will also be presented. Conclusions: TIGIT, CD226, PD-1, and PD-L1 were commonly expressed in MM bone marrow, but with different patterns. Among CD8+ T cells, the frequency of TIGIT+ T cells was almost twice that of PD-1+ T cells, whereas the majority of CD4+ T cells expressed CD226. TIGIT blockade may complement anti-PD-L1/PD-1 immunotherapy by activating distinct T-cell/NK-cell subsets with synergistic clinical benefit. These results provide new insight into the immune microenvironment of MM and rationale for targeting both the PD-L1/PD-1 interaction and TIGIT in MM. Disclosures Yadav: Genentech, Inc.: Employment. Green:Genentech, Inc.: Employment. Ma:Genentech, Inc.: Employment. Robert:Genentech, Inc.: Employment. Glibicky:Makro Technologies Inc.: Employment; Genentech, Inc.: Consultancy. Nakamura:Genentech, Inc.: Employment. Sumiyoshi:Genentech, Inc.: Employment. Meng:Genentech, Inc.: Employment, Equity Ownership. Chu:Genentech Inc.: Employment. Wu:Genentech: Employment. Byon:Genentech, Inc.: Employment. Woodard:Genentech, Inc.: Employment. Adamkewicz:Genentech, Inc.: Employment. Grogan:Genentech, Inc.: Employment. Venstrom:Roche-Genentech: Employment.