Abstract 2376: Comprehensive multiplexed protein analysis of biomarkers of hypoxia and the immune microenvironment of diffuse large B-cell lymphoma with clinical outcome analysis
Authors: Makarenko Vladislav V. , Dresser Karen , Chen Benjamin J. 
Published: August 2019

Abstract
Background: The host immune response has been shown to play an important role in the pathogenesis and clinical outcome of diffuse large B-cell lymphoma (DLBCL). Immune checkpoint molecules, such as PDL1, TIM3, and LAG3, can modulate the immune microenvironment by downregulating T-cell activity. Hypoxia inducible factor (HIF) is a transcription factor that regulates genes involved in responses to hypoxia and can induce PDL1 expression. PDL1, TIM3, and HIF1α expression in DLBCL tumor cells have been associated with inferior survival. We performed a comprehensive multiplexed analysis of hypoxia and immune-related proteins to better understand the DLBCL microenvironment.
Design: Immunohistochemical (IHC) analysis was performed on 49 DLBCL cases and correlated with clinicopathologic data. A microarray with an additional 36 DLBCL cases was evaluated by IHC and by using the NanoString nCounter Digital Spatial Profiling (DSP) platform with a 30-protein immuno-oncology panel. This study was approved by the UMass IRB.
Results: Subsets of tumors were positive by IHC in tumor cells and/or infiltrating non-tumor cells for PDL1 (12%), PD1 (39%), TIM3 (73%), LAG3 (18%), HIF1α (31%) and HIF2α (47%). A positive correlation was found between IHC expression of tumor-associated PDL1, PD1, and TIM3 (r&gt;0.6, p&lt;0.001) but not LAG3 (r=0.27, p&gt;0.05). HIF1α, but not HIF2α, expression positively correlated with PDL1 IHC expression (p=0.011). Pairwise comparison between proteins using data from the DSP analysis showed positive correlations between PDL1/LAG3 (r=0.71, p&lt;0.001), PDL1/B7-H3 (r=0.78, p&lt;0.001), PDL1/TIM3 (r=0.46, p&lt;0.01), and VISTA/LAG3 (r=0.46, p&lt;0.01). Levels of pSTAT, AKT, CD44, β-catenin, β-2-microglobulin, and PTEN were also found to correlate with PDL1 expression. HIF1α IHC expression correlated with PDL1, LAG3, B7-H3 levels, but not with PD1, VISTA and TIM3. Patients with tumors expressing HIF1α, but not HIF2α, demonstrated inferior OS upon long term follow up (p=0.045 and 0.059, respectively).
Conclusions: A comprehensive protein-level analysis of the immune microenvironment of DLBCL reveals coordinated expression of several therapeutically-relevant immunomodulatory molecules. HIF1α expression strongly correlated with PDL1 and other immune checkpoint molecules and was associated with inferior patient outcome. Further studies are required to elucidate causative links between hypoxia and the immune environment of lymphoma, as well as potential clinical and therapeutic implications of these findings.
Citation Format: Vladislav V. Makarenko, Karen Dresser, Benjamin J. Chen. Comprehensive multiplexed protein analysis of biomarkers of hypoxia and the immune microenvironment of diffuse large B-cell lymphoma with clinical outcome analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2376.