Abstract 1189: Revisiting immune exhaustion in Hodgkin’s lymphoma

Clinical successes with immune check-point blockers have demonstrated the potency of the immune system in controlling cancers, most strikingly in Hodgkin lymphoma (HL), where overall response rates to PD1/L1 inhibitors approach 90%. Complete or durable responses, however, are uncommon, therefore targeting the PD1/L1 axis alone is not sufficient. Recent work analyzing the spatial arrangement of PD1 and PDL1 expressing cells has given us new insight into the mechanism of action of PD1/L1 inhibitors, however this work limited itself to studying a single check point marker on a subset of cells. We hypothesize that comprehensive profiling of the frequency and spatial arrangement of immune cells in the Hodgkin lymphoma tumor immune microenvironment (TME) will provide new insights into the mechanism of checkpoint blockers and identify novel targets for immune therapy. Until now, multiparameter spatial analysis of the immune microenvironment was limited by technical challenges. Flow and mass cytometry are able to identify immune subsets of interest but spatial information is lost. Multiplex tissue imaging methods are limited to 6-8 simultaneous markers and cannot capture the full complexity of the immune phenotypes. The Fluidigm Hyperion imaging mass cytometry (IMC) system combines a CyTOF mass cytometer with a laser ablation system allowing for 40+ parameter simultaneous immunophenotyping on a single slide of FFPE tissue, with sub-cellular resolution. We have developed a panel of 34 antibodies that allow for comprehensive characterization of CD4, CD8 and myeloid cells components in the TME of Hodgkin lymphoma using IMC. Here we report on our spatial analysis of TIM3 and LAG3 expressing CD4+ lymphocytes. Our data suggests LAG3+CD4+ and TIM3+CD4+ lymphocytes had shorter mean nearest distance to PDL1+Hodgkin Reed-Sternberg (HRS) cells upon comparison to PDL1- HRS cells (t-test, p=1.703e-08,p=1.126e-14). Future studies should explore multiple exhausted marker models that seeks to further understand the presence of simultaneous exhaustion signals in the niche environment. These data suggest that therapies that target TIM3 and/or LAG3 should be tested in Hodgkin Lymphoma and that spatial analysis of immune subsets by IMC should be explored as selective and pharmacodynamic biomarkers. Citation Format: Anthony R. Colombo, Monirath Hav, Erik Gerdtsson, Jose Bisnesto-Villasboas, Stephen Ansell, James Hicks, Peter Kuhn, Akil Merchant. Revisiting immune exhaustion in Hodgkin’s lymphoma [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 1189.

Comprehensive Cell Specific Transcriptome Profiling of a Pediatric Hodgkin Lymphoma Cohort

Genome-Scale High-Resolution Spatial Mapping of the Pro-Tumorigenic Cellular Niche in Classic Hodgkin Lymphoma

Quantitative Assessment of PD-L1 Expression in Classical Hodgkin Lymphoma Suggests a Critical Role for Tumor Associated Macrophages in Suppressing Anti-Tumor Immunity

Expression of Major Histocompatibility Complex (MHC) Class II, but Not MHC Class I, Predicts Outcome in Patients with Classical Hodgkin Lymphoma (cHL) Treated with Nivolumab (Programmed Death-1 [PD-1] Blockade)

Background: In classical Hodgkin lymphoma (cHL), the rare malignant Hodgkin Reed-Sternberg (HRS) cells are surrounded by an inflammatory infiltrate. Yet, the host anti-tumor immune response is ineffective. HRS cells have multifaceted mechanisms to evade the immune system including 9p24.1/PD-L1/PD-L2 genetic alterations leading to overexpression of PD-1 ligands and subsequent T cell exhaustion, aberrant antigen presentation and modulation of the tumor microenvironment (TME). The clinical success of PD-1 blockade in cHL suggests the TME contains reversibly exhausted T-effectors (Teff). Paradoxically, durable responses are observed in patients with β2M/MHC class I loss on HRS cells, raising the possibility of non-CD8+ mediated mechanisms of efficacy of PD-1 blockade. For this reason, we sought to characterize HRS cells and the surrounding TME. Methods: Using CyTOF technology, we evaluated 7 primary cHL suspensions and 10 reactive lymphoid tissue (RLT) samples at the single-cell protein level. We designed a custom panel of 39 isotope-conjugated antibodies. A combination of surface and intracellular markers distinguish T cell subsets according to lineage, differentiation, polarization, activation and exhaustion. Additional markers were incorporated to identify B cells, NK cells and macrophages. HRS cells were defined by CD15/CD30/Pax5 positivity. Inclusion of β2M and MHC class I allowed assessment of antigen presentation on HRS cells. The data was acquired on a Helios CyTOF and analyzed using a fast k-weighted nearest neighbor algorithm, X-shift. X-shift clustered cells with phenotypic similarities together. Then, samples were separated into cHL and RLT and the contribution of a sample to a given cluster was quantified. Results: Comparison of viable cell suspensions from RLT and cHL revealed loss of naïve T-cells and skewing towards differentiation of Teff in both CD4+ and CD8+ subsets in cHLs. This prompted a second X-shift analysis focused on CD3+ cells, which highlighted salient differences between cHL and RLT within the CD4+ subset. In cHL, we found expansion of Teff and regulatory T cells (Treg) with a reduction of follicular helper T cells. Furthermore, both Treg and Teff populations were largely Th1 (T-bet+/CCR5+) polarized. Evaluation of PD-1 expression showed Tregs had little/no PD-1 while Teff had intermediate/high expression. Hence, Tregs retain functionality in contrast to Teff, which are exhausted, providing two mechanisms of immunosuppression. Manual gating identified HRS cells with a characteristic phenotype: CD15, CD30, Pax5, rosetted by CD4+ T cells. Importantly, we found loss or decrease of β2M and MHC class I in 5/7 cases. Conclusions: The TME in cHL is CD4+ T cell rich with frequent loss of MHC class I on HRS cells. Differential PD-1 expression results in functional CD4+ Tregs and exhausted Teff, a synergistic bases for the observed immunosuppression in cHL. Citation Format: Fathima Z. Cader, Ron C. Schackmann, Xihao Hu, Kirsty Wienand, Robert A. Redd, Bjoern Chapuy, Jing Ouyang, Nicole E. Paul, Evisa Gjini, Mikel Lipschitz, Laura M. Selfors, Philippe Armand, David Wu, Jonathan R. Fromm, Donna Neuberg, Xiaole S. Liu, Scott J. Rodig, Margaret A. Shipp. Single-cell mass cytometry of classical Hodgkin lymphoma defines an exhausted and immunosuppressive microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5675.

A Subset of Classical Hodgkin Lymphoma Cases Demonstrate CD19 Expression on Hodgkin Reed-Sternberg Cells By Immunohistochemistry

Gene Expression Profiling of Microdissected Hodgkin Reed Sternberg Cells: Molecular Subtypes and Treatment Outcome Correlations.

Mutations in CD58 and Other Immune System Related Genes in Hodgkin Lymphoma

Comprehensive Genomic Analysis of Flow-Sorted Hodgkin Reed Sternberg Cells Reveals Additional Genetic Bases of Immune Evasion

Disordered differentiation and cellular senescence in pediatric Hodgkin Reed-Sternberg cells.

CSF1R Expression of Hodgkin Reed Sternberg Cells Is Associated with the Number of Macrophages in the Tumor Microenvironment and Is Correlated with Treatment Outcome

A Critical Role for Intratumoral and Circulating LAG3 in Classical Hodgkin Lymphoma: Analysis from the Rathl Prospective Phase III International Clinical Trial

Approximately 20% of Hodgkin Lymphoma (HL) patients do not achieve a durable remission or fail to respond to front-line chemotherapy. Despite the attempt to improve clinical outcomes by using the risk adaptive therapy, a significant number of patients die as a results of relapsed/refractory (rel/ref) disease.1 Advances in understanding the etiology and molecular biology of HL are leading the development of novel therapeutic strategies that could be applied to improve clinical outcome of rel/ref HL patients. The pathologic features of HL reflect a defect in immune responses resulting from various cytokines and chemokines secreted partially by Hodgkin Reed-Sternberg (HRS) cells. HRS cells are unique in the way that they lost typical B cell gene expression pattern but retain the expression of surface molecules involving in antigen presentation (tumor necrosis factor receptor (CD30, CD40), CD80, MHC class II, and CD86). Aberration of Notch signaling pathway may contribute to their reprogramming.2,3 Multiple genetic lesions, deregulated signaling pathway and transcription factors play important role in pathogenesis of HL including constitutive activation of nuclear factor kappa B (NFκB) and the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway.4-6 Moreover, the role of the microenvironment in HL has been increasingly recognized. The majority of the cell population in HL-affected tissue is composition of the inflammatory cellular infiltrate, not the HRS cells that represents only small population (<1%). Understanding the complex relationship between the HRS cells and the microenvironment and chemokines milieu involved in its formation is crucial for the development of new therapeutic strategies.

Contribution of Deregulated miRNAs to the Phenotypic Characteristic of Hodgkin Lymphoma.