Abstract 499: NanoString®GeoMx®digital spatial profiling further defines the role of B cells in the response to immune checkpoint blockade
Authors: Helmink Beth A. , Reddy Sangeetha , Blando Jorge , Liang Yan , Warren Sarah , Gopalakrishnan Vancheswaran , Tawbi Hussein A. , Amaria Rodabe N. , Davies Michael , Gershenwald Jeffrey E. , Burton Elizabeth , Basar Rafet , Lazar Alexander J. , Hudgens Courtney W. , Rezvani Katy , Allison James P. , Sharma Padmanee , Beechem Joseph M. , Wargo Jennifer A. , Tetzlaff Michael T. 
Published: August 2019

Abstract
Background: While treatment with immune checkpoint blockade (ICB) has markedly improved outcomes in advanced melanoma patients and other malignancies, predicting response remains a challenge. Biomarkers including tumor mutational burden (TMB), T-cell infiltration, and PD-L1 expression, have been identifiedbut remain inadequate. Other components of innate and adaptive immunity, including B-cells and tertiary lymphoid structures (TLS), have been implicated in the response to other cancer therapies, andpreclinical data suggests B cells may contribute in the response to immunotherapy. Here, we use targeted protein expression profiling-via NanoString digital spatial profiling (DSP) technology (research use only)-to demonstrate a role for B cells in the response to ICB in patients with high-risk resectable melanoma; furthermore, we characterize the B cell subsets that enable this function.
Methods: We conducted a phase 2 clinical trial of neoadjuvant ICB therapy in patients with high-risk resectable melanoma (PD-1 blockade monotherapy or combined CTLA-4/PD-1 blockade) (NCT02519322). Longitudinal tumor samples were taken during therapy. Formalin-fixed paraffin embedded tissue sections from tumor samples (n=10 responders [R], n=10 non-responders [NR]) were analyzed by NanoString DSP technology and stained with a cocktail of S100B, CD45, CD19 and a 40-protein cocktail of antibodies conjugated to UV-photocleavable DNA barcodes. Regions of interest (ROI) were delineated using immunofluorescence followed by UV excitation of the defined ROIs, releasing the DNA barcodes for downstream quantitation on the NanoString nCounter®platform. Utilizing masking strategies, we define the unique expression pattern within discrete subsets of immune cells. These same tumors have concurrently been analyzed by RNAseq, immunohistochemistry, CYTOF, and single-cell RNAseq.
Results: Biomarker counts are highly concordant across samples from the same patient’s tumor. High concordance between DSP and quantitative fluorescence is seen as a validation for the DSP method. We identify B cells as components of TLS; the B cells are closely integrated with CD4 and CD8 T cells and follicular dendritic cells. There are significantly more TLS identified in R compared to NR patients. Utilizing CYTOF, we concurrently identify specific subsets of B cells present within the TLS associated with response. We characterize the spatial relationship of these same B cell subsets with other components of the TLS and define the protein expression patterns of these cells.
Conclusion: NanoString DSP data complement our deep molecular and immune profiling of tumors from melanoma patients treated with ICB; together, they provide a novel predictive role for B-cells and TLS in the response to ICB and, importantly, provide mechanistic insight into their potential contribution in the response to cancer therapy.
Citation Format: Beth A. Helmink, Sangeetha Reddy, Jorge Blando, Yan Liang, Sarah Warren, Vancheswaran Gopalakrishnan, Hussein A. Tawbi, Rodabe N. Amaria, Michael Davies, Jeffrey E. Gershenwald, Elizabeth Burton, Rafet Basar, Alexander J. Lazar, Courtney W. Hudgens, Katy Rezvani, James P. Allison, Padmanee Sharma, Joseph M. Beechem, Jennifer A. Wargo, Michael T. Tetzlaff. NanoString®GeoMx®digital spatial profiling further defines the role of B cells in the response to immune checkpoint blockade [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 499.