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51 Tissue-based spatial analysis of DLBCL tumor microenvironment using multiplex immunofluorescence AQUA® (Automated Quantitative Analyses) algorithms
  1. Jaspreet Kaur1,
  2. Lisa Adams1,
  3. Jimmie Lim2,
  4. Sophia Kekchidou1,
  5. Xun Li1,
  6. Sarah Choi2 and
  7. Jennifer Bordeaux1
  1. 1Navigate BioPharma Services, Inc., A Novartis Subsidiary, Carlsbad, CA, USA
  2. 2Novartis Institutes for BioMedical Research, Cambridge, MA, USA


Background Spatial architecture within the tumor microenvironment (TME) can potentially impact disease pathology, progression and treatment response of DLBCL. Additionally, activation of host’s immune system greatly determines response to immunotherapy treatments. Multiplex imaging using fluorescence immunohistochemistry (FIHC) has found to enable characterization and quantification of tissue-specific proteins involved in immune activation, which in turn have enabled identification of distinct cell types, their functional status, and their organization in TME. We developed and validated two complementary quantitative multiplex fluorescence IHC (mFIHC) panels using hypothesis-driven spatial profiling algorithms (Automated Quantitative Analysis known as AQUA® Technology) to evaluate T and B cell populations and their functional status to generate detailed spatial information of tumor microenvironment of DLBCL. mFIHC assays when combined with the use of AQUA® Technology were able to predict immunotherapies in a systematic meta-analysis of over 8000 patients treated with PD1/L1 pathway blockers.1 Implementation of mFIHC assays coupled with robust image analysis may assist in risk stratification of patients or enable efficacy assessment of immunomodulating cancer therapies.

Methods We designed two novel mFIHC assays to (1) characterize various T (CD2, CD3, CD8, CD28) and B cell populations (CD2, CD3, CD19, PAX5), and (2) evaluate T cell functional status (CD8, Granzyme B, PD1). We successfully validated clinical grade mFIHC assays using automated staining (Leica Bond RX), imaging (PhenoImager HT) and analysis (AQUA® Technology) workflow.

Results Sensitivity, accuracy and specificity were confirmed for all mFIHC assays on known positive and negative controls. Excellent reproducibility (less than 35% CV) and precision were observed across instruments, operators and independent experiments for all markers. Between these two panels, over 200 unique parameters were evaluated. The prevalence of CD2, CD3, CD19, and PAX5 ranged from 20% to >90% positive for the DLBCL samples tested and were overall highly concordant with each other.

Conclusions The validated mFIHC assays examined B and T cell antigen expression in DLBCL and their interaction with PAX5+ and CD58+ cells within the TME. Implementation of mFIHC assays coupled with robust image analysis may assist in risk stratification of patients or enable efficacy assessment of immunomodulating cancer therapies. These assays may be used to further understand the complex immune cell and tumor cell spatial biology in the context of clinical trials.


  1. Lu, Steve, Julie E Stein, David L Rimm, Daphne W Wang, J Michael Bell, Douglas B Johnson, Jeffrey A Sosman, et al. ‘Comparison of biomarker modalities for predicting response to PD-1/PD-L1 checkpoint blockade: a systematic review and meta-analysis.’ JAMA oncology 5. 2019;8:1195–1204.

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