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Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma
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Multiplex immunofluorescence staining and image analysis assay for diffuse large B cell lymphoma.

Chung-Wein Lee1, Yan J Ren1, Mathieu Marella1

  • 1Department of Non-clinical Drug Safety, Celgene Corporation, 10300 Campus Point Dr., San Diego, CA 92121, USA.

Journal of Immunological Methods
|November 30, 2019
PubMed
Summary

Multiplex immunofluorescence (mIF) assays can now analyze immune cells and checkpoint proteins in diffuse large B-cell lymphoma (DLBCL) tissue. This method reveals immune cell spatial patterns crucial for understanding immuno-oncology therapies.

Keywords:
Digital pathologyImage analysisImmunophenotypingLymphomaMultiplex immunofluorescenceMultispectral imaging

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Area of Science:

  • Oncology
  • Immunology
  • Biotechnology

Background:

  • Immuno-oncology and immune checkpoint therapies are rapidly advancing.
  • Understanding the tumor microenvironment (TME) and patient immune status is critical.
  • Multiplex immunofluorescence (mIF) offers spatial insights into tumor-immune cell interactions.

Purpose of the Study:

  • To develop and optimize a reliable mIF workflow for diffuse large B-cell lymphoma (DLBCL).
  • To characterize infiltrating immune cells and immune checkpoint proteins within the DLBCL TME.
  • To address challenges in analyzing DLBCL tissue, such as high tumor density and indistinct boundaries.

Main Methods:

  • Utilized Akoya Opal staining kits to label 6 markers per slide.
  • Developed five panels totaling 30 markers, including PD1, PD-L1, ICOS, SIRP-alpha, and Lag3.
  • Applied an image analysis workflow using InForm and Matlab for DLBCL sections.
  • Scanned multiplexed sections with an Akoya multispectral scanner.

Main Results:

  • Quantified densities of infiltrating immune cell subsets in DLBCL tissues.
  • Observed spatial patterns of immune cells within the tumor microenvironment.
  • Highlighted heterogeneous distribution of cytotoxic T cells, even in tumors with similar T cell densities.

Conclusions:

  • The developed mIF workflow is reliable for analyzing immune cells and checkpoint proteins in DLBCL.
  • Spatial context is essential for understanding the efficacy of immunotherapies in DLBCL.
  • This approach provides critical insights into the DLBCL tumor microenvironment for therapeutic development.