Abstract
Introduction
Immunohistochemistry provides biologically and clinically useful information on protein expression and cellular localization in cancer cells, but quantification of the expression by visual scores on light microscopy is subjective and suffers from poor reproducibility. There is increasing need for multiplex immunohistochemistry to study the co-localization of antigens in cell of interest. However, interpretation and quantification of co-localizing signals using conventional IHC is challenging. Novel methods in automated microscopy using machine learning algorithms have been utilized in epithelial cancers to quantitate the signals from immunohistochemistry. However, these have not been well tested in lymphoid malignancies, where IHC plays a critical role in the diagnosis and classification. Here we describe the application of quantitative analysis of immunofluorescence in lymphomas, using an example of a T- cell lymphoma, which can be challenging in terms of diagnosis and treatment.
Methods
We developed a multiplexed panel of immunofluorescent stains including CD4, CD8 and CD20 (T and B-lymphocyte markers) along with T-follicular helper markers (TFH) such as BCL6 and PD1. These were optimized and validated using benign tonsillar tissue to demonstrate appropriate cellular localization of each of these markers. (Figure1.) We then show the applicability of such a multiplexed panel in Angioimmunoblastic T-cell Lymphoma (AITL), a peripheral T-cell lymphoma characterized by cellular heterogeneity in the tumor microenvironment which often impedes IHC interpretation and quantification (figure not shown)
Results and Discussion
In AITL, we generated a tissue map which allows visualization of the spatial relationship of the different lymphocyte subsets and the quantification of TFH markers in tumor and non-tumor cell populations. We demonstrate the feasibility of an automated and quantitative imaging of this multiplexed panel of stains in formalin-fixed paraffin embedded tissue sections, which allows the study of the distribution of tumor cells in relationship to surrounding immune cells and the quantification of the expression of different biomarkers of interest in tumor compared to non-tumor cells in the microenvironment. Quantitative multiplex immunofluorescence and/or immunohistochemistry is likely to improve diagnostic accuracy and reproducibility compared to visual scoring in conventional single marker IHC. Furthermore, the ability to quantify the expression of multiple immune markers in tumor and surrounding immune cells and correlation with treatment response will play a vital role in the selection of patients for immunotherapy ( such as anti PD1 or anti CD30 antibodies) which is becoming an increasingly important modality in lymphoma treatment.
Figure legend. ( Figure 1)
Multiplexed composite immunofluorescence of CD20, CD8, CD4 and BCL6 in benign tonsil tissue (A). Unmixed images of CD20 (B, green, cell membrane), CD8 (C, magenta, cell membrane), CD4 (D, yellow, cell membrane), BCL6 (E, red, nuclear) and CD4/BCL6 (F) demonstrating the normal distribution of B and T cells and localization of BCL6-positive cells within germinal centres. The number of CD4-positive (G, red), BCL6-positive (H, green) and CD4/BCL6 double positive cells (I, yellow) are obtained after the images are segmented and scored using the InForm 2.0 software.
Chng: Janssen China R&D: Research Funding.
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