Semi-automated validation and quantification of CTLA-4 in 90 different tumor entities using multiple antibodies and artificial intelligence

David Dum,Frank Jacobsen,Tim Mandelkow,Katharina Möller ,Maximilian Lennartz,Stefan Steurer,T Henke ,Franziska Büscheck ,Sarah Minner,Anne Menz,Patrick Lebok,Andrea Hinsch,Christoph Fraune,Doris Höflmayer ,Ronald Simon,Sören Weidemann ,Waldemar Wilczak,Ria Uhlig,Cheng Yang,Elena Bady,Jonas B Raedler ,Till S Clauditz,Christian Bernreuther,Guido Sauter,Andreas M Luebke,Eike Burandt,Niclas C Blessin

doi:10.1038/s41374-022-00728-4

Abstract

AbstractCTLA-4 is an inhibitory immune checkpoint receptor and a negative regulator of anti-tumor T-cell function. This study is aimed for a comparative analysis of CTLA-4+ cells between different tumor entities. To quantify CTLA-4+ cells, 4582 tumor samples from 90 different tumor entities as well as 608 samples of 76 different normal tissue types were analyzed by immunohistochemistry in a tissue microarray format. Two different antibody clones (MSVA-152R and CAL49) were validated and quantified using a deep learning framework for automated exclusion of unspecific immunostaining. Comparing both CTLA-4 antibodies revealed a clone dependent unspecific staining pattern in adrenal cortical adenoma (63%) for MSVA-152R and in pheochromocytoma (67%) as well as hepatocellular carcinoma (36%) for CAL49. After automated exclusion of non-specific staining reaction (3.6%), a strong correlation was observed for the densities of CTLA-4+ lymphocytes obtained by both antibodies (r = 0.87; p < 0.0001). A high CTLA-4+ cell density was linked to low pT category (p < 0.0001), absent lymph node metastases (p = 0.0354), and PD-L1 expression in tumor cells or inflammatory cells (p < 0.0001 each). A high CTLA-4/CD3-ratio was linked to absent lymph node metastases (p = 0.0295) and to PD-L1 positivity on immune cells (p = 0.0026). Marked differences exist in the number of CTLA-4+ lymphocytes between tumors. Analyzing two independent antibodies by a deep learning framework can facilitate automated quantification of immunohistochemically analyzed target proteins such as CTLA-4.

Highlights

CTLA-4 is an important inhibitory immune checkpoint receptor
CTLA-4 in normal tissues Using both antibodies, a strong and distinct, predominantly membranous CTLA-4 immunostaining was seen in a subset of T-lymphocytes
For MSVA-152R, an intense granular cytoplasmic staining could be seen in adrenocortical cells and decidua cells while a less

Summary

Introduction

CTLA-4 (cytotoxic T-lymphocyte-associated protein 4, CD152) is an important inhibitory immune checkpoint receptor. It is expressed on various subtypes of T-lymphocytes including CD4+ and CD8+ T-cells as well as regulatory T-cells[1]. CTLA-4 can compete with its stimulating counterpart CD28 for ligand binding to CD80 and CD862,3. CD28 co-stimulation is required for T-cell activation, whereas CTLA-4 inhibits T-cell response by opposing the actions of CD28-mediated co-stimulation[2,3]. Even though CTLA-4 is expressed on activated CD8+ cytotoxic T-cells, the major physiologic role of CTLA-4 appears to be through downmodulation of non-regulatory T-cell activity and supportively enhancement of regulatory T-cell suppressive activity[1,4,5,6].

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