Abstract P069: Semi-automated validation and quantification of CTLA-4 in 90 different Tumor entities using multiple antibodies and artificial intelligence

Abstract

Abstract Introduction: CTLA-4 is an inhibitory immune checkpoint receptor and a negative regulator of anti-tumor T-cell function. This study aimed at a comparative analysis of CTLA-4+ cells between different tumor entities. Methods: To quantify CTLA-4+ cells, 4,582 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. Results: Comparing both CTLA-4 antibodies revealed a clone dependent cytoplasmic cross-reactivity 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). The mean density of CTLA-4+ cells was 674±1482 cells/mm2 and ranged from 71±175 cells/mm2 in leiomyoma to 5916±3826 cells/mm2 in Hodgkin's lymphoma. Within epithelial tumors, the density of CTLA-4+ lymphocytes were higher in squamous cell (421±467 cells/mm2) and urothelial carcinomas (419±347 cells/mm2) than in adenocarcinomas (269±375 cells/mm2) and renal cell neoplasms (256±269 cells/mm2). 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). Conclusion: Marked differences exist in the number of CTLA-4+ lymphocytes between tumors. Analyzing two independent antibodies by a deep learning framework identifies clone-specific cross-reactivity and facilitates automated quantification of target proteins such as CTLA-4. Citation Format: David Dum, Tjark L. C. Henke, Tim Mandelkow, Elena Bady, Jonas B. Raedler, Ronald Simon, Guido Sauter, Maximilian Lennartz, Waldemar Wilczak, Eike Burandt, Stefan Steurer, Niclas C. Blessin. Semi-automated validation and quantification of CTLA-4 in 90 different Tumor entities using multiple antibodies and artificial intelligence [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P069.