Background 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.

Materials and 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 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). 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).

Conclusions 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.

Disclosure Information D. Dum: None. T.L.C. Henke: None. T. Mandelkow: None. E. Bady: None. R. Simon: None. G. Sauter: None. S. Steuerer: None. W. Wilczak: None. E. Burandt: None. J. Raedler: None. M. Lennartz: None. N.C. Blessin: None.