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

CTLA-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.