Abstract 4116: Immune cell checkpoint profiling of solid tumors by multiplex immunofluorescence

• Published in: Cancer research (Chicago, Ill.) Ill.), 2019-07, Vol.79 (13_Supplement), p.4116-4116
• Main Authors: Finan, Amanda, Goulange, Nicolas, Motte, Manon, Tliba, Maroua, Mace, Alexandra, Coton, Jean-Philippe, Lazzaro, Domenico, Burrer, Renaud
• Format: Article
• Language: English
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/1538-7445.AM2019-4116

Immune checkpoint proteins are important regulators in self-tolerance. However, these same molecules also allow cancer cells to evade immune destruction. Checkpoint inhibitor (CKI) blockade therapies that aim at restoring antitumoral immunity have resulted in long-lasting remission in patients with cancers that were previously seen as incurable. However, only a fraction of the patients respond to these treatments. Research is currently focusing on identifying biomarkers that can predict a response to CKI therapies and stratify patient populations. As it is likely that several CKI need to be targeted at once to restore immune responses, numerous clinical trials associating more than one CKI are under evaluation. We have developed a multiplex immunohistochemistry panel, validated by in-depth image analysis, for immune cell checkpoint profiling. The base of the panel consists of CD3/CD8/PD-1/PD-L1 with the capability of adding additional targets including other immune checkpoint (e.g. TIGIT, SIRPα, CTLA-4) or tumor markers. Here we present our validation assay which uses the base panel and the ability to interchange the additional targets. Assay conditions for the individual biomarkers, including antigen retrieval, antibody concentration, panel position, and efficiency of stripping, are optimized. Simplex protocols are analyzed to ensure accuracy and specificity of the staining, success of stripping, and a balance of signal intensities. Validation includes the comparison of staining (% positive cells or % positive area) between a reference protocol and the position of interest. Multiplex optimization consists of staining serial, single stained slides of the individual biomarkers and the multiplex slide in parallel. The staining concordance (% positive cells) is evaluated with imaging software. Repeatability and robustness of the panels are validated by image analysis. This technique gives clinicians the potential to determine candidate patients for CKI single or double therapies and their progress during treatment. The established, rigorous workflow that we have developed allows our customers to adapt the panels to their needs and to have the highest confidence in the consequent images and results related to the profiling of immune cell checkpoints in solid tumors. Citation Format: Amanda Finan, Nicolas Goulange, Manon Motte, Maroua Tliba, Alexandra Mace, Jean-Philippe Coton, Domenico Lazzaro, Renaud Burrer. Immune cell checkpoint profiling of solid tumors by