Background Therapies based on checkpoint inhibitors have emerged as promising strategies in the treatment of cancer patients. The selection of the most efficient combination of immunotherapies along with the accurate prediction of cancer prognosis are unmet endeavors. Thus, scientists and clinicians need deeper knowledge of the immune profile of each patient as well as a detailed characterization of the tumor microenvironment (TME) to develop targeted therapies. Multiplex immunofluorescence (mIF) has become an important tool in the immune profiling of the TME. Ideally, multiplex IF assays should allow for the study of multiple markers in the same tissue sample while preserving the spatial information of tissue morphology. Despite a fast-growing need of multiplex methods, the broad implementation of mIF still remains challenging due to several technical barriers. Assays require robust validation but also a large degree of flexibility to be used across multiple tissue types.

Materials and Methods Here we present COMET™, an innovative solution to easily develop mIF assays that work as a fully automated staining and imaging platform allowing for hyperplex staining with up to 40-markers in just a few hours, without human intervention. COMET™ performs sequential immunofluorescence assays, which consist of sequential cycles of staining, imaging, and elution of two markers per cycle. With the aim of phenotyping different immune and cancer cells and their functional states in epithelial tumors, we developed a core immuno-oncology panel that allows the simultaneous detection of ten biomarkers on the same tissue slide.

Results and Conclusions This core panel was initially developed and validated on formaldehyde-fixed paraffin-embedded tissue (FFPE) human tonsil as positive control tissue. In the next steps, we employed the same validated panel in different tissue contexts, such as breast, lung and head and neck carcinomas. Thanks to an assisted workflow of optimization and the fast staining cycles lasting approximately 30 minutes, the optimal staining conditions for each tumor type were rapidly identified in a few steps. Our data proves that immuno-oncology panels developed on COMET™ can be easily transferred from one tissue type to another, enabling researchers to identify core biomarkers across different tumors for the development of novel targeted immunotherapies.

Disclosure Information P. Pulimeno: None. J. Mignot: None. D. Guerrera: None. F. Rivest: None. M. Cassano: None. D. Dupouy: None.