Background Immuno-oncology and targeted molecular therapies have acquired a central role in the treatment of multiple cancers. Consequently, high-throughput biomarker analysis and tumor immune profiling have seen an increased demand. Multiplexed immuno-assays are a powerful tool to address these needs, but still time- and resource-consuming. Our goal is to develop a fast and automated high-plex fluorescent immunostaining procedure, using a microfluidic-based device, that can be easily implemented as routine assay.

Materials and Methods Protocol optimization has been performed on FFPE sections of human tonsil. Slides were manually deparaffinized before being entirely processed (antigen-retrieval, staining, elution and counterstaining) by Lunaphore’s autostainer, LabSat^TM. The OPAL® tyramide signal amplification (TSA) system was used as detection method. Signal analysis was done on Mantra® workstation. The 6-plex panel was composed of FoxP3, PD-L1, PD-1, CD68, CD8 and pan-CK, plus DAPI counterstaining. Protocols were subsequently transferred on NSCLC representative specimens and finally assessed on a TMA cohort.

Results Our platform allowed to reduce drastically the incubation times due to active transport of reagents across the tissue. Thereby, the automated 6-plex assay could be performed in less than 4h30min, within the timeframe of a single IHC standard assay. Protocol optimization resulted in high signal-to-background ratio for each marker and removal of previous step antibodies over 99%. LabSat^TM also guaranteed remarkable signal uniformity, even over large tissue sections with less than 10% signal gradient over 1 cm. On NSCLC samples, the detected pattern and expression level for all six biomarkers were comparable to the standard chromogenic stainings performed with standard automated tissue stainer.

Conclusions LabSat^TM autostainer enables multistaining runs in a timely manner, opening the perspective of rapid simultaneous detection of multiple markers in their morphological context on a routine-based approach. This versatile analysis tool can offer a better and more quantitative understanding of tumor heterogeneity and microenvironmental interactions, allowing advances in targeted therapy for lung cancer as well as broader spectrum of malignancies.

Disclosure Information A. Kehren: A. Employment (full or part-time); Significant; Lunaphore Technologies SA. C. Other Research Support (supplies, equipment, receipt of drugs or other in-kind support); Significant; Akoya Biosciences. M.G. Procopio: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; KTI grant (Schweiz). B. Pelz: A. Employment (full or part-time); Significant; Lunaphore Technologies SA. C. Other Research Support (supplies, equipment, receipt of drugs or other in-kind support); Significant; Akoya Biosciences. Z. Siddiqui: A. Employment (full or part-time); Modest; Lunaphore Technologies SA. K. Roman: A. Employment (full or part-time); Significant; Akoya Biosciences. S. Adnane: A. Employment (full or part-time); Modest; Lunaphore Technologies SA. S. Brajkovic: A. Employment (full or part-time); Significant; Lunaphore Technologies SA. C. Hoyt: A. Employment (full or part-time); Significant; Akoya Biosciences. D.G. Dupouy: A. Employment (full or part-time); Significant; Lunaphore Technologies SA. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Lunaphore Technologies SA. A. Soltermann: A. Employment (full or part-time); Significant; Institut für Klinische Pathologie Universitätsspital Zürich.