Background: Emerging data suggests that predictive biomarkers based on the spatial arrangement of multiple cell types in FFPE tissue sections will be an important component of precision medicine in immune-oncology. Multiplexed immunofluorescence (mIF) facilitates such assessments. If mIF is to play a translational role in research and ultimately clinical practice, it is vital to refine, standardize, and validate an end-to-end workflow that supports large scale multi-site trials and clinical laboratory processes. To this end, six institutions collaborated to develop an automated 6-plex assay focused on the PD-1/PD-L1 axis and assessed its inter- and intra-site reproducibility. Specific attention was paid to assessment of %PD-L1 expression by immune cells (ICs), as pathologists have poor concordance for this parameter as noted in the Blueprint 2 and multi-institutional NCCN studies on PD-L1 IHC.

Methods: A 7-color mIF panel (PD-L1, PD-1, CD8, CD68, FoxP3, Cytokeratin, and DAPI) was optimized on a Leica Bond Rx autostainer. Serial sections of tonsil and a lung cancer tissue-microarray (TMA), antibodies and TSA-Opal detection reagents (Akoya Biosciences) were distributed. Cell pellet arrays were also distributed and used to normalize batch variation in intensity measurements. Tonsil and TMA sections were stained at each site and imaged at 20x using a Vectra Polaris. Cells were segmented and phenotyped using image analysis algorithms. In tonsil sections, the average intensity of the top quartile of cells positive for each marker was assessed to identify potential variation in staining intensity. In lung TMAs, cell densities and %PD-L1 expression in immune cells (CD68+ and CD8+ cells) was determined.

Results: The average staining intensity coefficients of variation (CV) for all markers within sites was 10% in tonsil samples. Inter-site concordance for tumor cell and immune cell subset densities in TMAs, had an average R2 value of 0.86 and slope of 0.96. Inter-site concordance for %PD-L1+ ICs had an average R2 value of 0.81, and slope of 0.82, in contrast to ICC values of <0.3 in the NCCN and Blueprint 2 studies.

Conclusions: We demonstrate a reproducible end-to-end process for mIF characterization of the PD-1/PD-L1 axis including automated staining, multispectral imaging, and machine-learning-trained image analysis algorithms. This approach improved reproducibility of %PD-L1 IC assessment and brought it in line with %PD-L1 tumor cell assessment by pathologists. Sources of variation were identified and will be discussed. The described approach may serve as a template for assessing reproducibility of emerging mIF panels for other investigative teams, with an eye toward translating such approaches into clinical trials and ultimately into the clinic.

Citation Format: Clifford Hoyt, Kristin Roman, Liz Engle, Chichung Wang, Carmen Ballesteros-Merino, Shawn M. Jensen, John McGuire, Yi Zheng, Carla Coltharp, Mei Jiang, Justin Lucas, Edwin Parra, Ignacio Wistuba, Darren Locke, Bernard A. Fox, David L. Rimm, Janis Marie Taube. Multi-institutional TSA-amplified Multiplexed Immunofluorescence Reproducibility Evaluation (MITRE study): Reproducibility assessment of an automated multiplexed immunofluorescence slide staining, imaging, and analysis workflow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-318.