Background: PD-L1 expression and tumor-mutational burden enrich for patients that respond to checkpoint blockade, but these evaluations are only a component of the entire story. Recently, our lab reported that evaluation of specific cell-cell relationships provided a powerful biomarker for overall survival in patients with HPV- head and neck cancer (HNSCC). However, the areas selected for analysis were operator selected “hot spots”. This approach introduces the potential for unconscious bias in the selection process. To address this, we have sought to perform whole slide evaluations of sections to compare with hot spot analysis. This study is a preliminary report applying a novel set of fluorophores and filters that allow the visualization of seven colors on a whole slide.

Methods: Tissue samples included pellets of cultured lymphocytes and tumor specimens. A sample of the cultured lymphocytes that were fixed and embedded were analyzed by flow cytometry for immune markers. Formalin-fixed paraffin embedded (FFPE) sections were stained with antibodies for CD8, CD68, FoxP3, PD-1, PD-L1, cytokeratin and DAPI. PerkinElmer Opal reagents were used to identify markers and included standard and a new set of fluorophores that included Opal 480, 520, 570, 620, 690, and 780. Slides were imaged using a new scanning approach on a Vectra Polaris (PerkinElmer, Inc, Waltham, MA).

Results: Preliminary comparison of cells that were used to produce FFPE blocks by flow cytometry and multiplex IHC provided similar results for some markers. Determining optimal staining, exposure times and thresholds for analysis for this new method needs work, but the potential exists for effective evaluation of a whole slide with 7 different markers.

Conclusions: Our preliminary results provide reason to be optimistic that this approach can assess 7 colors in a whole slide. Whole sections labelled with 7 colors and spectrally unmixed supports deeper analysis of immune-biology on multiple scales, including re-analysis of spatial metrics based on emerging hypotheses about how cellular and expression distributions relate to disease progression and response to therapy.