BACKGROUND: Immune checkpoint inhibitors (ICI) therapies have demonstrated 
significant benefit in the treatment of many tumors including high grade 
urothelial cancer (HGUC) of the bladder. However, variability in patients' 
clinical responses highlights the need for biomarkers to aid patient 
stratification. ICI relies on an intact host immune response. In this context, 
we hypothesize that key players in the antitumor immune response such as markers 
of activated cytotoxic T lymphocytes (CD8, granzyme-B) and immune suppression 
(FOXP3) may help to identify patients who will derive the greatest therapeutic 
benefit from ICI. A major obstacle for deployment of such a strategy is the 
limited quantities of tumor-derived biopsy material. Therefore, in this 
technical study, we develop a multiplex biomarker with digital workflow. We 
explored the (1) concordance of conventional single stain results using digital 
image analysis, and (2) agreement between digital scoring versus manual 
analysis.
METHODS: (1) For concordance study of single and multiplex stains, triplicate 
core tissue microarrays of 207 muscle invasive, HGUC of bladder had sequential 
4-micron sections cut and stained with CD8, FOXP3 and granzyme-B. An inhouse 
developed tri-chromogen multiplex immunohistochemistry (m-IHC) assay consisting 
of CD8 (green), granzyme B (brown), and FOXP3 (red) was used to stain the next 
sequential tissue section. (2) Agreement between manual and digital analysis was 
performed on 19 whole slide sections of HGUC cystectomy specimens. All slides 
were scanned using Aperio ScanScope AT Digital Scanner at 40X. Quantitative 
digital image analysis was performed using QuPath version 0.2.3 open-source 
software. Scores from triplicate cores were averaged for each HGUC specimen for 
each marker. Intraclass correlation coefficients were used to compare percent 
positive cells between the single- and multi-plex assays. Lin's concordance 
correlation coefficients were used for manual versus digital analysis.
RESULTS AND CONCLUSIONS: m-IHC offers significant advantages in characterizing 
the host immune microenvironment particularly in limited biopsy tissue material. 
Utilizing a digital image workflow resulted in significant concordance between 
m-IHC and individual single stains (p < 0.001 for all assessments). Moderate to 
good agreements were achieved between manual and digital scoring. Our technical 
work demonstrated potential uses of multiplex marker in assessing the host 
immune status and could be used in conjunction with PD-L1 as a predictor of 
response to ICI therapy.