Multiplex immunofluorescence assay
tumor, mesothelioma cancer, cholangiocarcinoma
TUMOR, PATHOLOGY, CHOLANGIOCARCINOMA
MultiOmyxTM technology was used to evaluate the expression of a panel of 14 biomarkers, including TIGIT, CD155, LAG3, CD4, CD56, CD3, CTLA4, CD8, PD-L1, PD-1, FoxP3, CD68 and TIM-3 and tumor segmentation marker PanCK on ovarian, cholangiocarcinoma and mesothelioma cancer FFPE samples. FFPE slides were evaluated by pathology (at NeoGenomics) for tissue annotation and selection. The selected tumor-relevant areas by pathology were used for staining and analysis. The staining was performed using a single 5-µM FFPE slide. Within each staining round, two cyanine dye-labeled (Cy3 and Cy5) antibodies were paired together and recognized two markers. The staining signal was then imaged and followed by novel dye inactivation, enabling repeated rounds of staining. For cell classification for all individual markers, proprietary deep-learning-based workflows were applied to identify individual cells and perform individual cell classification results. These results were combined to generate co-expression summaries and compute spatial distribution statistics for phenotypes of interest. For area quantification of PD-L1 or CD155, ImageJ software (NIH) was used to measure area of fluorescent marker expression within three high-powered fields divided by the total area. For visualization of each marker, individually established fluorescent minimum and maximum values were set and applied equally across all samples using the fluorescent look-up table (LUT).