Utilizing Image Analysis to Characterize an 8-plex Immunofluorescence Immune Checkpoint Biomarker Panel within the Tumor Tissue Microenvironment

OracleBio are proud to present a poster on quantitative analysis of an 8-plex Immunofluorescence panel at the AACR 2020 Virtual Annual Meeting.

The poster, produced in collaboration with Ultivue, focuses on the analysis of an immune checkpoint biomarker panel in NSCLC and CRC samples. The poster is entitled ‘Utilizing Image Analysis to Characterize an 8-plex Immunofluorescence Immune Checkpoint Biomarker Panel within the Tumor Tissue Microenvironment’ and is available to view and download by clicking on the poster below:

Utilizing Image Analysis to Characterize an 8-plex Immunofluorescence Immune Checkpoint Biomarker Panel within the Tumor Tissue Microenvironment

Background

Multiplex staining and image analysis are being increasingly deployed for immuno-oncology (I/O) evaluations to investigate and understand the complexity of immune cell / tumor cell interactions in the tumor microenvironment (TME) and in discovering novel predictive biomarkers for targeted immunotherapy. Immune checkpoint inhibitor treatment, guided by immunohistochemically labelled tissue expression of PD-L1 and PD-1, have already been shown to improve outcomes, in the metastatic setting, for non-small cell lung carcinoma (NSCLC) and colorectal cancer (CRC) patients. The purpose of the current study was to phenotype and compare checkpoint and immune cell interactions within NSCLC and CRC tumor types.

Methods

The study included samples from NSCLC, CRC, and normal tonsil tissues. Sections were stained using InSituPlex® multiplex immunofluorescence (mIF), for studying PD-L1/PD-1 checkpoint expression including immune cell markers for CD3, CD4, CD8, CD68, FoxP3, and pan-CK as an epithelial tumor indicator. Slides were scanned at 20X magnification on the ZEISS Axio Scan.Z1 platform, with individual .tiff format files being generated per marker. A stacked OME.tif file was then created from the individual .tiff format files, per section. Image analysis was performed using the Visiopharm Oncotopix® platform. Individual marker image layers were aligned, tumor/stroma regions of interest (ROI) classified, and cell formations detected. Markers were quantified across the image layers to evaluate positive cell staining within both cancer indications.

Results

Quantitative image analysis data generated included total positive and negative cell counts per marker, per mm2 ROI.  Co-localization analysis provided relevant immune cell phenotypes (T-cytotoxic, T-helper, T-regs, and macrophages) and immune cell functional status. Cell spatial analysis relationships were generated, including CD8+/PD-1- and CD8+/PD-1+ to CK+/PD-L1+ tumor cells.

Conclusions

This study highlighted the benefits of utilizing an 8-plex staining protocol and detailed image analysis approach for the comprehensive assessment of the immune cell types and interactions within the TME. The ability to carry out multiparameter cellular analysis facilitates the delivery of key phenotypic data, thereby leading to greater understanding of tumor biology.

 

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