Optimizing immunofluorescence with high-dynamic-range imaging to enhance PD-L1 expression evaluation for 3D pathology assessment from NSCLC tumor tissue.
Journal
Scientific reports
Journal Volume
14
Journal Issue
1
Start Page
15176
ISSN
2045-2322
Date Issued
2024-07-02
Author(s)
Hung, Yu-Ling
Yang, Chia-Hung
Hsieh, Yu-Han
Lin, Yu-Chieh
Chang, Margaret Dah-Tsyr
Lin, Yen-Yin
Abstract
Assessing programmed death ligand 1 (PD-L1) expression through immunohistochemistry (IHC) is the golden standard in predicting immunotherapy response of non-small cell lung cancer (NSCLC). However, observation of heterogeneous PD-L1 distribution in tumor space is a challenge using IHC only. Meanwhile, immunofluorescence (IF) could support both planar and three-dimensional (3D) histological analyses by combining tissue optical clearing with confocal microscopy. We optimized clinical tissue preparation for the IF assay focusing on staining, imaging, and post-processing to achieve quality identical to traditional IHC assay. To overcome limited dynamic range of the fluorescence microscope's detection system, we incorporated a high dynamic range (HDR) algorithm to restore the post imaging IF expression pattern and further 3D IF images. Following HDR processing, a noticeable improvement in the accuracy of diagnosis (85.7%) was achieved using IF images by pathologists. Moreover, 3D IF images revealed a 25% change in tumor proportion score for PD-L1 expression at various depths within tumors. We have established an optimal and reproducible process for PD-L1 IF images in NSCLC, yielding high quality data comparable to traditional IHC assays. The ability to discern accurate spatial PD-L1 distribution through 3D pathology analysis could provide more precise evaluation and prediction for immunotherapy targeting advanced NSCLC.
Subjects
High dynamic range
Immunofluorescence
Non-small cell lung cancer
Programmed death ligand 1
Three-dimensional pathology
SDGs
Type
journal article