Computer-assisted tumor grading, validation of PD-L1 scoring, and quantification of CD8-positive immune cell density in urothelial carcinoma, a visual guide for pathologists using QuPath

Background Advances in digital imaging in pathology and the new capacity to scan high-quality images have change the way to practice and research in surgical pathology. QuPath is an open-source pathology software that offers a reproducible way to analyze quantified variables. We aimed to present the functionality of biomarker scoring using QuPath and provide a guide for the validation of pathologic grading using a series of cases of urothelial carcinomas. Methods Tissue microarrays of urothelial carcinomas were constructed and scanned. The images stained with HE, CD8 and PD-L1 immunohistochemistry were imported into QuPath and dearrayed. Training images were used to build a grade classifier and applied to all cases. Quantification of CD8 and PD-L1 was undertaken for each core using cytoplasmic and membrane color segmentation and output measurement and compared with pathologists semi-quantitative assessments. Results There was a good correlation between tumor grade by the pathologist and by QuPath software (Kappa agreement 0.73). For low-grade carcinomas (by the report and pathologist), the concordance was not as high. Of the 32 low-grade tumors, 22 were correctly classified as low-grade, but 11 (34%) were diagnosed as high-grade, with the high-grade to the low-grade ratio in these misclassified cases ranging from 0.41 to 0.58. The median ratio for bona fide high-grade carcinomas was 0.59. Some of the reasons the authors list as potential mimickers for high-grade cases are fulguration artifact, nuclear hyperchromasia, folded tissues, and inconsistency in staining. The correlation analysis between the software and the pathologist showed that the CD8 marker showed a moderate (r = 0.595) and statistically significant (p < 0.001) correlation. The internal consistency of this parameter showed an index of 0.470. The correlation analysis between the software and the pathologist showed that the PDL1 marker showed a robust (r = 0.834) and significant (p < 0.001) correlation. The internal consistency of this parameter showed a CCI of 0.851. Conclusions We were able to demonstrate the utility of QuPath in identifying and scoring tumor cells and IHC quantification of two biomarkers. The protocol we present uses a free open-source platform to help researchers deal with imaging and data processing in the surgical pathology field. Keywords: Digital pathology, QuPath, CD8, Tumor infiltrating lymphocytes, Programmed death-ligand 1 immunotherapy, Biomarker, Bladder cancer