Digital quantification of somatostatin receptor subtype 2a immunostaining: a validation study

Objective: The aim of this study was to develop an open-source and reproducible digital quantitative analysis (DIA) of somatostatin receptor subtype 2a (SST2) staining in formalin-fixed paraffin-embedded tissues of pancreatic neuroendocrine tumors (panNETs) and growth hormone (GH)-secreting pituitary adenomas (GHomas). Design: SST2 immunostaining of 18 panNETs and 39 GHomas was assessed using a novel DIA protocol and compared with a widely used semi-quantitative immunoreactivity score (IRS). Methods: The DIA software calculates the staining intensity/area and the percentage of positive cells (%PC). Four representative images were selected for each sample by two independent selectors (S-1 and S-2), with the analysis performed by two independent analyzers (A(1) and A(2)). Agreement between observers was calculated using the concordance correlation coefficient (CCC). Results: In panNETs, the CCC ranged 0.935-0.977 for intensity/area and 0.942-0.983 for %PC. In GHomas, the CCC ranged 0.963-0.997 for intensity/area and 0.979-0.990 for %PC. In both panNETs and GHomas, the DIA staining intensity was strongly correlated with the IRS (Spearman rho: 0.916-0.969, P < 0.001), as well as the DIA %PC with the IRS %PC (Spearman rho: 0.826-0.881, P < 0.001). In GHomas, the biochemical response to somatostatin receptor ligands correlated with SST2 expression, evaluated both as DIA intensity/area (Spearman rho: -0.448 to -0.527, P = 0.007-0.004) and DIA %PC (Spearman rho: -0.558 to -0.644, P <= 0.001). Conclusions: The DIA has an excellent inter-observer agreement and showed a strong correlation with the widely used semi-quantitative IRS. The DIA protocol is an open-source, highly reproducible tool and provides a reliable quantitative evaluation of SST2 immunohistochemistry.