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

Abstract

Objective

The aim of this study was to develop an open-source and reproducible digital quantitative analysis (DIA) of somatostatin receptor subtype 2a (SST ₂) staining in formalin-fixed paraffin-embedded tissues of pancreatic neuroendocrine tumors (panNETs) and growth hormone (GH)-secreting pituitary adenomas (GHomas).

Design

SST ₂ 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 rh: 0.826–0.881, P < 0.001). In GHomas, the biochemical response to somatostatin receptor ligands correlated with SST ₂ 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 SST ₂ immunohistochemistry.