Immunohistochemical assessment of mitotic count in uveal melanoma

Background/Aims: The mitotic count of uveal melanomas correlates with the risk of metastatic death, but with haematoxylin and eosin (H&E)-stained sections, it can be difficult to identify mitotic figures (MF) reliably. We investigated whether this measurement could be enhanced by immunohistochemistry, using the mitosis-specific marker Phospho-Histone H3 Ser10 (PHH3). Methods: Formalin-fixed, paraffin-embedded choroidal melanomas from patients treated by enucleation or trans-scleral local resection between 2006 and 2008 were used in this study. Sections from 132 tumours were stained with H&E and PHH3. Mitotic count was defined as the sum of MF counted in 40 high-power fields (HPF). In both H&E and PHH3-stained sections, mitotic count was determined independently by an experienced pathologist (SEC) and by a trainee ophthalmologist (MA). Data were assessed using the Kolmogorov-Smirnov (KS) statistical test and the Bland-Altman (BA) analysis. Results: The mitotic count determined by SEC in H&E and PHH3-stained sections had median values of 4/40 HPF and 8/40 HPF, respectively (KS, Z = 2.585 p < 0.001; BA, mean difference -5.95 [CI -22.15-10.22]), indicating poor reproducibility between these two methods. Similarly, the mean difference between the H&E and the PHH3 methods for calculating the mitotic count by MA was -7.03 (BA) [CI -23.38-9.31]. In contrast, the median mitotic counts determined by SEC & MA using PHH3 were 8/40 HPF and 9/40 HPF, respectively (KS, Z = 0.308 p = 1; BA, mean difference -0.29 [CI -2.65-2.06]), indicating good reproducibility between examiners. Conclusions: MF were more easily identified following immunohistochemical staining with anti-PHH3. This resulted in higher mitotic counts than obtained with H&E sections. For PHH3, the mitotic count determined by a MA was virtually the same as those obtained by an experienced pathologist. PHH3 is now routinely used in our centre, and new prognostic thresholds for uveal melanoma will be defined in further studies.