Abstract
Purpose :
Anaplasia has been demonstrated to be a useful characteristic in identifying patients with retinoblastoma that may have higher risk of metastasis but do not exhibit the classical associated high-risk histopathological features. To further our understanding of anaplasia in retinoblastoma, we performed morphometric analysis of retinoblastomas with varying degrees of cellular anaplasia.
Methods :
Glass slides of haematoxylin and eosin-stained tumor tissue were digitally scanned at 200×. Images were imported to the QuPath Bioimage analysis software. 2 pathologists identified 2 circular regions per tumor that were representative of its highest grade of cellular anaplasia (mild, moderate or severe). A workflow for morphometric analysis was then created for each tumor. Measurements of 33 different parameters related to the shape, size and staining patterns of each tumor cell nucleus and cytoplasm within the regions were recorded and analyzed. In tests of the null hypothesis in tumors with severe versus non-severe anaplasia, Mann-Whitney U-test and Student’s T-tests were used.
Results :
Of the 33 morphometric variables analyzed in our 28 tumors, 15 variables relating to staining characteristics differed significantly: The sum of hematoxylin staining intensity in tumor cell nuclei was significantly higher in tumors with severe anaplasia (p=0.008). Similarly, the maximum (p=0.036) and minimum (p=0.045) hematoxylin staining intensities, mean of eosin staining intensity (p=0.022) and sum of eosin staining intensity (p=0.014) were all significantly higher in tumor cell nuclei in tumors with severe versus non-severe anaplasia. In entire cells (nuclei + cytoplasms), the mean eosin staining intensity was significantly lower in tumors with severe anaplasia (p=0.039). Conversely, the minimum eosin and hematoxylin staining intensities in entire cells and cytoplasms were significantly higher in tumors with severe anaplasia (p=0.036 and p=0.007, respectively).
Conclusions :
The staining characteristics of retinoblastoma tumor cell nuclei and cytoplasms correlated to the degree of anaplasia. Hematoxylin staining intensities were significantly higher and eosin staining intensities significantly lower in severely versus non-severely anaplastic tumor cells. Previously, similar findings were described by pathologists as hyperchromasia. For the first time, we have here quantified what would otherwise be a subjective description, with a semi-automatic digital approach.
This is a 2020 ARVO Annual Meeting abstract.