Spatial intratumor heterogeneity in uveal melanoma: Tumor cell subtypes with a presumed invasive potential exhibit a particular epigenetic staining reaction.

Cancer evolves from a combination of genetic and epigenetic abnormalities resulting in aberrant gene expression profiles as well as altered epigenomic patterns. Epigenetic alterations such as DNA methylation and histone modification play an important role in tumorigenesis. While in the pathobiology of uveal melanoma (UM) genetic changes have been well characterized, there is growing evidence suggesting that epigenetic changes are also involved. We investigated whether epigenetic modifications (global levels of histone acetylation, DNA methylation, ubiquitination) are detectable in UM tissues compared to healthy controls with respect to inter- and intratumoral heterogeneity. Formalin-fixed paraffin-embedded tissues of primary UM (n = 15), UM metastasis (n = 13), and control choroid (n = 12) were immunohistochemically investigated by two ophthalmic pathologists for global levels of histone acetylation (Histone 3 acetylation, H3Ac; Histone 4 acetylation, H4Ac), DNA methylation (5-methylcytosine, 5-MeC; 5'-hydroxymethylcytosine, 5-hMeC), global ubiquitination (UBC) as well as Ubiquityl-Histone H2A (H2Aub). The nuclear staining intensity of primary tumors, metastases and control choroids was evaluated using a score from 0 to 3, which was multiplied with the percentage of stained cells (score from 0 to 4). The control choroid and the choroid next to the tumor showed a more intense nuclear staining than the primary tumor tissue. The choroid next to the tumor was stained less than the control choroid. The nuclear staining intensity in the tumor was comparable to that in the metastases. The tumor tissue itself often exhibited a heterogeneous staining pattern, as nuclei in the tumor center were less intensely stained than in the periphery. Cells with a presumed invasive potential (extraocular extension, growth along emissary canals) showed also an intense staining reaction. Although no prognostically relevant pattern of global epigentic markers could be identified, our results suggest that epigenetic changes play a role in UM pathogenesis and metastasis. In particular the staining reaction of tumor cell subtypes with a presumed invasive potential warrants further attention. The role of epigenetically relevant interactions with the tumor micromilieu should be further investigated as immune cells are predominantly located in the tumor periphery which showed a different staining intensity than the tumor center. However, as considerable epigenetic diversity exists in primary tumors, studies on biopsy tissue are not recommended for the immunohistochemical investigation of epigenetic markers.