Prognostic impact of HERC2 protein and pink-eyed dilution protein in uveal melanoma
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RESEARCH ARTICLE
Prognostic impact of HERC2 protein and pink‑eyed dilution protein in uveal melanoma Seema Kashyap1 · Mithalesh Kumar Singh1 · Jayanti Jha1 · Lata Singh1 · Neelam Pushker2 · Seema Sen1 · Pradeep Venkatesh2 · Rachna Meel2 · Neiwete Lomi2 Received: 9 May 2020 / Accepted: 8 July 2020 © Japan Human Cell Society 2020
Abstract Biological understanding of pigmentation and its association with clinicopathological implications in uveal melanoma (UM) risk is still unexplored. HECT and RLD Domain Containing E3 Ubiquitin Protein Ligase 2 (HERC2) and Pink-eyed dilution protein (P-protein) are the important markers that regulate pigmentation in eye. Therefore, our aim of the study was to investigate the expression of HERC2 and P-protein in the UM patients and correlate with patient outcome. Fiftytwo formalin-fixed paraffin-embedded UM tissue samples were included to detect the expression of HERC2 and P-protein by immunohistochemistry and validated by western blot. Cox proportional hazard model and log-rank test were used to determine the prognostic potential of these proteins. High pigmentation was seen in 67% of the UM cases. The expression of HERC2 and P-protein was present in 44% and 71% cases, respectively. On statistical analysis, increased pigmentation, epithelioid cell type, and ciliary body invasion were significant with the protein expressions (p 100/20 HPF) [18].
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Grading of pigmentation Fundus examination for clinical grading of pigmentation was done by indirect ophthalmoscopy. ‘Low pigmentation’ was considered when choroidal vessels were darker than the surrounding choroid due to less pigmentation between vessels, whereas ‘high pigmentation’ was considered when choroidal vessels were lighter than the surrounding dark choroidal pigment between vessels, as shown in Fig. 1a, c [4]. Histopathological grading of pigmentation was also performed on hematoxylin and eosin slides by JJ and MKS under the supervision of experienced pathologist (SK). Grading of pigmentation was defined as the percentage presence of melanin in the melanoma cells of the analyzed tumor sections: 1—melanin was present in ≤ 30% of melanoma cells, 2—melanin was present in 31–70% of melanoma cells, and 3—melanin was present in > 70% of melanoma cells. Pigmentation grading of ‘1’ was considered as low pigmentation, while pigmentation grading of ‘2’ and ‘3’ together grouped as high pigmentation in Fig. 1b, d [19].
Immunohistochemistry and analysis Immunohistochemistry was performed on all 52 formalinfixed paraffin-embedded UM tissue samples. Following deparaffinization and heat-induced antigen retrieval in 0.01 M citrate buffer (pH-6) for 25 min, the tissue sections were treated with protein block and washed with the 1X PBS buffer. After frequent washing, tissue sections were incubated overnight with anti-HERC2 (mouse monoclonal; 1:100 dilution; catalogue# sc-515891) and anti P-protein (rabbit polyclonal; dilution 1:100; catalogue# ab196500). Sections were incubated with horseradish peroxidase (HRP) conjugated se
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