## 2 Materials and Methods  
### Patients and Tissue Samples  
A retrospective study was performed on 55 primitive choroid and/or ciliary body melanomas after surgical treatment consisting of enucleation in cases not eligible for radiotherapy, such as plaque brachytherapy or proton beam radiotherapy [8], at the Eye Clinic, University of Catania, from October 2009 to October 2017. No written informed consent was necessary because of the retrospective nature of the study. The research protocols were conformed to the ethical guidelines of the Declaration of Helsinki. The patients were 28 males and 27 females at an average age of 67 years (range 29-85). In particular, the patients with metastatic UM were 11 males and 12 females at an average age of 72 years (range 50-85): disease progression caused death in 13 of the 23 patients. The patients with nonmetastatic UM were 17 males and 15 females at an average age of 64 years (range 29-84) (Tables 1 and 2). The size and site of tumor onset were studied by ophthalmoscopy and A and B scan ultrasonography, while the study of metastases was performed by physical examination, hepatic ultrasound, and computerized tomography. The median follow-up period was 60 months (range 8-138 months). Forty melanomas were localized only in the choroid, while 15 involved both the choroid and the ciliary body: only in one case was an extrascleral extension found. As concerns histotypes, 15 cases were classified as epithelioid cells and 12 as spindle cells, and 28 cases were diagnosed as mixed type. Based on the eighth TNM classification, 23 metastatic UMs were distributed as follows: pT1a (1 case, 4.3%), pT2a (9 cases, 39.1%), pT2b (2 cases, 8.7%), pT2d (1 case, 4.3%), pT3a (4 cases, 17.4%), pT3b (5 cases, 21.7%) and pT4a (1 case, 4.3%). TNM staging in 32 patients with metastatic UMs included pT1a (6 cases, 18.7%), pT1b (1 case, 3.1%), pT2a (12 cases, 37.5%), pT2b (6 cases, 18.7%), pT3a (4 cases, 12.5%), and pT4b (3 cases, 9.4%). We also tested mH2A in two cases of metastasis from uveal melanoma. The cases were collected from the files of the Anatomic Pathology Department of Medical, Surgical, and Advanced Technologies, [PERSON], University of Catania. Some cases were excluded from the study for the following reasons: (1) if it was not possible to obtain sections from paraffin blocks for immunohistochemical staining, (2) the absence of representative tumor tissue, (3) the presence of exclusively necrotic material, and (4) preoperatively treated UMs. Five sections were cut from each paraffin block. Briefly, the deparaffinized slides were pretreated with 10 mg/mL of ovalbumin in phosphate-buffered saline (PBS) followed by 0.2% biotin+ in PBS, each for 15 min at room temperature, and they were rinsed for 20 min with PBS (Bio-Optica, Milan, Italy) in order to reduce the usually seen nonspecific immunoreactivity due to endogenous biotin. Microwave pretreatment was performed to unmask antigenic sites. Then the slides were incubated overnight at 4 \({}^{\circ}\)C with rabbit polyclonal anti-macroH2A.1 antibody (ab37264; Abcam, Cambridge, UK) diluted 1:200 in PBS (Sigma, Milan, Italy). Sections were counterstained with hematoxylin, dehydrated, mounted (Zymed Laboratories, San Francisco, CA, USA), and observed with a light microscope ([PERSON], Oberkochen, Germany). \begin{table}
\begin{tabular}{c c c c c c c c c c c} \hline \hline \multirow{2}{*}{**Sex**} & **Age** & \multirow{2}{*}{**Location**} & **Thickness** & **Largest** & **Cell** & **Pathological** & **DFS** & **Follow-Up** & \multicolumn{3}{c}{**MacroH2A**} \\  & & & **(mm)** & **Diameter** & **Type** & & & **(Months)** & **(Months)** & **IS** & **ES** & **IRS** \\ \hline F & 58 & Ch & 6.04 & 17.8 & Mc & pT2a & 63 & 64 (t) & 3 & 3 & 9 & L \\ M & 69 & Ch & 7.21 & 15.8 & Mc & pT2a & 54 & 81 (t) & 3 & 4 & 12 & H \\ F & 75 & Ch/CB & 1  
The immunohistochemical expression of mH2A.1 was evaluated as positive if brown chromogen was observed in the nucleus.