## 3 Results
### mH2A.1 Expression and Clinicopathological Features in UIMs  
Immunohistochemistry showed only mH2A.1 nuclear staining: no immunohistochemical expression of mH2A.1 was observed in non-neoplastic ocular tissue. Interobserver agreement was excellent (kappa = 0.943).  
Considering the whole group (\(n\) = 55), the median mH2A.1 value was 12: H-IRS was observed in 28 (50.9%) melanomas and L-IRS in 27 (49.1%).  
In 32 nonmetastatic primary UMs, mH2A.1 IS was intense/intermediate in 18 cases (56.3%) and mild in only one case (3.1%), while in 13 (40.6%) cases no immunoreactivity was observed (Figure 1). ES was \(>\)50% in 10 cases (31.3%) and 5%-30% in 9 cases (28.1%). Only 8/32 cases (25%) showed H-IRS, while 24 cases (75%) showed L-IRS (Table 1) ([PERSON]'s exact test, \(p<0.001\), Table 4).  
In 23 metastatic primary UMs, mH2A.1 IS was intense/intermediate in 21 cases (91.4%) and mild in 1 case (4.3%). Only 1 case (4.3%) showed an absence of immunoreactivity (Figure 2). ES was \(>\)75% in 20 cases (87%), 50%-75% in 1 case (4.3%), and 5%-30% in 1 case (4.3%). Here, 20/23 cases (87%) showed H-IRS, while only 3 cases (13%) had L-IRS (Table 2) ([PERSON]'s exact test, \(p<0.001\), Table 4).  
\begin{table}
\begin{tabular}{c c c} \hline
**mH2A** & **Metastasis (\(n\) = 23)** & **Metastasis-Free (\(n\) = 32)** \\ \hline Low & 3 (13\%) * & 24 (75\%) \\ High & 20 (87\%) & 8 (25\%) \\ \hline \end{tabular} Abbreviations: mH2A, macroH2A. \(p\) ([PERSON]’s exact test). * \(p<0.0001\).  
\end{table}
Table 4: Number of UMs (with and without metastasis) with low and high macroH2A.  
Figure 1: Immunohistochemical expression of mH2A.1 in nonmetastatic uveal melanomas. Melanoma cells showed weak staining for macroH2A.1 (low magnification in (**A**) and high magnification in (**B**)).  
In two cases of metastasis from uveal melanoma, mH2A.1 showed diffuse and intense immunoreactivity with H-IRS (Figure 3).  
Univariate analysis based on a Cox proportional hazards regression model revealed that factors related to the presence of metastasis were age (\(p\) = 0.053), greater tumor size (\(p\) = 0.009), pT (pathological Tumor) stage (\(p\) = 0.016), the epithelioid variant (\(p\) = 0.011), and mH2A.1 expression (\(p\) < 0.001). Factors significantly related to the presence of metastasis in the multivariate analysis were mH2A.1 expression (\(p\) = 0.002), greater tumor size (\(p\) = 0.026), and the epithelioid variant (\(p\) = 0.019).  
Here, mH2A.1 expression was not related to histological type (Spearman's rho, \(p\) = 0.173). A Kaplan-Meier survival analysis showed that the estimated survival times free from metastasis were greater in patients with low mH2A.1 expression in UMs: mean values (SE (Standard error), with 95% CI (confidential interval)) were 110.3 (6.80) (CI: 97.0 to 123.7) in patients with low mH2A.1 expression and 56.7 (10.0) (CI: 37.2 to 76.2) in patients with high mH2A.1 expression (\(p\) < 0.001, log-rank (Mantel-Cox) test, Figure 4).  
Figure 3: Immunohistochemical expression of mH2A.1 in metastases from uveal melanomas. Metastatic cells revealed diffuse (ES 4) and strong (IS 4) immunoreactivity, respectively, at low (**A**) and high magnifications (**B**).  
Figure 2: Immunohistochemical expression of mH2A.1 in metastatic uveal melanomas. Tumor cells revealed moderate (IS 2) immunoreactivity in (**A**). (**B**) and (**C**) show strong staining (IS 3), respectively, at low and high magnifications.