The average change (
n-fold) in genes with increased expression in liver metastases from UM at FDR 5 was calculated in a variety of microarray datasets as a measure of similarity of gene expression across other tissues (see the Methods section). This analysis demonstrated marked increased expression of UM metastasis–associated genes according to microarray analysis (FDR 5) in normal liver tissue (average change = 3.1-fold). Such increased expression was not identified in other normal tissues
(Fig. 4A) . Increased expression was also detected in several malignancies—among them, leukemia (average = 2.5-fold) and skin melanoma (average = 2-fold)—but not in other malignancies, including colon and gastric carcinomas
(Fig. 4B) . Such increased expression was not detected in metastases from variety of malignancies (data not shown). None of the genes with metastasis-associated expression in our study were found to be differentially expressed between primary UMs with high or low risk for development of metastases, respectively, according to Tschentscher et al.
12 and Onken et al.
13
Several facts suggest that the metastasis tissue which was included in this study did not contained liver tissue. UM metastases were identified by gross appearance of pigmented tissues which was clearly distinct from the surrounding nonpigmented liver. This gross distinction between metastasis and normal liver tissue was validated by histology. Typical histology of UM metastasis was demonstrated in the pigmented area with a distinct border from the nonpigmented area composed of normal liver tissue
(Fig. 5) .
Bioinformatics further excluded potential bias that might be introduced by normal liver tissue. Our analysis showed that 14 genes that were represented on the array and which normally show liver enriched expression pattern (http://expression.gnf.org/cgi-bin/index.cgi#Q/ Novartix GNF) including APOF, MPST, CFHL3, CYP2D6, SARDH, IQCE, SLC6A12, SLC25A20, CCL16, CYP4F11, PGM1, IQCE, KIAA0841, and SEC14L2 did not manifest increased expression levels in UM liver metastasis. Finally, expression of ASGR1, a liver hepatocyte-specific gene, as measured by semiquantitative PCR, demonstrated expression levels ranging between 0 and 3.7 OD/mm2 in the metastases samples that were used for microarray analysis, compared with 177 OD/mm2 in normal liver sample. Four of the six metastasis samples that were tested demonstrated no expression of ASGR1. These results demonstrate that only a negligible quantity of liver tissue was present in a minority of the metastases samples.