250k nsp chip

SNP microarray analysis was used to determine chromosomal aberrations. Two types of SNP microarray chips were used, the Affymetrix 250K_NSP-chip, with ~250,000 probes across the genome and the Affymetrix Cytoscan HD chip, with ~750,000. The first 28 samples were analyzed with the Affymetrix 250K_NSP chip, and the remaining 36 samples with the Affymetrix Cytoscan HD chip. Analysis of the Affymetrix 250K_NSP chips was performed with the ‘Genotyping Console’ to determine the copy number values and the ‘GCT Browser’ to visualize the data (both from Affymetrix, Santa Clara, USA). Affymetrix Cytoscan HD chips were analyzed with ‘ChAS’. Different loci per chromosome were evaluated to adjust for partial gains or deletions. ~200 probes per gene locus were averaged to determine eventual copy number.

The Leiden University Medical Centre (LUMC) cohort includes clinical, histopathological, and genetic information on 64 UM cases, enucleated between 1999 and 2008. Clinical information was collected from the Integral Cancer Center West patient records and updated in 2019. For each sample, part of the tumor was snap frozen with 2-methyl butane and used for mRNA and DNA isolation, while the remainder was embedded in paraffin after 48 hours of fixation in 4% neutral-buffered formalin and sent for histological analysis. Chromosome status was determined with the Affymetrix 250K_NSP-chip and Affymetrix Cytoscan HD chip (Affymetrix, Santa Clara, California, United States of America). RNA was isolated with the RNeasy mini kit (Qiagen, Venlo, The Netherlands) and mRNA expression was determined with the HT-12 v4 chip (Illumina, San Diego, California, United States of America). Statistical analyses of the LUMC cohort were carried out in SPSS, version 25 (IBM Corp). For survival analysis, Kaplan-Meier and log-rank test were performed with death due to metastases as endpoint. Cases that died of another or unknown cause were censored. The two subpopulations that were compared in each analysis were determined by splitting the total cohort along the median value of mRNA expression for each analyzed gene.

The QIAmp DNA Mini Kit was used to isolate DNA for single nucleotide polymorphism (SNP) analysis according to guidelines of the manufacturer (Qiagen, Venlo, The Netherlands). Status of chromosome 3 was determined with SNP analysis performed with the Affymetrix 250K_NSP chip and the Affymetrix Cytoscan HD chip (Affymetrix, Santa Clara, CA, USA) [14 (link)]. The copy number of chromosome 8q was identified with ddPCR. A threshold of >2.1 was defined as gain of 8q [14 (link)]. The RNeasy Mini Kit was used to isolate mRNA for gene expression analysis (Qiagen, Venlo, The Netherlands). Gene expression levels were obtained using the Illumina HT-12 v4 chip (Illumina, San Diego, CA, USA). Angiogenesis-related factors were selected based on literature regarding angiogenesis. Only these predefined genes were assessed in the current analysis (Table S5).