Chromosomal Aberrations and Immune Profiling in Uveal Melanoma

DNA and gene expression analysis were performed on 54 tumor specimens from Leiden, in which the BAP1 status was known. The QIAmp DNA Mini kit was used to isolate DNA for single-nucleotide polymorphism (SNP) analysis (Qiagen, Venlo, The Netherlands). SNP analysis was then performed with the Affymetrix 250K_NSP microarray and Affymetrix Cytoscan HD chip (Affymetrix, Santa Clara, California, United States of America) to detect aberrations of chromosome 3 as described previously [20 (link)]. Information on chromosome 8q was obtained by digital polymerase chain reaction (dPCR) [20 (link)]. A threshold of >2.1 was defined as having extra copies of chromosome 8q. The RNeasy mini kit was used to isolate RNA for gene expression analyses (Qiagen, Venlo, The Netherlands). Gene expression levels of CD3 and CD8 (T cells), CD68 (macrophages), and pro-inflammatory cytokines, specifically macrophage inflammatory protein 1α (CCL3), vascular endothelial growth factor A (VEGFA), stromal cell-derived factor 1 (CXCL12), CCL7, CSF-1, monocyte chemoattractant protein-1 (CCL2), RANTES (CCL5), interferon gamma-induced protein 10 (CXCL10), CCR7, and CXCR4 were obtained using the Illumina HT-12 v4 chip (Illumina, San Diego, California, United States of America). The pro-inflammatory cytokines were selected based on our previous papers [31 (link)–33 (link)]. We could validate the probes for CD3,CD8 and CD68 in 24 tumors in which gene expression levels had been determined with an Illumina HT12 v4 array and in which the number of infiltrating cells was analyzed by IF. In addition, data on RNA sequencing and Affymetrix SNP 6.0 array from 80 samples of UM were obtained from the TCGA Research Network: http://cancergenome.nih.gov/. Copy numbers for 8q were determined by Affymetrix SNP 6.0 array and analyzed with the GISTIC 2.0 algorithm [34 (link), 35 (link)]. Copy numbers >2 were categorized as extra copies of chromosome 8q. BAP1, CD68, CD3, and CD8 expression were obtained by RNA sequencing and quantified as log2(RSEM+1). BAP1 expression was dichotomized into BAP1-positive and BAP1-negative expression at the median.

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Gezgin G., Dogrusöz M., van Essen T.H., Kroes W.G., Luyten G.P., van der Velden P.A., Walter V., Verdijk R.M., van Hall T., van der Burg S.H, & Jager M.J. (2017). Genetic evolution of uveal melanoma guides the development of an inflammatory microenvironment. Cancer Immunology, Immunotherapy, 66(7), 903-912.

Publication 2017

Ccl2 Ccl3 Ccl5 Ccl7 Cd8 t cells Chip Chromosome Chromosome aberrations Csf 1 Cxcl12 Cxcr4 Cytokines Digital Gene expression Gene expression analyses Inflammatory Interferon gamma Macrophage inflammatory protein 1α Macrophages Microarray Monocyte chemoattractant protein 1 Polymerase chain reaction Protein Rantes Single nucleotide polymorphism Tumors Vascular endothelial growth factor a

Corresponding Organization :

Other organizations : Leiden University Medical Center, Penn State Milton S. Hershey Medical Center, Erasmus MC

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Variable analysis

independent variables

BAP1 status
Chromosome 3 aberrations
Chromosome 8q copy number

dependent variables

Gene expression levels of CD3, CD8 (T cells), CD68 (macrophages)
Gene expression levels of pro-inflammatory cytokines (CCL3, VEGFA, CXCL12, CCL7, CSF-1, CCL2, CCL5, CXCL10, CCR7, CXCR4)

control variables

Tumor specimens from Leiden
QIAmp DNA Mini kit for DNA isolation
Affymetrix 250K_NSP microarray and Affymetrix Cytoscan HD chip for SNP analysis
Digital polymerase chain reaction (dPCR) for chromosome 8q information
RNeasy mini kit for RNA isolation
Illumina HT-12 v4 chip for gene expression analysis
Validation of CD3, CD8 and CD68 probes in 24 tumors
TCGA Research Network data (80 samples of UM) for RNA sequencing and Affymetrix SNP 6.0 array

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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