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Original Investigation |

Analysis of the Expression and Single-Nucleotide Variant Frequencies of the Butyrophilin-like 2 Gene in Patients With Uveal Melanoma ONLINE FIRST

Adriana Amaro, PhD1,2; Federica Parodi, PhD1,3; Konrad Diedrich, MSc1; Giovanna Angelini, PhD1; Cornelia Götz, MSc4; Silvia Viaggi, PhD2,5; Irena Maric, PhD5; Domenico Coviello, MD, PhD5; Maria Pia Pistillo, PhD6; Anna Morabito6; Mario Mandalà, MD7; Paola Ghiorzo, PhD8; Paola Visconti, PhD9; Marina Gualco, MD10; Luca Anselmi, MD11; Roberto Puzone, PhD12; Francesco Lanza, MD5; Carlo Mosci, MD5; Federica Raggi, PhD13; Maria Carla Bosco, PhD13; Luigi Varesio, PhD13; Michael Zeschnigk, MD14; Laura Spano, MD15; Paola Queirolo, MD15; Ulrich Pfeffer, PhD1
[+] Author Affiliations
1Laboratory of Molecular Pathology, Istituto di Ricovero e Cura a Carattere Scientifico Azienda Ospedaliera Universitaria San Martino–IST Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy
2Department of Earth Sciences, Environment, and Life, Università Degli Studi di Genova, Genova, Italy
3Intergruppo Melanoma Italiano, Genova, Italy
4TIB-Molbiol Syntheselabor, Berlin, Germany
5Ente Ospedaliero Galliera, Genova, Italy
6Department of Tumor Epigenetics, Istituto di Ricovero e Cura a Carattere Scientifico Azienda Ospedaliera Universitaria San Martino–IST Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy
7Azienda Socio Sanitaria Territoriale Ospedale Papa Giovanni XXIII, Bergamo, Italy
8Department of Genetics of Rare Tumors, Istituto di Ricovero e Cura a Carattere Scientifico Azienda Ospedaliera Universitaria San Martino–IST Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy
9Department of Blood Transfusion Center, Istituto di Ricovero e Cura a Carattere Scientifico Azienda Ospedaliera Universitaria San Martino–IST Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy
10Department of Anatomy and Cytohistopathology, Istituto di Ricovero e Cura a Carattere Scientifico Azienda Ospedaliera Universitaria San Martino–IST Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy
11Ospedale Villa Scassi, Genova, Italy
12Department of Clinical Epidemiology, Istituto di Ricovero e Cura a Carattere Scientifico Azienda Ospedaliera Universitaria San Martino–IST Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy
13Laboratory of Molecular Biology, Istituto Giannina Gaslini, Genova, Italy
14Institute of Human Genetics, Faculty of Medicine, University Duisburg-Essen, West German Cancer Center and the German Cancer Consortium, Essen, Germany
15Department of Medical Oncology, Istituto di Ricovero e Cura a Carattere Scientifico Azienda Ospedaliera Universitaria San Martino–IST Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy
JAMA Ophthalmol. Published online August 11, 2016. doi:10.1001/jamaophthalmol.2016.2691
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Importance  Chromosome 6p amplification is associated with more benign behavior for uveal melanomas (UMs) with an otherwise high risk of metastasis conferred by chromosome 3 monosomy. Chromosome 6p contains several members of the B7 family of immune regulator genes, including butyrophilin-like 2 (BTNL2; OMIM, 606000), which is associated with prostate cancer risk and autoimmune diseases.

Objective  To investigate the expression and variant allele frequencies of BTNL2, a candidate gene for chromosome 6 amplification, in patients with UM.

Design, Setting, and Participants  In this case-control study, we analyzed the expression of BTNL2 in UM cell lines and human macrophages in patients with UM. Variants of BTNL2 were analyzed using probes for polymerase chain reaction and high-resolution melting. The association of missense variants rs28362679 and rs41441651 with tumor risk was analyzed in 209 patients with UM and 116 matched control patients as well as 12 UM and 64 other tumor cell lines. Genes that were differentially expressed in M1- and M2-polarized macrophages were identified by microarray analysis of 111 patients with UM, and the association of the expression of these genes with disease-free survival was analyzed by Cox regression analysis. Data were collected from September 2013 to November 2015.

Main Outcomes and Measures  Butyrophilin-like 2 single-nucleotide variants were associated with UM risk; M1 and M2 macrophage-specific gene expression was associated with disease-free survival.

Results  We genotyped a total of 325 patients. Of the 209 patients with UM, 124 (59.3%) were male, 114 (54.5%) were Italian, and 95 (45.5%) were German; the mean (range) age was 65 (27-94) years. Of the 116 Italian control patients, 67 (57.8%) were female, and the mean (range) age was 39 (21-88) years. Butyrophilin-like 2 is expressed in patients with UM and macrophages. The frequency of the rs28362679 variant was higher in patients with UM (16 of 209 [7.7%]; 95% CI, 4.7-12.2) than frequencies from European Variation Archive and Exome Aggregation Consortium data (2134 of 118 564 [1.8%]; 95% CI, 1.7-1.9) and Exome Sequencing Project data (100 of 4540 [2.2%]; 95% CI, 1.8-2.7) but were not higher compared with Italian control patients (10 of 116 [8.6%]; 95% CI, 4.6-15.4). The rs41441651 variant was present in 5 patients with UM (2.4%; 95% CI, 0.9-5.7), 2 Italian control patients (1.7%; 95% CI, 0.1-6.5), 2846 patients from European Variation Archive and Exome Aggregation Consortium data (2.4%; 95% CI, 2.3-2.5), and 23 patients from Exome Sequencing Project data (0.5%; 95% CI, 0.3-0.8). Human UM cells express M1 and M2 macrophage-specific genes, whose expression is associated with disease-free survival.

Conclusions and Relevance  Butyrophilin-like 2, expressed at various levels by UM cells and macrophages, might interfere with the immune control of the tumor. Butyrophilin-like 2 variants showed highly variable frequencies among ethnically related cohorts. There was no enrichment of BTNL2 variants in patients with UM compared with control patients.

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Figure 1.
Butyrophilin-like 2 (BTNL2) Expression

A, BTNL2 expression in patients with uveal melanoma. B, BTNL2 expression in uveal melanoma cell lines. C, BTNL2 expression in human macrophages generated from 5 donors. Δct indicates difference of threshold cycles. Error bars indicate standard deviation.

aPatients with amplification of chromosome 6.

bPatients who progressed to metastasis.

cCell lines with monosomy of chromosome 3.

dCell lines derived from the same patient.

eP < .05 by paired t test.

fP < .001 by paired t test.

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Figure 2.
Expression of M1- and M2-Polarized Macrophage-Specific Genes in Uveal Melanoma Samples

Hierarchical clustering shows 2 gene clusters with differential expression relative to the expression data for each single gene in all samples analyzed; blue indicates the value is below the mean, red indicates it is above the mean, and white indicates it is at the mean. One cluster is enriched for patients who progressed to metastasis; green indicates metastasis and yellow indicates no metastasis.

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Figure 3.
Survival According to the M1- and M2-Polarization Multigene Score

A multigene score was calculated by Cox regression analysis using differentially expressed genes, and samples were classified according to the median value of the multigene score in high-risk (blue) and low-risk (orange) patients.

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