A genome-wide scan identifies NFIB as important for metastasis in osteosarcoma patients. L. Mirabello1, R. Koster1, L. Spector2, O. A. Panagiotou1, P. S. Meltzer3, B. Moriarty2, D. Largaespada2, N. Pankratz2, J. M. Gastier-Foster4, R. Gorlick5, C. Khanna3, A. M. Flanagan6,7, R. Tirabosco7, I. L. Andrulis8, N. Gokgoz8, J. S. Wunder8, A. Patiño-Garcia9, F. Lecanda9, L. Sierrasesúmaga9, S. R. C. de Toledo10, A. S. Petrilli10, M. Serra11, C. Hattinger11, P. Picci11, S. Wacholder1, L. Helman3, M. Yeager12, R. N. Hoover1, S. J. Chanock1, S. A. Savage1 1) Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA; 2) University of Minnesota, 420 Delaware Street, Minneapolis, MN, USA; 3) Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA; 4) Nationwide Children's Hospital, and The Ohio State University Department of Pathology and Pediatrics, 700 Children's Dr., C0988A, Columbus, OH, USA; 5) Albert Einstein College of Medicine, The Children's Hospital at Montefiore, 3415 Bainbridge Avenue, Rosenthal Room 300, Bronx, NY, USA; 6) UCL Cancer Institute, Huntley Street, London WC1E 6BT, UK; 7) Royal National Orthopaedic Hospital NHS Trust, Stanmore, Middlesex HA7 4LP, UK; 8) University of Toronto, Lunenfeld-Tanenbaum Research Institute, Mt Sinai Hospital, 600 University Ave., Toronto, Ontario, Canada, M5G 1X5; 9) Department Of Pediatrics, University Clinic of Navarra, Universidad de Navarra, Pío XII 36, 31080 Pamplona, Spain; 10) Pediatric Oncology Institute, GRAACC/UNIFESP, Rua Botucatu, 743 8º andar Laboratorio de Genética, CEP 04023-061 São Paulo SP Brazil; 11) Laboratory of Experimental Oncology, Orthopaedic Rizzoli Institute, Bologna, Italy; 12) Cancer Genomics Research Laboratory, Leidos Biomedical Research, Frederick National Laboratory for Cancer Research, Frederick, MD, USA.

   Osteosarcoma (OS), the most common primary bone malignancy, typically occurs in adolescents and young adults. We recently completed the first international, multi-institutional genome-wide association study (GWAS) of OS and identified two novel loci associated with susceptibility to OS. A defining feature of OS is the high rate of metastasis to distant secondary sites, and patients presenting with metastases have a very poor prognosis. We have assembled clinical outcome data on OS cases accrued for our previous GWAS, and independent replication sets, to conduct the first GWAS of metastasis in 860 OS cases. Genotyping of germline genomic DNA was conducted using the Illumina OmniExpress SNP chip and quality control filters applied. Associations between SNPs and the risk of metastasis at diagnosis were estimated using an adjusted logistic regression log-additive genetic model. We identified one locus associated with metastasis that approached genome-wide significance in our discovery analysis of 541 European cases. Specifically, six linked intronic SNPs in the nuclear factor I/B gene (NFIB) on chromosomes 9p24.1 were significantly associated with an increased risk of metastasis (rs2890982, OR 2.65, P=5.8x10-7). To further explore this locus, we imputed SNPs across a 1 Mb region centered on the index SNP. A subsequent adjusted association analysis identified new signals that were substantially stronger than the genotyped SNPs. The top SNPs and the imputed region were replicated in three independent OS populations (319 cases). In the fixed effects meta-analysis of 860 cases, the 9p24.1 locus was strongly associated with susceptibility to metastasis at diagnosis (OR 2.49, 95% CI 1.78-3.52, P=4.6x10-8). We genotyped two of the linked NFIB SNPs and examined NFIB expression in 15 human OS cell lines. The variant allele was associated with a significant change in expression (P=0.006). We further performed proliferation and migration assays using human OS cell lines and siRNA knockdown of NFIB, and observed a significant change in migration and proliferation after knockdown of NFIB. We have identified a novel locus associated with risk of metastasis at diagnosis in OS patients, and our data suggests that variation in the NFIB gene is important for metastasis. We are further evaluating the function of this region and other top regions to advance our understanding of the role of germline genetics in OS metastasis.

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