Elsevier

Cancer Genetics

Volume 205, Issues 7–8, July–August 2012, Pages 391-404
Cancer Genetics

Original article
Molecular inversion probe analysis detects novel copy number alterations in Ewing sarcoma

https://doi.org/10.1016/j.cancergen.2012.05.012Get rights and content

Ewing sarcoma (ES) is the second most common bone tumor in children and young adults, with dismal outcomes for metastatic and relapsed disease. To better understand the molecular pathogenesis of ES and to identify new prognostic markers, we used molecular inversion probes (MIPs) to evaluate copy number alterations (CNAs) and loss of heterozygosity (LOH) in formalin-fixed paraffin-embedded (FFPE) samples, which included 40 ES primary tumors and 12 ES metastatic lesions. CNAs were correlated with clinical features and outcome, and validated by immunohistochemistry (IHC). We identified previously reported CNAs, in addition to SMARCB1 (INI1/SNF5) homozygous loss and copy neutral LOH. IHC confirmed SMARCB1 protein loss in 7–10% of clinically diagnosed ES tumors in three separate cohorts (University of Utah [N = 40], Children's Oncology Group [N = 31], and University of Michigan [N = 55]). A multifactor copy number (MCN)-index was highly predictive of overall survival (39% vs. 100%, P < 0.001). We also identified RELN gene deletions unique to 25% of ES metastatic samples. In summary, we identified both known and novel CNAs using MIP technology for the first time in FFPE samples from patients with ES. CNAs detected by microarray correlate with outcome and may be useful for risk stratification in future clinical trials.

Section snippets

Cell lines

Genomic DNA was extracted from five ES cell lines (A673, EWS502, SKNMC, TC32, TC71) and from six healthy individuals as controls. Cell line DNA was run on the Agilent aCGH 44B array (44K tiled probes; Agilent Technologies, Santa Clara, CA) per manufacturer's directions, and 37 ng of cell line and control DNA was submitted for MIP analysis using an initial customized panel of 24,000 cancer gene probes (Affymetrix Research Services Laboratory, Santa Clara, CA). Data from the Agilent aCGH and MIP

Cell lines

To establish the validity of the MIP array, we compared ES cell lines (n = 5) run on the Agilent 44K aCGH to the same cell lines run on the original MIP 24K platform. The same gains and losses were seen across both platforms (Supplementary Materials Figure 1). The cell lines demonstrated similar CNAs as reported in the literature. Trisomy 8 appeared in 3/5 of cell lines with an additional focal high gain encompassing the MYC gene. 1q amplification was seen in 2/5 of cell lines. Trisomies 12 and

Discussion

The introduction of genome-wide, high-resolution SNP microarray technology has led to the discovery of CNAs in a variety of common tumors such as breast cancer (43), ovarian cancer (44), lung cancer (45), glioblastoma (46), neuroblastoma 47, 48, pediatric astrocytoma 28, 49, and acute lymphoblastic leukemia (ALL) 29, 50, 51. These copy number studies identified specific genes and molecular pathways that have emerged as prognostic markers and therapeutic targets, such as the IKZF1 gene and the

Acknowledgments

J.D.S. received support for this research from St. Baldrick's Foundation, Sarcoma Alliance for Research through Collaboration (SARC) Career Development Award, Damon Runyon Clinical Investigator Award, and the Eunice Kennedy Shriver Children's Health Research Career Development Award NICHD 5K12HD001410. S.L.L. is supported by the NIH (R21 CA138295, R01 CA140394), the Terri Anna Perine Sarcoma Fund, and the University of Utah Department of Pediatrics and Huntsman Cancer Institute/Huntsman Cancer

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