High Resolution Genome-Wide Copy Number Analysis of Pediatric Burkitt Lymphoma Identifies Copy Number Alterations In the Majority of Patients

Abstract 3123

Although cure rates are high for pediatric Burkitt lymphoma (BL), a subset of patients relapses and succumbs to the disease. BL is characterized by translocation of the MYC gene with an immunoglobulin gene, but secondary changes including gain of 1q, gain of 13q, loss of 13q, loss of 17p, and others have been described by both conventional cytogenetic and oligo array CGH approaches. Secondary changes may contribute to the clinical heterogeneity of BL as evidenced by the fact that loss of 13q is associated with a worse prognosis in pediatric BL (Poirel et al., Leukemia 23:323, 2009). However, high resolution, genome-wide copy number analysis has not yet been reported in pediatric BL. The objective of this study was to identify copy number alterations (CNAs) in pediatric BL using a genome-wide approach, and to examine the relationship between CNAs and clinical parameters including outcome. After institutional review board approval, we identified 30 pediatric BL patients treated at Primary Children's Medical Center (n=25, Salt Lake City, UT) and Penn State Hershey Medical Center (n=5, Hershey, PA) with available formalin-fixed, paraffin-embedded (FFPE) diagnostic biopsy specimens. Age, site, and gender data were available for all specimens, and 22/24 of the Utah patients were treated according to the COG 5961 protocol with full clinical information and follow-up available. DNA was isolated from FFPE biopsies containing at least 80% tumor. In addition, germline DNA was isolated from negative staging bone marrow clot sections on Utah patients (n=25) to serve as a pooled normal reference and to provide germline copy number variation data on individual patients. Tumor and paired normal DNA was submitted for Molecular Inversion Probe (MIP) assay (330K Cancer Panel, Affymetrix, Santa Clara, CA). The Nexus Copy Number (BioDiscovery, El Segundo, CA) software package was used to analyze the MIP data with the following stringent call criteria: SNPRank segmentation, 5.0E-4 significance threshold, 1000 kb maximum contiguous probe spacing, minimum of 5 probes per segment, gains ≥ 2.7 copy number value, and loss ≤ 1.3 copy number value. Patients included 23 males and 7 females with a mean age of 8.0 years (range 2 – 18). At presentation, lactate dehydrogenase (LDH) levels ranged from 443 – 13851 U/L and uric acid levels ranged from 1.5 – 13 mg/dL. Patients were Murphy stage I (n=1), II (n=9), III (n=13), and IV (n=2). A total of three patients relapsed (one stage II patient and two stage III patients) and three died (the two stage III patients that relapsed and a different stage II patient who did not have a complete clinical response). 27 of the 30 tumor samples and 22/25 paired normals had adequate DNA for MIP analysis. The 3 tumor samples without adequate DNA were clinically similar to the others. We identified a total of 103 CNAs (defined as change seen in the same cytoband in 1 or more patients), which included 63 gains and 40 copy number losses. 23/27 cases (85%) had at least one gain or loss. We identified 21 recurrent CNAs (same cytoband affected in 2 or more patients), which included 14 gains and 7 losses. We found gains of 1q in 10/27 patients (37%), gains of 13q in 4/27 patients (15%) and losses of 17p in 3/27 patients (11%). Despite a relatively small sample size, deletion of 17p13 was significantly associated with relapse (p=0.041). To our knowledge, this is the first report of high-resolution genome-wide copy number analysis of pediatric BL. Furthermore, we show for the first time that FFPE archived materials can be used for high resolution gene copy number analysis in lymphomas. We identified CNAs in 85% of the pediatric BL cases, which include previously described changes of 1q, 13q, and 17p. Although sample size limited statistical power, deletion of the 17p13 locus was associated with relapse. We plan to extend these studies in a larger sample of patients to evaluate the potential prognostic significance of both the 17p13 locus deletion as well as additional recurrent CNAs.