Molecular Inversion Probes (MIPs) Identify Novel Areas of Allelic Imbalance in Childhood Leukemia.

Abstract Background: Leukemia accounts for over 30% of newly diagnosed childhood malignancies, and is the leading cause of death for children with cancer. Genomic instability events contribute to tumorigenesis and have been used to classify and risk stratify adult and pediatric cancers. Molecular Inversion Probes (MIPs) analyze genetic target sequences in parallel at the highest genomic resolution, and can detect both gene copy number and loss of heterozygosity (LOH) events in clinical samples. Studying pediatric leukemia samples with MIP technology may identify new molecular alterations that could prove useful in risk stratification and discovery of new therapeutic targets for childhood leukemia. Objective: To use MIP technology to identify novel areas of allelic imbalance in childhood leukemia. Methods: DNA was extracted from leukemia blasts at diagnosis (n=45, 23 pre-B ALL, 14 AML, 7 pre-T ALL, 1 Burkitt's). DNA was also extracted from normal peripheral blood collected at remission to use as paired germline controls. The MIP assay was run with a customized Affymetrix 24K Cancer Panel (representing oncogenes, tumor suppressor, DNA repair, cell growth, and metabolism genes). DNA required for this assay was limited to 75 ng per sample. Copy number changes and LOH were identified by comparing probe signal intensity between leukemia and normal germline samples. Clinical cytogenetic data (karyotype and FISH analysis) was used as a control to confirm findings from known areas of allelic imbalance. Results: Each leukemia sample had unique patterns of allelic imbalance distributed across all chromosomes. MIPs identified all clinically reported cytogenetic copy number changes for each sample, in addition to areas of allelic imbalance not clinically reported. Samples had recurring areas of copy number changes and LOH events shared by all leukemia types. MIPs detected areas of allelic imbalance in both previously described and novel genes. Areas of recurring genomic deletions included: ATR (3q23), TLX3 (5q35.1), ADRB3 (8p12), CDKN2A (9p21.3), DOCK8 (9p24.3), PAX5 (9p13.2), PTPN11 (12q24.13), C3AR1 (12p13.31), TCRA (14q11.2), AKT1 (q14q32.33). Areas of recurring genomic amplification included: SLC2A9 (4p16.1), RAI14 (5p13.2), CDH12 (5p14.3), PMCHL1(5p14.3), AURKB (17p13.1). These findings are being validated with Quantitative Real-Time PCR. Conclusions: MIPs represent a novel genomic technology that can identify previously unreported gene copy number and LOH events in childhood leukemia. Unique and overlapping areas of allelic imbalance were found in both childhood ALL and AML clinical samples. The shared genomic regions of allelic imbalance between different leukemia types may represent a common molecular mechanism of leukemogenesis that warrants further investigation. Analysis of more childhood leukemia samples through Pediatric Cooperative Groups may help to determine how common these areas of allelic imbalance are in children and whether they are of prognostic significance. Further exploration of these copy number and LOH events may help us to better understand the biology of childhood leukemia and its clinical behavior.