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RESEARCH PRODUCT

Abstract 4107: Targeted re-sequencing of neuroblastoma tumors reveals chromosomal rearrangements that involve the Anaplastic Lymphoma Kinase (ALK) gene.

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Abstract Neuroblastoma (NBL) is a cancer of early childhood arising from the developing sympathetic nervous system. NBL tumors display a broad clinical and biological heterogeneity, ranging from highly aggressive tumors with fatal outcome to tumors with spontaneous regression. Recurrent mutations are mainly only observed in Anaplastic Lymphoma Kinase (ALK), which is involved in the pathogenesis of both familiar and sporadic NBL. ALK encodes a tyrosine kinase receptor with importance in neuronal development and was initially characterized in anaplastic large cell lymphoma from a translocation leading to the NPM-ALK fusion protein. Subsequent studies show that additional ALK chimeras have been generated with different fusion partners in various cancers. To date no ALK-containing rearrangement has been reported in primary NBL tumors although one study has described a NBL cell line with an ALK intragenic deletion leading to constitutive ALK kinase activity. This led us to screen for and further investigate possible ALK containing translocations in our NBL material. Genomic profiling of 350 NBL samples though SNP microarrays indicated ALK-rearrangement in four primary tumors and two cell lines (IMR32 and CLB-BAR) which were further analyzed through targeted re-sequencing of the common MYCN amplicon region (Chr2;14-18Mb) and the entire ALK gene. Briefly, detection of rearrangements was performed through solution-based enrichment followed pair-end sequencing of 400-500bp libraries. Junction sites were identified by manual sequence viewing on IGV and validated through PCR followed by Sanger sequencing. With this strategy we were able to determine translocation sites for all but one sample. In one tumor ALK was found to be translocated to GAB2 (chr11q14) causing loss of the first exon of respective gene. However, ALK and GAB2 were placed head-to-head with respect to transcription direction and this novel translocation is therefore unlikely to cause a chimeric protein. Another tumor showed translocation between ALK intron 4 and chr 4 subtelomeric region without known genes. Two NBL cell lines and one tumor had both MYCN and ALK amplification and sequencing revealed high level complexity of respective amplicon with multiple rearrangements. IMR32 show involvement of at least six different 2p-regions including MYCN and ALK exon 3-4. CLB-BAR has also multiple rearrangements including an ALK intragenic translocation causing loss of exon 4-11, ultimately resulting in an ALK protein with truncated extracellular domain. Here we show that ALK translocation sites could be deduced from targeted re-sequencing without previous knowledge about fusion partner. However, no translocations resulting in chimeric protein were detected. We were also able to detect MYCN amplicon junctions, which are highly unique for each patient and could be used for detection of minimal residual disease. Citation Format: Susanne M. Fransson, Anna Djos, Niloufar Javanmardi, Rosa Noguera, Ana Berbegall, Magnus Hansson, Per Kogner, Kristina Ruuth, Ruth Palmer, Bengt Hallberg, Tommy Martinsson. Targeted re-sequencing of neuroblastoma tumors reveals chromosomal rearrangements that involve the Anaplastic Lymphoma Kinase (ALK) gene. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4107. doi:10.1158/1538-7445.AM2013-4107