Abstract 4977: Targeted next-generation sequencing of DNA regions proximal to a conserved GXGXXG signaling motif enables discovery of a novel C6orf204-PDGFRβ fusion in a patient with T-ALL and eosinophilia
Authors: Chmielecki Juliann , Peifer Martin , Viale Agnes , Hutchinson Kathernie , Giltnane Jennifer , Socci Nicholas D. , Yenamandra Ashwini , Jagasia Madan , Kim Annette S. , Dave Utpal , Thomas Roman K. , Pao William 
Published: June 2012

Tyrosine kinase (TK) fusion proteins result from genomic rearrangements that juxtapose TK signaling domains with various upstream partners. Since tumor cells can become dependent upon the resultant constitutive kinase activity for tumorigenesis and survival, TK fusions serve as attractive therapeutic targets. However, identification of TK fusions in cancers has been hindered by experimental techniques that lack sufficient sensitivity and throughput. To overcome these challenges, we developed a screen to systematically identify TK fusions in tumor DNA. This method involves selective capture of regions upstream of GXGXXG kinase motifs where TK fusion breakpoints are likely to occur followed by next generation sequencing and custom computational analysis. This approach was previously validated using cell line DNA (Chmielecki et al, ‘10). Here, we used an improved version of our platform to identify a novel TK fusion using DNA isolated from whole blood of a patient with recurrent precursor T lymphoblastic lymphoma (T-ALL) who presented with new leukocytosis and bone marrow findings compatible with a myeloproliferative neoplasm (MPN) with eosinophilia. Cytogenetic analysis of a bone marrow aspirate prior to treatment for the patient's MPN revealed a karyotype with a t(5;6)(q33-34;q23). At recurrence, a 5q33/PDGFRβ rearrangement with a breakpoint proximal to the MYB locus on chromosome 6 was identified with fluorescence in situ hybridization (FISH). Prior to allogeneic hematopoietic cell transplantation, a brief trial of imatinib confirmed responsiveness of eosinophilia. To identify the exact fusion breakpoint, 3 µg of whole blood genomic DNA was subjected to genomic capture followed by paired-end SOLiD sequencing. Fusion candidates were validated by direct sequencing of PCR products generated with breakpoint spanning primers. Analysis of the recovered sequences identified a fusion involving c6orf204 upstream of the PDGFRβ TK domain. 5’-RACE confirmed expression of c6orf204-PDGFRβ mRNA in the patient's blood cells. Motif analysis of the 5’ c6orf204 sequence present within the fusion revealed a coiled-coil domain, which likely facilitates dimerization of the kinase, as observed in other TK fusions (e.g. BCR-ABL). Interestingly, retrospective FISH studies of the patient's diagnostic lymph node involved by T-ALL also revealed the presence of a PDGFRβ rearrangement. These data suggest that the rearrangement occurred in a multi-potent progenitor cell that gave rise to both the T-ALL and MPN. This finding is analogous to 8p MPNs involving FGFR1, but has not yet been reported with PDGFRβ. Further biochemical analysis of this TK fusion is currently underway, and additional tumor samples are undergoing screening for other novel TK fusions. This work was supported by an SU2C-AACR Innovative Research Grant to WP.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4977. doi:10.1158/1538-7445.AM2011-4977