Next Generation Sequencing Identifies Novel Oncogene Alterations In CRLF2-rearranged Precursor B-Cell Acute Lymphoblastic Leukemia

Abstract 472

We and others recently identified locus rearrangements involving the type I cytokine receptor subunit CRLF2 (also known as thymic stromal lymphopoietin receptor (TSLPR)) in 5–7% of all adult and pediatric B-cell precursor acute lymphoblastic leukemia (B-ALL). CRLF2 rearrangement places full-length CRLF2 under alternate transcriptional control, and can either result from an intrachromosomal CRLF2-P2RY8 deletion or from a CRLF2-IGH translocation. Prognosis for CRLF2-rearranged B-ALL is particularly poor, suggesting that a sizable fraction of relapsed and ultimately fatal B-ALL harbor CRLF2 rearrangements. Approximately 50% of CRLF2-rearranged B-ALL harbor mutations in JAK2 (and rarely JAK1) that cluster around JAK2 Arg683. A separate 15% have a CRLF2 F232C mutation that promotes constitutive homodimerization and signaling. In the remaining cases, the driver of CRLF2 signaling is not known. To identify additional genetic alterations that contribute to CRLF2-mediated leukemogenesis, we performed next-generation sequencing on 3 CRLF2-rearranged B-ALL specimens, including one with CRLF2 F232C (#536). Exome libraries were assembled from bone marrow specimens with greater than 90% B-ALL involvement and from paired remission bone marrows. Transcriptome sequencing was also performed on cDNA isolated from the involved marrow of patient #536 to: 1) confirm the findings from exome sequencing, 2) focus on transcribed genes and 3) identify possible RNA editing events. Sequencing utilized the SOLiD (Sequencing by Oligonucleotide Ligation and Detection; Applied BioSystems) platform, with a target recovery of greater than 40 million (transcriptome) and 25 million (exome) uniquely mapping reads per sample. Mutations were considered high confidence if a minimum of 3 individual reads identified the same mutation within the involved specimen and the mutation was not identified in any reads from the remission specimen (minimum 10 reads at that base-pair). Paired exome sequencing of germline and involved marrow specimens identified 129, 297 and 630 high-confidence, non-synonymous, somatic mutations in the three samples. Of the total 1056 mutations, 1023 (96.9%) were missense and the remaining 33 (3.1%) were nonsense mutations. The expected mutation in CRLF2 F232C (#536) was recovered, as was a novel JAK2 mutation from a sample previously thought to be JAK2 wild-type (#002). Comparison between transcriptome and exome sequence markedly reduced the number of potential “driver” alterations, i.e., nonsynonymous coding mutations expressed in the tumor but not present in the remission exome. For example, from the 129 somatic alterations in the involved exome from #536, only 15 were recovered from the involved transcriptome. Sequence alterations affected genes known to be involved in histone modification, oxygen metabolism, and steroid responsiveness. To our knowledge, none of the identified mutations have previously been described. One mutation in a gene involved in E2F transcription was observed in two of three cases. In addition, 2,428 coding sequence mutations were identified from the involved marrow transcriptome of #536 that were not present in the involved marrow exome, possibly indicating a high rate of RNA editing. Additional sequence analysis and molecular epidemiology of the novel sequence alterations in CRLF2-rearranged and —unrearranged B-ALL is underway.