CSF3R T618I co-occurs with mutations of splicing and epigenetic genes and with a new PIM3 truncated fusion gene in chronic neutrophilic leukemia

Mutations in CSF3R have been recently defined as the common genetic event in patients with myeloid neoplasms, including the rare entity known as chronic neutrophilic leukemia (CNL), becoming a potentially useful biomarker for diagnosing and therapy target. CSF3R encodes the transmembrane receptor for granulocyte colony-stimulating factor (G-CSF; CSF3), which provides the proliferative and survival signal for granulocytes and also contributes to their differentiation and function. Although there are several studies on massive next-generation sequencing of myeloid disorders, not a single comprehensive study has been reported in CNL. Here, we used whole-exome sequencing (WES) and RNA sequencing (RNA-seq) to identify new candidate genes to the disease pathogenesis of an index CNL patient. A 66-year-old man was diagnosed with CNL, according to the 2008 World Health Organization (WHO) classification. At diagnosis, the patient presented peripheral blood leukocytosis (66 10/l), segmented neutrophils and band forms were 91.5% of the white blood cells counts (WBCs), immature granulocytes were o10% of WBCs and myeloblasts were o1%. The aspirate showed a hypercellular bone marrow (BM) with neutrophilic granulocytes increased in number and percentage and myeloblasts 0.5% of WBCs. No dysplastic features were observed in the myeloid lineages. His Zubrod Performance Status (ECOG) was 1. A GTG-banding chromosome analysis revealed a normal karyotype (46,XY[20]), and molecular biology studies were negative for BCR-ABL1 transcripts and JAK2 V617F mutation. The patient was treated with hydroxyurea but, unfortunately, died 7 months after the diagnosis due to an intensification of the disease. To improve our understanding of the genes involved in the pathogenesis of CNL, WES was performed on matched tumor and oral mucosa cell (germline) samples from the patient. Candidate somatic mutations were identified using RUbioSeq software. The bioinformatics analysis and the filtering steps to identify the coding variants are detailed in the Supplementary Material. In total, we found 1437 candidate variants; among them, 797 were somatic mutations (412 were intronic, intergenic, affecting non-conding-RNA or untranscribed regions and 385 were exonic). From the 385 exonic variants, we selected only those variants within coding regions that, after passing sequencing depth and quality filters, were, frameshift, stop gain/loss and non-synonymous amino acid changes predicted to produce a deleterious effect in the protein structure, resulting in 56 single-nucleototide variants (SNVs) and small insertions/deletions (indels). We selected 24 for further validation by Haloplex/Ion Torrent. In addition to the CSF3R p.Thr618Ile mutation, we validated mutations in U2AF1, TET2, LUC7L2 and ASXL1 (Figure 1a and Table 1). The current study, first, confirms the observations by Maxon et al. and Pardanani et al. regarding the association between CNL and CSF3R mutations. Second, it presents a complete picture of the mutational profiling of CNL, certainly more complex than expected from these previous reports. In fact, we found and validated mutations affecting both splicing machinery and epigenetic genes. Kosmider et al., very recently, showed that CSF3R somatic mutations can be identified in B4% of chronic myelomonocytic leukemias. These mutations, which affect distinct residues in CSF3R as compared with CNL, are frequently associated with mutations in the ASXL1 gene and have a poor prognostic impact on overall and acute myeloid leukemia (AML)-free survival. Together, these data indicate that CNL genome had a combination of few mutations with a pattern of cooperation with a strong biological relationship among genes and categories, similar to AML. Along this line of cooperating mutations on epigenetic genes, we also found in our CNL patient mutated copies of ASXL1 and TET2 genes. The variant allelic frequency of LUC7L2 mutation was found to be high (more than 95%). We previously described mutations of this gene in myelodysplastic syndrome (MDS). As this gene is located in the 7q region, a frequently deleted chromosomal region in myeloid leukemias, we decided to investigate whether a critical deletion or a loss of heterozygosity (LOH) affecting this genomic region was also present in the patient. To study this phenomenon, we interrogate our WES data for the LOH across the whole genome of the sample. Interestingly, we found an LOH of 53.2Mb in chromosome 7q including the locus of the LUC7L2 gene. Allele frequencies of each SNP along chromosome 7 are shown in Figure 1b and Supplementary Figure 4. As no del(7q) was detected with metaphase cytogenetics, our study demonstrates for the first time mutations in LUC7L2 accompanied by a copy-neutral LOH (uniparental disomy) in 7q in a patient with an aggressive CNL phenotype. To further evaluate the biological consequences of this homozygous mutation, we explored LUC7L2 expression in the BM cells of the patient and in some myeloid leukemia cell lines (Figure 1c). By using real-time PCR, we observed a downregulation in the expression of LUC7L2 in the patient cells compared with normal granulocytes, as well as a general downregulation in myeloid leukemia cell lines. To determine the functional consequences of the mutations in LUC7L2 and U2AF1, genes involved in the splicing machinery, in the proper splicing process, we performed RNA-seq in our index patient as well as in CD34þ cells from a normal control BM. Although no clear genome-wide increase in intron retention was observed in the patient, as previously reported by us in some MDS cases, we found an altered pattern of splicing in the mRNA species transcribed from the RUNX1 gene. At the 30 splice site of RUNX1 intron 5, the un-spliced reads were almost three times more frequent in the mutated patient than in the normal control (Supplementary Figure 1). Because of the large numbers of diverse mutations in the splicing machinery, larger studies will be needed to fully evaluate the impact of these mutations in splicing. In relation with the effects of an aberrant RNA splicing due to the presence of two mutations in the genes responsible for these processes, we used RNA-seq data to investigate the presence of aberrant fusion transcripts in our CNL patient. In fact, we identified a chimeric transcript involving the PIM3 and SCO2 genes (both were located on 22q13.33), which was the result of an Citation: Blood Cancer Journal (2013) 3, e158; doi:10.1038/bcj.2013.55 & 2013 Macmillan Publishers Limited All rights reserved 2044-5385/13