Genome-wide association study identifies novel susceptibility loci for KIT D816V positive mastocytosis.

Mastocytosis is a rare myeloid neoplasm characterized by uncontrolled expansion of mast cells, driven in >80% of affected individuals by acquisition of the KIT D816V mutation. To explore the hypothesis that inherited variation predisposes to mastocytosis, we performed a two-stage genome-wide association study, analyzing 1,035 individuals with KIT D816V positive disease and 17,960 healthy control individuals from five European populations. After quality control, we tested 592,007 SNPs at stage 1 and 75 SNPs at stage 2 for association by using logistic regression and performed a fixed effects meta-analysis to combine evidence across the two stages. From the meta-analysis, we identified three intergenic SNPs associated with mastocytosis that achieved genome-wide significance without heterogeneity between cohorts: rs4616402 (pmeta = 1.37 × 10-15, OR = 1.52), rs4662380 (pmeta = 2.11 × 10-12, OR = 1.46), and rs13077541 (pmeta = 2.10 × 10-9, OR = 1.33). Expression quantitative trait analyses demonstrated that rs4616402 is associated with the expression of CEBPA (peQTL = 2.3 × 10-14), a gene encoding a transcription factor known to play a critical role in myelopoiesis. The role of the other two SNPs is less clear: rs4662380 is associated with expression of the long non-coding RNA gene TEX41 (peQTL = 2.55 × 10-11), whereas rs13077541 is associated with the expression of TBL1XR1, which encodes transducin (ß)-like 1 X-linked receptor 1 (peQTL = 5.70 × 10-8). In individuals with available data and non-advanced disease, rs4616402 was associated with age at presentation (p = 0.009; beta = 4.41; n = 422). Additional focused analysis identified suggestive associations between mastocytosis and genetic variation at TERT, TPSAB1/TPSB2, and IL13. These findings demonstrate that multiple germline variants predispose to KIT D816V positive mastocytosis and provide novel avenues for functional investigation.

Limited duration of complete remission on ruxolitinib in myeloid neoplasms with PCM1-JAK2 and BCR-JAK2 fusion genes.

Rearrangements of chromosome band 9p24 are known to be associated with JAK2 fusion genes, e.g., t(8;9)(p22;p24) with a PCM1-JAK2 and t(9;22)(p24;q11) with a BCR-JAK2 fusion gene, respectively. In association with myeloid neoplasms, the clinical course is aggressive, and in absence of effective conventional treatment options, long-term remission is usually only observed after allogeneic stem cell transplantation (ASCT). With the discovery of inhibitors of the JAK2 tyrosine kinase and based on encouraging in vitro and in vivo data, we treated two male patients with myeloid neoplasms and a PCM1-JAK2 or a BCR-JAK2 fusion gene, respectively, with the JAK1/JAK2 inhibitor ruxolitinib. After 12 months of treatment, both patients achieved a complete clinical, hematologic, and cytogenetic response. Non-hematologic toxicity was only grade 1 while no hematologic toxicity was observed. However, remission in both patients was only short-term, with relapse occurring after 18 and 24 months, respectively, making ASCT indispensable in both cases. This data highlight (1) the ongoing importance of cytogenetic analysis for the diagnostic work-up of myeloid neoplasms as it may guide targeted therapy and (2) remission under ruxolitinib may only be short-termed in JAK2 fusion genes but it may be an important bridging therapy prior to ASCT.

Inactivation of polycomb repressive complex 2 components in myeloproliferative and myelodysplastic/myeloproliferative neoplasms.

The polycomb repressive complex 2 (PRC2) is a highly conserved histone H3 lysine 27 methyltransferase that regulates the expression of developmental genes. Inactivating mutations of the catalytic component of PRC2, EZH2, are seen in myeloid disorders. We reasoned that the other 2 core PRC2 components, SUZ12 and EED, may also be mutational targets in these diseases, as well as associated factors such as JARID2. SUZ12 mutations were identified in 1 of 2 patients with myelodysplastic syndrome/myeloproliferative neoplasms with 17q acquired uniparental disomy and in 2 of 2 myelofibrosis cases with focal 17q11 deletions. All 3 were missense mutations affecting the highly conserved VEFS domain. Analysis of a further 146 myelodysplastic syndrome/myeloproliferative neoplasm patients revealed an additional VEFS domain mutant, yielding a total mutation frequency of 1.4% (2 of 148). We did not find mutations of JARID2 or EED in association with acquired uniparental disomy for chromosome 6p or 11q, respectively; however, screening unselected cases identified missense mutations in EED (1 of 148; 1%) and JARID2 (3 of 148; 2%). All 3 SUZ12 mutations tested and the EED mutation reduced PRC2 histone methyltransferase activity in vitro, demonstrating that PRC2 function may be compromised in myeloid disorders by mutation of distinct genes.

Ponatinib as targeted therapy for FGFR1 fusions associated with the 8p11 myeloproliferative syndrome.

The 8p11 myeloproliferative syndrome is a rare, aggressive myeloproliferative neoplasm characterized by constitutively active FGFR1 fusion proteins that arise from specific chromosomal translocations and which drive aberrant proliferation. Although FGFR1 inhibitors have shown in vitro activity against FGFR1 fusions, none are in use clinically and there is a need to assess additional compounds as potential therapy. Here we use cell lines and primary cells to investigate ponatinib (AP24534). Ponatinib-treated Ba/F3 cells transformed by ZMYM2-FGFR1 and BCR-FGFR1 and the FGFR1OP2-FGFR1 positive KG1A cell line showed reduced proliferation and decreased survival when compared to control cells. Inhibition induced apoptosis and reduced phosphorylation of the FGFR1 fusion proteins and substrates. Ponatinib-treated cells from 8p11 myeloproliferative syndrome patients (n=5) showed reduced colony growth compared to controls. In one evaluable patient, ponatinib specifically reduced numbers of FGFR1-fusion gene positive colonies. Ponatinib, therefore, shows considerable promise for the treatment of patients with 8p11 myeloproliferative syndrome.

Ruxolitinib as potential targeted therapy for patients with JAK2 rearrangements.

JAK2 fusion genes are rare but recurrent abnormalities associated with diverse, clinically heterogeneous hematologic malignancies. Here we assess the JAK1/2 inhibitor ruxolitinib as therapy for patients with JAK2-rearrangement associated myeloproliferative neoplasms (MPN). Ruxolitinib-treated Ba/F3 cells transformed to IL3 independence by ETV6-JAK2 showed reduced proliferation and survival (IC(50) = 370 nM) compared with KG1A or Ba/F3 cells transformed by BCR-ABL1, SPBN1-FLT3 and ZMYM2-FGFR1 (IC(50) > 10 µM for all). Inhibition was associated with reduced phosphorylation of ETV6-JAK2, ERK, STAT5 and AKT. Primary cell growth from 2 patients with JAK2 rearrangement and one patient with JAK2 amplification was assessed in methylcellulose assays. Reduced colony growth was seen for all patients in ruxolitinib-treated cultures compared with healthy controls (n=7). Fluorescence in situ hybridization showed reduced growth of JAK2-rearrangement positive colonies compared to JAK2-rearrangement negative colonies. Our data, therefore, provide evidence that ruxolitinib is a promising therapy for treatment of patients with JAK2 fusion genes.

The JAK2 46/1 haplotype predisposes to MPL-mutated myeloproliferative neoplasms.

The 46/1 JAK2 haplotype predisposes to V617F-positive myeloproliferative neoplasms, but the underlying mechanism is obscure. We analyzed essential thrombocythemia patients entered into the PT-1 studies and, as expected, found that 46/1 was overrepresented in V617F-positive cases (n = 404) versus controls (n = 1492, P = 3.9 x 10(-11)). The 46/1 haplotype was also overrepresented in cases without V617F (n = 347, P = .009), with an excess seen for both MPL exon 10 mutated and V617F, MPL exon 10 nonmutated cases. Analysis of further MPL-positive, V617F-negative cases confirmed an excess of 46/1 (n = 176, P = .002), but no association between MPL mutations and MPL haplotype was seen. An excess of 46/1 was also seen in JAK2 exon 12 mutated cases (n = 69, P = .002), and these mutations preferentially arose on the 46/1 chromosome (P = .029). No association between 46/1 and clinical or laboratory features was seen in the PT-1 cohort either with or without V617F. The excess of 46/1 in JAK2 exon 12 cases is compatible with both the "hypermutability" and "fertile ground" hypotheses, but the excess in MPL-mutated cases argues against the former. No difference in sequence, splicing, or expression of JAK2 was found on 46/1 compared with other haplotypes, suggesting that any functional difference of JAK2 on 46/1, if it exists, must be relatively subtle.

Frequent CBL mutations associated with 11q acquired uniparental disomy in myeloproliferative neoplasms.

Recent evidence has demonstrated that acquired uniparental disomy (aUPD) is a novel mechanism by which pathogenetic mutations in cancer may be reduced to homozygosity. To help identify novel mutations in myeloproliferative neoplasms (MPNs), we performed a genome-wide single nucleotide polymorphism (SNP) screen to identify aUPD in 58 patients with atypical chronic myeloid leukemia (aCML; n = 30), JAK2 mutation-negative myelofibrosis (MF; n = 18), or JAK2 mutation-negative polycythemia vera (PV; n = 10). Stretches of homozygous, copy neutral SNP calls greater than 20Mb were seen in 10 (33%) aCML and 1 (6%) MF, but were absent in PV. In total, 7 different chromosomes were involved with 7q and 11q each affected in 10% of aCML cases. CBL mutations were identified in all 3 cases with 11q aUPD and analysis of 574 additional MPNs revealed a total of 27 CBL variants in 26 patients with aCML, myelofibrosis or chronic myelomonocytic leukemia. Most variants were missense substitutions in the RING or linker domains that abrogated CBL ubiquitin ligase activity and conferred a proliferative advantage to 32D cells overexpressing FLT3. We conclude that acquired, transforming CBL mutations are a novel and widespread pathogenetic abnormality in morphologically related, clinically aggressive MPNs.

Pathogenic variants causing ABL1 malformation syndrome cluster in a myristoyl-binding pocket and increase tyrosine kinase activity.

ABL1 is a proto-oncogene encoding a nonreceptor tyrosine kinase, best known in the somatic BCR-ABL fusion gene associated with chronic myeloid leukaemia. Recently, germline missense variants in ABL1 have been found to cause an autosomal dominant developmental syndrome with congenital heart disease, skeletal malformations and characteristic facies. Here, we describe a series of six new unrelated individuals with heterozygous missense variants in ABL1 (including four novel variants) identified via whole exome sequencing. All the affected individuals in this series recapitulate the phenotype of the ABL1 developmental syndrome and additionally we affirm that hearing impairment is a common feature of the condition. Four of the variants cluster in the myristoyl-binding pocket of ABL1, a region critical for auto-inhibitory regulation of the kinase domain. Bio-informatic analysis of transcript-wide conservation and germline/somatic variation reveals that this pocket region is subject to high missense constraint and evolutionary conservation. Functional work to investigate ABL1 kinase activity in vitro by transient transfection of HEK293T cells with variant ABL1 plasmid constructs revealed increased phosphorylation of ABL1-specific substrates compared to wild-type. The increased tyrosine kinase activity was suppressed by imatinib treatment. This case series of six new patients with germline heterozygous ABL1 missense variants further delineates the phenotypic spectrum of this condition and recognises microcephaly as a common finding. Our analysis supports an ABL1 gain-of-function mechanism due to loss of auto-inhibition, and demonstrates the potential for pharmacological inhibition using imatinib.

Fusion of PDGFRB to two distinct loci at 3p21 and a third at 12q13 in imatinib-responsive myeloproliferative neoplasms.

We identified four patients who presented with BCR-ABL1 negative myeloproliferative neoplasms and cytogenetically visible abnormalities of chromosome band 5q31-35. Fluorescence in situ hybridization indicated that the platelet-derived growth factor receptor beta gene (PDGFRB) was disrupted in all four cases and 5' rapid amplification of cDNA ends identified in-frame mRNA fusions between PDGFRB and WDR48 (3p21), GOLGA4 (3p21) and BIN2 (12q13). Strikingly, all three genes encode proteins involving intracellular trafficking. Imatinib, a known inhibitor of PDGFRbeta, selectively blocked the growth of t(3;5) myeloid colonies and produced clinically significant responses in all patients. We conclude that PDGFRB fuses to diverse partner genes in atypical myeloproliferative neoplasms (MPNs). Although very rare, identification of these fusions is critical for proper management of affected individuals.

A polymorphism associated with STAT3 expression and response of chronic myeloid leukemia to interferon α.

Interferon alpha (IFN) induces variable responses in chronic myeloid leukemia (CML), with 8-30% of early chronic phase cases achieving a complete cytogenetic response. We hypothesized that polymorphic differences in genes encoding IFN signal transduction components might account for different patient responses. We studied 174 IFN-treated patients, of whom 79 achieved less than 35% Philadelphia-chromosome (Ph) positive metaphases (responders) and 95 failed to show any cytogenetic response (more than 95% Ph-positive metaphases; non-responders). We compared 17 single nucleotide polymorphisms (SNPs) at IFNAR1, IFNAR2, JAK1, TYK2, STAT1, STAT3 and STAT5a/b between the two groups and found a significant difference for rs6503691, a SNP tightly linked to STAT5a, STAT5b and STAT3 (minor allele frequency 0.16 for non-responders; 0.06 for responders, P=0.007). Levels of STAT3 mRNA correlated with rs6503691 genotype (P<0.001) as assessed by real time quantitative PCR and therefore we conclude that rs6503691 is associated with the STAT3 expression levels and response of CML patients to IFN.

TFG, a target of chromosome translocations in lymphoma and soft tissue tumors, fuses to GPR128 in healthy individuals.

BACKGROUND: The formation of fusion genes plays roles in both oncogenesis and evolution by facilitating the acquisition of novel functions. Here we describe the first example of a human polymorphic in-frame fusion of two unrelated genes associated with a copy number variant. DESIGN AND METHODS: Array comparative genomic hybridization was used to identify cryptic oncogenic fusion genes. Fusion gene structure and origin was examined using molecular biological and computational methods. Phenotype associations were examined using PopGen cohorts. RESULTS: Targeted array comparative genomic hybridization to identify cryptic oncogenic fusion genes in patients with atypical myeloproliferative neoplasms identified a 111 kb amplification with breakpoints within the TRK-fused gene (TFG, a target of translocations in lymphoma and thyroid tumors) and G-protein-coupled receptor 128 (GPR128) resulting in an expressed in-frame TFG-GPR128 fusion transcript. The fusion gene was also identified in healthy individuals at a frequency of 0.02 (3/120). Normally both genes are in identical orientations with TFG immediately downstream of GPR128. In individuals with a copy number variant amplification, one or two copies of the TFG-GPR128 fusion are found between the two parental genes. The breakpoints share a region of microhomology, and haplotype and microsatellite analysis indicate a single ancestral origin. Analysis of PopGen cohorts showed no obvious phenotype association. An in silico search of EST databases found no other copy number variant amplification-associated fusion transcripts, suggesting that this is an uncommon event. Conclusions The finding of a polymorphic gene fusion in healthy individuals adds another layer to the complexity of human genome variation and emphasizes the importance of careful discrimination of oncogenic changes found in tumor samples from non-pathogenic normal variation.

Transcription factor mutations in myelodysplastic/myeloproliferative neoplasms.

BACKGROUND: Aberrant activation of tyrosine kinases, caused by either mutation or gene fusion, is of major importance for the development of many hematologic malignancies, particularly myeloproliferative neoplasms. We hypothesized that hitherto unrecognized, cytogenetically cryptic tyrosine kinase fusions may be common in non-classical or atypical myeloproliferative neoplasms and related myelodysplastic/myeloproliferative neoplasms. DESIGN AND METHODS: To detect genomic copy number changes associated with such fusions, we performed a systematic search in 68 patients using custom designed, targeted, high-resolution array comparative genomic hybridization. Arrays contained 44,000 oligonucleotide probes that targeted 500 genes including all 90 tyrosine kinases plus downstream tyrosine kinase signaling components, other translocation targets, transcription factors, and other factors known to be important for myelopoiesis. RESULTS: No abnormalities involving tyrosine kinases were detected; however, nine cytogenetically cryptic copy number imbalances were detected in seven patients, including hemizygous deletions of RUNX1 or CEBPA in two cases with atypical chronic myeloid leukemia. Mutation analysis of the remaining alleles revealed non-mutated RUNX1 and a frameshift insertion within CEBPA. A further mutation screen of 187 patients with myelodysplastic/myeloproliferative neoplasms identified RUNX1 mutations in 27 (14%) and CEBPA mutations in seven (4%) patients. Analysis of other transcription factors known to be frequently mutated in acute myeloid leukemia revealed NPM1 mutations in six (3%) and WT1 mutations in two (1%) patients with myelodysplastic/myeloproliferative neoplasms. Univariate analysis indicated that patients with mutations had a shorter overall survival (28 versus 44 months, P=0.019) compared with patients without mutations, with the prognosis for cases with CEBPA, NPM1 or WT1 mutations being particularly poor. CONCLUSIONS: We conclude that mutations of transcription and other nuclear factors are frequent in myelodysplastic/myeloproliferative neoplasms and are generally mutually exclusive. CEBPA, NPM1 or WT1 mutations may be associated with a poor prognosis, an observation that will need to be confirmed by detailed prospective studies.

EZH2 in Myeloid Malignancies.

Our understanding of the significance of epigenetic dysregulation in the pathogenesis of myeloid malignancies has greatly advanced in the past decade. Enhancer of Zeste Homolog 2 (EZH2) is the catalytic core component of the Polycomb Repressive Complex 2 (PRC2), which is responsible for gene silencing through trimethylation of H3K27. EZH2 dysregulation is highly tumorigenic and has been observed in various cancers, with EZH2 acting as an oncogene or a tumor-suppressor depending on cellular context. While loss-of-function mutations of EZH2 frequently affect patients with myelodysplastic/myeloproliferative neoplasms, myelodysplastic syndrome and myelofibrosis, cases of chronic myeloid leukemia (CML) seem to be largely characterized by EZH2 overexpression. A variety of other factors frequently aberrant in myeloid leukemia can affect PRC2 function and disease pathogenesis, including Additional Sex Combs Like 1 (ASXL1) and splicing gene mutations. As the genetic background of myeloid malignancies is largely heterogeneous, it is not surprising that EZH2 mutations act in conjunction with other aberrations. Since EZH2 mutations are considered to be early events in disease pathogenesis, they are of therapeutic interest to researchers, though targeting of EZH2 loss-of-function does present unique challenges. Preliminary research indicates that combined tyrosine kinase inhibitor (TKI) and EZH2 inhibitor therapy may provide a strategy to eliminate the residual disease burden in CML to allow patients to remain in treatment-free remission.

Mutational mechanisms of EZH2 inactivation in myeloid neoplasms.

EZH2, a component of the polycomb repressive complex 2, catalyses the trimethylation of histone H3 lysine 27, a chromatin mark associated with transcriptional repression. EZH2 loss-of-function mutations are seen in myeloid neoplasms and are associated with an adverse prognosis. Missense mutations in the SET/CXC domain abrogate catalytic activity as assessed by in vitro histone methylation assays, but missense mutations clustering in the conserved DI and DII regions retain activity. To understand the role of DI and DII mutations, we initially developed a cell-based histone methylation assay to test activity in a cellular context. Murine induced pluripotent stem cells lacking EZH2 were transiently transfected with wild type or mutant EZH2 (n = 15) and any resulting histone methylation was measured by flow cytometry. All DI mutations (n = 5) resulted in complete or partial loss of methylation activity whilst 5/6 DII mutations retained activity. Next, we assessed the possibility of splicing abnormalities induced by exon 8 mutations (encoding DII) using RT-PCR from primary patient samples and mini-gene assays. Exon 8 mutations resulted in skipping of exon 8 and an out-of-frame transcript. We have therefore shown that mutations within regions encoding EZH2 domains DI and DII are pathogenic by loss of function and exon skipping, respectively.

The t(1;9)(p34;q34) and t(8;12)(p11;q15) fuse pre-mRNA processing proteins SFPQ (PSF) and CPSF6 to ABL and FGFR1.

We have investigated two patients with acquired chromosomal rearrangements, a male presenting with a t(1;9)(p34;q34) and B cell progenitor acute lymphoid leukemia and a female presenting with a t(8;12)(p11;q15) and the 8p11 myeloproliferative syndrome. We determined that the t(1;9) fused ABL to SFPQ (also known as PSF), a gene mapping to 1p34 that encodes a polypyrimidine tract-binding protein-associated splicing factor. The t(8;12) fused CPSF6, a cleavage and polyadenylation specificity factor, to FGFR1. The fusions were confirmed by amplification of the genomic breakpoints and RT-PCR. The predicted oncogenic products of these fusions, SFPQ-ABL and CPSF6-FGFR1, are in-frame and encode the N-terminal domain of the partner protein and the entire tyrosine kinase domain and C-terminal sequences of ABL and FGFR1. SFPQ interacts with two FGFR1 fusion partners, ZNF198 and CPSF6, that are functionally related to the recurrent PDGFRalpha partner FIP1L1. Our findings thus identify a group of proteins that are important for pre-mRNA processing as fusion partners for tyrosine kinases in hematological malignancies.

PRR14L mutations are associated with chromosome 22 acquired uniparental disomy, age-related clonal hematopoiesis and myeloid neoplasia.

Acquired uniparental disomy (aUPD, also known as copy-neutral loss of heterozygosity) is a common feature of cancer cells and characterized by extended tracts of somatically-acquired homozygosity without any concurrent loss or gain of genetic material. The presumed genetic targets of many regions of aUPD remain unknown. Here we describe the association of chromosome 22 aUPD with mutations that delete the C-terminus of PRR14L in patients with chronic myelomonocytic leukemia (CMML), related myeloid neoplasms and age-related clonal hematopoiesis (ARCH). Myeloid panel analysis identified a median of three additional mutated genes (range 1-6) in cases with a myeloid neoplasm (n = 8), but no additional mutations in cases with ARCH (n = 2) suggesting that mutated PRR14L alone may be sufficient to drive clonality. PRR14L has very limited homology to other proteins and its function is unknown. ShRNA knockdown of PRR14L in human CD34+ cells followed by in vitro growth and differentiation assays showed an increase in monocytes and decrease in neutrophils, consistent with a CMML-like phenotype. RNA-Seq and cellular localization studies suggest a role for PRR14L in cell division. PRR14L is thus a novel, biallelically mutated gene and potential founding abnormality in myeloid neoplasms.

Recurrent activating STAT5B N642H mutation in myeloid neoplasms with eosinophilia.

Determining the underlying cause of persistent eosinophilia is important for effective clinical management but remains a diagnostic challenge in many cases. We identified STAT5B N642H, an established oncogenic mutation, in 27/1715 (1.6%) cases referred for investigation of eosinophilia. Of the 27 mutated cases, a working diagnosis of hypereosinophilic syndrome (HES; n = 7) or a myeloid neoplasm with eosinophilia (n = 20) had been made prior to the detection of STAT5B N642H. Myeloid panel analysis identified a median of 2 additional mutated genes (range 0-4) with 4 cases having STAT5B N642H as a sole abnormality. STAT5B N642H was absent in cultured T cells of 4/4 positive cases. Individuals with SF3B1 mutations (9/27; 33%) or STAT5B N642H as a sole abnormality had a markedly better overall survival compared to cases with other additional mutations (median 65 months vs. 14 months; hazard ratio = 8.1; P < 0.001). The overall survival of STAT5B-mutated HES cases was only 30 months, suggesting that these cases should be reclassified as chronic eosinophilic leukemia, not otherwise specified (CEL-NOS). The finding of STAT5B N642H as a recurrent mutation in myeloid neoplasia with eosinophilia provides a new diagnostic and prognostic marker as well as a potential target for therapy.

Evolutional change of karyotype with t(8;9)(p22;p24) and HLA-DR immunophenotype in relapsed acute myeloid leukemia.

The rare recurrent translocation of (8;9)(p22;p24) with PCM1-JAK2 fusion was recently characterized in diverse hematological malignancies. Most of them are atypical chronic myeloid leukemia (CML) or other myeloproliferative disorders (MPD), and are predominantly in the male. We report a female patient with acute myeloid leukemia (AML) initially presenting with normal karyotype and negative HLA-DR expression who achieved complete remission after standard chemotherapy. The disease relapsed 7 months later with cytogenetic change of t(8;9)(p22;p24). Flow cytometry analysis showed evolutional change of immunophenotype from negative to positive HLA-DR expression and fluorescence in situ hybridization (FISH) analysis demonstrated a PCM1-JAK2 fusion gene. We speculate that the cytogenetic change of t(8;9)(p22;p24) may induce HLA-DR immunophenotypic switch and a coordination of the two evolutional changes may play a role in leukemic cell progression.

A constitutively active SPTBN1-FLT3 fusion in atypical chronic myeloid leukemia is sensitive to tyrosine kinase inhibitors and immunotherapy.

OBJECTIVES: To determine the consequences and significance of an acquired 46XX,t(2;13;2;21)(p13;q12;q33;q11.2) in atypical chronic myeloid leukemia (aCML). METHODS: Translocation breakpoints were identified by fluorescence in situ hybridization and a novel fusion gene identified by rapid amplification of cDNA ends polymerase chain reaction. Functional analysis of the fusion was performed using the Ba/F3 transformation assay and specific inhibition demonstrated using small molecule inhibitors. RESULTS: Fluorescence in situ hybridization indicated that FLT3 at 13q12 was disrupted and 5'-rapid amplification of cDNA ends polymerase chain reaction identified a novel in-frame mRNA fusion between exon 3 of SPTBN1 (spectrin, beta, nonerythrocytic 1) at chromosome 2p16 and exon 13 of FLT3. Expression of SPTBN1-FLT3 transformed Ba/F3 cells to growth factor independence and was accompanied by constitutive phosphorylation of the fusion protein and the downstream substrate extracellular signal-regulated kinase 1/2. The growth of transformed cells was inhibited in a dose-dependent fashion by SU11657, PKC412, and TKI258 (CHIR-258), but not by imatinib. To determine if FLT3 might be involved more widely in BCR-ABL-negative aCML, we analyzed 40 cases and found two were internal tandem duplication-positive, but D835 mutations were not observed. The t(2;13;2;21) patient was initially treated with hydroxyurea and subsequently underwent an unrelated donor bone marrow transplantation. She relapsed cytogenetically at 4 years, but responded to donor lymphocyte infusion, achieving sustained cytogenetic and molecular (nested reverse transcription polymerase chain reaction) remission. CONCLUSION: Although FLT3 abnormalities are uncommon in aCML, SPTBN1-FLT3 is a novel constitutively active tyrosine kinase that appears to responsive to both targeted signal transduction therapy and immunotherapy.