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.

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.

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.

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.

p53-Binding protein 1 is fused to the platelet-derived growth factor receptor beta in a patient with a t(5;15)(q33;q22) and an imatinib-responsive eosinophilic myeloproliferative disorder.

We describe the fusion of TP53BP1 to PDGFRB in a patient with a chronic myeloid leukemia-like disorder associated with eosinophilia and a t(5;15)(q33;q22). TP53BP1 encodes 53BP1, a p53-binding protein that plays a role in cellular responses to DNA damage. The 53BP1-PDGFRbeta fusion protein is predicted to retain the kinetochore-binding domain of 53BP1 fused to the transmembrane and intracellular tyrosine kinase domain of PDGFRbeta. The presence of the fusion was confirmed by two-color fluorescence in situ hybridization, reverse transcription-PCR, and by characterizing the genomic breakpoints. The reciprocal fusion, which would contain the p53-binding 53BP1 BRCA1 COOH-terminal domains, was not detectable by fluorescence in situ hybridization or nested PCR. Imatinib, a known inhibitor of PDGFRbeta, blocked the growth of patient colony-forming unit, granulocyte-macrophage in vitro and produced a clinically significant response before relapse and subsequent death with imatinib-resistant disease. We conclude that TP53BP1-PDGFRB is a novel imatinib target in atypical chronic myeloid leukemia.

NUP98-LEDGF fusion and t(9;11) in transformed chronic myeloid leukemia.

The molecular basis for disease progression in chronic myeloid leukaemia (CML) is poorly understood, but is believed to be a consequence of additional acquired genetic lesions. We describe here a case of CML who presented de novo in transformation with a t(9;11)(p21;p15) and NUP98-LEDGF fusion in addition to the t(9;22). The t(9;11) was present in only 2/45 (4%) of bone marrow metaphases, but 17/20 (85%) of metaphases from peripheral blood, suggesting an extramedullary or focal origin. This is the first description of NUP98-LEDGF in CML and strengthens the association between disease progression in and NUP98 abnormalities.

Inactivating mutations of the histone methyltransferase gene EZH2 in myeloid disorders.

Abnormalities of chromosome 7q are common in myeloid malignancies, but no specific target genes have yet been identified. Here, we describe the finding of homozygous EZH2 mutations in 9 of 12 individuals with 7q acquired uniparental disomy. Screening of a total of 614 individuals with myeloid disorders revealed 49 monoallelic or biallelic EZH2 mutations in 42 individuals; the mutations were found most commonly in those with myelodysplastic/myeloproliferative neoplasms (27 out of 219 individuals, or 12%) and in those with myelofibrosis (4 out of 30 individuals, or 13%). EZH2 encodes the catalytic subunit of the polycomb repressive complex 2 (PRC2), a highly conserved histone H3 lysine 27 (H3K27) methyltransferase that influences stem cell renewal by epigenetic repression of genes involved in cell fate decisions. EZH2 has oncogenic activity, and its overexpression has previously been causally linked to differentiation blocks in epithelial tumors. Notably, the mutations we identified resulted in premature chain termination or direct abrogation of histone methyltransferase activity, suggesting that EZH2 acts as a tumor suppressor for myeloid malignancies.

Activity of TKI258 against primary cells and cell lines with FGFR1 fusion genes associated with the 8p11 myeloproliferative syndrome.

The 8p11 myeloproliferative syndrome (EMS) is an aggressive, atypical stem cell myeloproliferative disorder associated with chromosome translocations that disrupt and constitutively activate FGFR1 by fusion to diverse partner genes. To explore the possibility of targeted therapy for EMS, we have investigated the use of TKI258, a multitargeted receptor tyrosine kinase inhibitor with activity against FGFR, VEGFR, PDGFR, FLT3, and KIT that is currently being assessed for the treatment of a variety of malignancies in phase 1 clinical studies. The viability of Ba/F3 cells transformed to IL3 independence by ZNF198-FGFR1 or BCR-FGFR1 was specifically inhibited by TKI258 with IC(50) values of 150 nM and 90 nM, respectively. Inhibition was accompanied by dose-dependent inhibition of phosphorylation of each fusion gene, ERK, and STAT5. TKI258 also specifically inhibited proliferation and survival of the FGFR1OP2-FGFR1-positive KG1 and KG1A cell lines, resulting in increased levels of apoptosis. Primary cells from EMS patients showed significant, dose-dependent responses in liquid culture and in methylcellulose colony assays compared with controls. This work provides evidence that targeted therapy may be beneficial for patients with EMS.

Two novel imatinib-responsive PDGFRA fusion genes in chronic eosinophilic leukaemia.

We identified two patients with a t(2;4)(p24;q12) and a t(4;12)(q2?3;p1?2), respectively, in association with BCR-ABL and FIP1L1-PDGFRA negative chronic eosinophilic leukaemia. Molecular analysis revealed a novel STRN-PDGFRA fusion for the t(2;4) and ETV6-PDGFRA for the t(4;12). The fusions were confirmed by specific amplification of the genomic breakpoints, reverse transcription polymerase chain reaction and fluorescence in situ hybridisation. Both patients were treated with imatinib and, following a rapid haematological response, achieved cytogenetic remission and a major molecular response. In conclusion, PDGFRA fuses to diverse partner genes in myeloid disorders. Identification of these fusions is important as they are particularly sensitive to imatinib.

Der(6)t(1;6)(q21-23;p21.3): a specific cytogenetic abnormality in myelofibrosis with myeloid metaplasia.

Chromosome anomalies are detected in approximately half of patients with myelofibrosis with myeloid metaplasia (MMM) although none of the most prevalent lesions are specific to the disease. In a prospective cytogenetic study of 81 patients with MMM, we encountered three with an unbalanced translocation between chromosomes 1 and 6 with specific breakpoints; der(6)t(1;6)(q21-23;p21.3). A subsequent Mayo Clinic cytogenetic database search identified 12 patients with this chromosome anomaly among 17 791 consecutive patients. A similar database search from Royal Hallamshire Hospital in Sheffield, UK revealed two additional patients among 8000 cases. The clinical phenotype and survival for each of these 14 patients was typical of MMM. These findings suggested that der(6)t(1;6)(q21-23;p21.3) is a highly specific cytogenetic anomaly that may harbour gene(s) specifically associated with MMM. In a preliminary fluorescence in situ hybridization study, the breakpoints on chromosome 6 in two additional cases were found to be telomeric to the gene for 51 kDa FK506-binding protein (FKBP51).

Widespread occurrence of the JAK2 V617F mutation in chronic myeloproliferative disorders.

The analysis of rare chromosomal translocations in myeloproliferative disorders has highlighted the importance of aberrant tyrosine kinase signaling in the pathogenesis of these diseases. Here we have investigated samples from 679 patients and controls for the nonreceptor tyrosine kinase JAK2 V617F mutation. Of the 480 myeloproliferative disorder (MPD) samples, the proportion of positive cases per disease subtype was 30 (20%) of 152 for atypical or unclassified MPD, 2 of 134 (2%) for idiopathic hypereosinophilic syndrome, 58 of 72 (81%) for polycythemia vera, 24 of 59 (41%) essential thrombocythemia (ET), and 15 of 35 (43%) for idiopathic myelofibrosis. V617F was not identified in patients with systemic mastocytosis (n = 28), chronic or acute myeloid leukemia (n = 35), secondary erythrocytosis (n = 4), or healthy controls (n = 160). Homozygosity for V617F was seen in 43% of mutant samples and was closely correlated with chromosome 9p uniparental disomy. Homozygosity was significantly less common in ET compared with other MPD subtypes. In 53 cases analyzed, the median level of PRV1 expression was significantly higher in V617F-positive cases compared with cases without the mutation. We conclude that V617F is widespread in MPDs. Detection of this acquired mutation is likely to have a major impact on the way patients with MPD are diagnosed, as well as serving as an obvious target for signal transduction therapy.

Identification of a novel gene, FGFR1OP2, fused to FGFR1 in 8p11 myeloproliferative syndrome.

The 8p11 myeloproliferative syndrome (EMS) is an aggressive hematological malignancy caused by the fusion of diverse partner genes to fibroblast growth factor receptor 1 (FGFR1). The partner proteins promote dimerization and ligand-independent activation of FGFR1-encoded tyrosine kinase, deregulating hemopoiesis in a manner analogous to BCR-ABL in chronic myeloid leukemia. Here, we describe the identification of a new FGFR1 fusion gene in a patient who presented with T-cell lymphoblastic lymphoma in conjunction with an acquired ins(12;8)(p11;p11p22). Initial FISH analysis and Southern blotting confirmed that FGFR1 was disrupted. Using 5'-RACE PCR, we identified part of a novel gene, FGFR1OP2, at chromosome band 12p11 that was fused to exon 9 of FGFR1.FGFR1OP2 is predicted to be translated into an evolutionarily conserved protein containing coiled-coil domains but no other recognizable motifs. The presence of the chimeric gene was confirmed by RT-PCR, genomic DNA PCR, and FISH. These data further support the central role of deregulated FGFR1 in the pathogenesis of EMS.