Francis H. Grand

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.

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.

JAK2 haplotype is a major risk factor for the development of myeloproliferative neoplasms.

Chronic myeloproliferative neoplasms (MPNs) are a group of related conditions characterized by the overproduction of cells from one or more myeloid lineages. More than 95% of cases of polycythemia vera, and roughly half of essential thrombocythemia and primary myelofibrosis acquire a unique somatic 1849G>T JAK2 mutation (encoding V617F) that is believed to be a critical driver of excess proliferation. We report here that JAK2V617F-associated disease is strongly associated with a specific constitutional JAK2 haplotype, designated 46/1, in all three disease entities compared to healthy controls (polycythemia vera, n = 192, P = 2.9 × 10−16; essential thrombocythemia, n = 78, P = 8.2 × 10−9 and myelofibrosis, n = 41, P = 8.0 × 10−5). Furthermore, JAK2V617F specifically arises on the 46/1 allele in most cases. The 46/1 JAK2 haplotype thus predisposes to the development of JAK2V617F-associated MPNs (OR = 3.7; 95% CI = 3.1–4.3) and provides a model whereby a constitutional genetic factor is associated with an increased risk of acquiring a specific somatic mutation.

Identification of a novel imatinib responsive KIF5B-PDGFRA fusion gene following screening for PDGFRA overexpression in patients with hypereosinophilia

Idiopathic hypereosinophilic syndrome (IHES) is a disease that is difficult to classify, and diagnosis is one of exclusion. The identification of a cytogenetically invisible interstitial deletion resulting in the fusion of FIP1-Like-1 (FIP1L1) to platelet-derived growth factor receptor alpha (PDGFRA) has enabled many IHES cases to be reclassified as chronic eosinophilic leukemia. As it is likely that PDGFRA may fuse to other partner genes, we established a reverse transcriptase-PCR test to detect specific overexpression of the PDGFRA kinase domain as an indicator of the presence of a fusion gene. Overexpression was detected in 12/12 FIP1L1-PDGFRA-positive patients, plus 9/217 (4%) patients with hypereosinophilia who had tested negative for FIP1L1-PDGFRA. One of the positive cases was investigated in detail and found to have a complex karyotype involving chromosomes 3, 4 and 10. Amplification of the genomic breakpoint by bubble PCR revealed a novel fusion between KIF5B at 10p11 and PDGFRA at 4q12. Imatinib, a known inhibitor of PDGFRα, produced a complete cytogenetic response and disappearance of the KIF5B-PDGFRA fusion by PCR, from both genomic DNA and mRNA. This study demonstrates the utility of screening for PDGFRA kinase domain overexpression in patients with IHES and has identified a third PDGFRA fusion partner in chronic myeloproliferative disorders.

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, FGFR1OP, 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.

p53-Binding Protein 1 Is Fused to the Platelet-Derived Growth Factor Receptor β 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-PDGFRβ fusion protein is predicted to retain the kinetochore-binding domain of 53BP1 fused to the transmembrane and intracellular tyrosine kinase domain of PDGFRβ. 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 PDGFRβ, 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.

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.

Disruption and aberrant expression of HMGA2 as a consequence of diverse chromosomal translocations in myeloid malignancies.

Chromosomal translocations that target HMGA2 at chromosome band 12q14 are seen in a variety of malignancies, notably lipoma, pleomorphic salivary adenoma and uterine leiomyoma. Although some HMGA2 fusion genes have been reported, several lines of evidence suggest that the critical pathogenic event is the expression of truncated HMGA2 isoforms. We report here the involvement of HMGA2 in six patients with myeloid neoplasia, dysplastic features and translocations or an inversion involving chromosome bands 12q13–15 and either 7p12, 8q22, 11q23, 12p11, 14q31 or 20q11. Breaks within or very close to HMGA2 were found in all six cases by molecular cytogenetic analysis, leading to overexpression of this gene as assessed by RT-PCR. Truncated transcripts consisting of HMGA2 exons 1–2 or exons 1–3 spliced to intron-derived sequences were identified in two patients, but were not seen in controls. These findings suggest that abnormalities of HMGA2 play an important and previously unsuspected role in myelodysplasia.

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 PDGFRα 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.

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).