Helena Kempski

Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients

X-linked SCID (SCID-X1) is amenable to correction by gene therapy using conventional gammaretroviral vectors. Here, we describe the occurrence of clonal T cell acute lymphoblastic leukemia (T-ALL) promoted by insertional mutagenesis in a completed gene therapy trial of 10 SCID-X1 patients. Integration of the vector in an antisense orientation 35 kb upstream of the protooncogene LIM domain only 2 (LMO2) caused overexpression of LMO2 in the leukemic clone. However, leukemogenesis was likely precipitated by the acquisition of other genetic abnormalities unrelated to vector insertion, including a gain-of-function mutation in NOTCH1, deletion of the tumor suppressor gene locus cyclin-dependent kinase 2A (CDKN2A), and translocation of the TCR-beta region to the STIL-TAL1 locus. These findings highlight a general toxicity of endogenous gammaretroviral enhancer elements and also identify a combinatorial process during leukemic evolution that will be important for risk stratification and for future protocol design.

Genetic variegation of clonal architecture and propagating cells in leukaemia

Little is known of the genetic architecture of cancer at the subclonal and single-cell level or in the cells responsible for cancer clone maintenance and propagation. Here we have examined this issue in childhood acute lymphoblastic leukaemia in which the ETV6–RUNX1 gene fusion is an early or initiating genetic lesion followed by a modest number of recurrent or ‘driver’ copy number alterations. By multiplexing fluorescence in situ hybridization probes for these mutations, up to eight genetic abnormalities can be detected in single cells, a genetic signature of subclones identified and a composite picture of subclonal architecture and putative ancestral trees assembled. Subclones in acute lymphoblastic leukaemia have variegated genetics and complex, nonlinear or branching evolutionary histories. Copy number alterations are independently and reiteratively acquired in subclones of individual patients, and in no preferential order. Clonal architecture is dynamic and is subject to change in the lead-up to a diagnosis and in relapse. Leukaemia propagating cells, assayed by serial transplantation in NOD/SCID IL2Rγnull mice, are also genetically variegated, mirroring subclonal patterns, and vary in competitive regenerative capacity in vivo. These data have implications for cancer genomics and for the targeted therapy of cancer.

Mutations of JAK2 in acute lymphoblastic leukaemias associated with Down's syndrome

BACKGROUND Children with Downs syndrome have a greatly increased risk of acute megakaryoblastic and acute lymphoblastic leukaemias. Acute megakaryoblastic leukaemia in Downs syndrome is characterised by a somatic mutation in GATA1. Constitutive activation of the JAK/STAT (Janus kinase and signal transducer and activator of transcription) pathway occurs in several haematopoietic malignant diseases. We tested the hypothesis that mutations in JAK2 might be a common molecular event in acute lymphoblastic leukaemia associated with Downs syndrome. METHODS JAK2 DNA mutational analysis was done on diagnostic bone marrow samples obtained from 88 patients with Downs syndrome-associated acute lymphoblastic leukaemia; and 216 patients with sporadic acute lymphoblastic leukaemia, Downs syndrome-associated acute megakaryoblastic leukaemia, and essential thrombocythaemia. Functional consequences of identified mutations were studied in mouse haematopoietic progenitor cells. FINDINGS Somatically acquired JAK2 mutations were identified in 16 (18%) patients with Downs syndrome-associated acute lymphoblastic leukaemia. The only patient with non-Downs syndrome-associated leukaemia but with a JAK2 mutation had an isochromosome 21q. Children with a JAK2 mutation were younger (mean [SE] age 4.5 years [0.86] vs 8.6 years [0.59], p<0.0001) at diagnosis. Five mutant alleles were identified, each affecting a highly conserved arginine residue (R683). These mutations immortalised primary mouse haematopoietic progenitor cells in vitro, and caused constitutive Jak/Stat activation and cytokine-independent growth of BaF3 cells, which was sensitive to pharmacological inhibition with JAK inhibitor I. In modelling studies of the JAK2 pseudokinase domain, R683 was situated in an exposed conserved region separated from the one implicated in myeloproliferative disorders. INTERPRETATION A specific genotype-phenotype association exists between the type of somatic mutation within the JAK2 pseudokinase domain and the development of B-lymphoid or myeloid neoplasms. Somatically acquired R683 JAK2 mutations define a distinct acute lymphoblastic leukaemia subgroup that is uniquely associated with trisomy 21. JAK2 inhibitors could be useful for treatment of this leukaemia. FUNDING Israel Trade Ministry, Israel Science Ministry, Jewish National Fund UK, Sam Waxman Cancer Research Foundation, Israel Science Foundation, Israel Cancer Association, Curtis Katz, Constantiner Institute for Molecular Genetics, German-Israel Foundation, and European Commission FP6 Integrated Project EUROHEAR.

Down syndrome acute lymphoblastic leukemia, a highly heterogeneous disease in which aberrant expression of CRLF2 is associated with mutated JAK2: a report from the International BFM Study Group.

We report gene expression and other analyses to elucidate the molecular characteristics of acute lymphoblastic leukemia (ALL) in children with Down syndrome (DS). We find that by gene expression DS-ALL is a highly heterogeneous disease not definable as a unique entity. Nevertheless, 62% (33/53) of the DS-ALL samples analyzed were characterized by high expression of the type I cytokine receptor CRLF2 caused by either immunoglobulin heavy locus (IgH@) translocations or by interstitial deletions creating chimeric transcripts P2RY8-CRLF2. In 3 of these 33 patients, a novel activating somatic mutation, F232C in CRLF2, was identified. Consistent with our previous research, mutations in R683 of JAK2 were identified in 10 specimens (19% of the patients) and, interestingly, all 10 had high CRLF2 expression. Cytokine receptor-like factor 2 (CRLF2) and mutated Janus kinase 2 (Jak2) cooperated in conferring cytokine-independent growth to BaF3 pro-B cells. Intriguingly, the gene expression signature of DS-ALL is enriched with DNA damage and BCL6 responsive genes, suggesting the possibility of B-cell lymphocytic genomic instability. Thus, DS confers increased risk for genetically highly diverse ALLs with frequent overexpression of CRLF2, associated with activating mutations in the receptor itself or in JAK2. Our data also suggest that the majority of DS children with ALL may benefit from therapy blocking the CRLF2/JAK2 pathways.

Specific JAK2 mutation (JAK2R683) and multiple gene deletions in Down syndrome acute lymphoblastic leukemia

Children with Down syndrome (DS) have a greatly increased risk of acute megakaryoblastic leukemia (AMKL) and acute lymphoblastic leukemia (ALL). Both DS-AMKL and the related transient myeloproliferative disorder (TMD) have GATA1 mutations as obligatory, early events. To identify mutations contributing to leukemogenesis in DS-ALL, we undertook sequencing of candidate genes, including FLT3, RAS, PTPN11, BRAF, and JAK2. Sequencing of the JAK2 pseudokinase domain identified a specific, acquired mutation, JAK2R683, in 12 (28%) of 42 DS-ALL cases. Functional studies of the common JAK2R683G mutation in murine Ba/F3 cells showed growth factor independence and constitutive activation of the JAK/STAT signaling pathway. High-resolution SNP array analysis of 9 DS-ALL cases identified additional submicroscopic deletions in key genes, including ETV6, CDKN2A, and PAX5. These results infer a complex molecular pathogenesis for DS-ALL leukemogenesis, with trisomy 21 as an initiating or first hit and with chromosome aneuploidy, gene deletions, and activating JAK2 mutations as complementary genetic events.

RAG-mediated recombination is the predominant driver of oncogenic rearrangement in ETV6-RUNX1 acute lymphoblastic leukemia

The ETV6-RUNX1 fusion gene, found in 25% of childhood acute lymphoblastic leukemia (ALL) cases, is acquired in utero but requires additional somatic mutations for overt leukemia. We used exome and low-coverage whole-genome sequencing to characterize secondary events associated with leukemic transformation. RAG-mediated deletions emerge as the dominant mutational process, characterized by recombination signal sequence motifs near breakpoints, incorporation of non-templated sequence at junctions, ∼30-fold enrichment at promoters and enhancers of genes actively transcribed in B cell development and an unexpectedly high ratio of recurrent to non-recurrent structural variants. Single-cell tracking shows that this mechanism is active throughout leukemic evolution, with evidence of localized clustering and reiterated deletions. Integration of data on point mutations and rearrangements identifies ATF7IP and MGA as two new tumor-suppressor genes in ALL. Thus, a remarkably parsimonious mutational process transforms ETV6-RUNX1–positive lymphoblasts, targeting the promoters, enhancers and first exons of genes that normally regulate B cell differentiation.

Long-term follow-up of relapsed childhood acute lymphoblastic leukaemia

Summary. We have reviewed the outcome after relapse in a cohort of 505 children with acute lymphoblastic leukaemia (ALL) seen at a single institution. The majority of relapses (74%) occurred within 3 years from diagnosis, and most involved the bone marrow alone or with overt extramedullary relapse. Early relapse was more common in children with T‐ALL and those with unfavourable cytogenetics. Factors influencing second remission included length of first remission and type of relapse. Children who had not received previous cranial irradiation had a superior survival. The German relapse score involving length of first remission, site of relapse and immunophenotype was highly predictive of outcome: event‐free survival with 95% confidence intervals at 6 years for patients who received modern treatment [intensive chemotherapy or bone marrow transplantation (BMT)] was 78% (51–92%) for standard risk, 41% (33–49%) for intermediate risk and 19% (10–31%) for highest risk. Retrospective comparison of BMT with chemotherapy showed no difference in the intermediate‐risk group but a possible advantage in the highest risk group. Follow‐up of 235 patients who relapsed after chemotherapy and received a third course of treatment showed an extremely high early attrition rate, but a small number of patients survived in third remission. We conclude that new approaches are needed to individualize therapy in intermediate‐risk patients and to improve the outcome for those in the highest risk group. Only a small number of children can be treated effectively in third remission.

Paediatric myelodysplastic syndromes and juvenile myelomonocytic leukaemia in the UK: a population‐based study of incidence and survival

Summary.  We aimed to identify and classify cases of paediatric myelodysplastic syndromes (MDS) occurring in Britain to estimate the incidence of this rare group of diseases, investigate the results of therapy and identify prognostic risk factors. Patients aged below 15 years at diagnosis were collected from England, Scotland and Wales, inclusively between 1990 and 1999. One hundred and thirty‐five patients were accepted as de novo MDS or juvenile myelomonocytic leukaemia (JMML). The incidence for this period was 1·35 per million (age standardized rate) which is below that reported outside the UK. The overall survival was 45%[standard error (SE) = 4%] at 5 years: 40% (SE = 6%) for JMML and 50% (SE = 6%) for other MDS. Significant adverse prognostic factors for JMML were a platelet count < 40 × 109/l, raised fetal haemoglobin, FPC score and age above 2 years at diagnosis, for other MDS only monosomy 7 was significant. To conclude, the incidence of MDS/JMML in children in the UK appears to be lower than that reported outside the UK. This may be either a real difference in incidence or variation in reporting. Monosomy 7 is associated with poor outcome in MDS other than JMML. The prognosis of JMML depends on age, platelet count and fetal haemoglobin.

Clonal origins of relapse in ETV6-RUNX1 acute lymphoblastic leukemia

B-cell precursor childhood acute lymphoblastic leukemia with ETV6-RUNX1 (TEL-AML1) fusion has an overall good prognosis, but relapses occur, usually after cessation of treatment and occasionally many years later. We have investigated the clonal origins of relapse by comparing the profiles of genomewide copy number alterations at presentation in 21 patients with those in matched relapse (12-119 months). We identified, in total, 159 copy number alterations at presentation and 231 at relapse (excluding Ig/TCR). Deletions of CDKN2A/B or CCNC (6q16.2-3) or both increased from 38% at presentation to 76% in relapse, suggesting that cell-cycle deregulation contributed to emergence of relapse. A novel observation was recurrent gain of chromosome 16 (2 patients at presentation, 4 at relapse) and deletion of plasmocytoma variant translocation 1 in 3 patients. The data indicate that, irrespective of time to relapse, the relapse clone was derived from either a major or minor clone at presentation. Backtracking analysis by FISH identified a minor subclone at diagnosis whose genotype matched that observed in relapse ∼ 10 years later. These data indicate subclonal diversity at diagnosis, providing a variable basis for intraclonal origins of relapse and extended periods (years) of dormancy, possibly by quiescence, for stem cells in ETV6-RUNX1(+) acute lymphoblastic leukemia.

t(7;12)(q36;p13), a new recurrent translocation involving ETV6 in infant leukemia.

The ETV6 gene is rearranged as a result of translocations involving a wide variety of chromosomal partners. To date, 12 partner genes for ETV6 have been cloned, and a further 23 chromosomal regions have been described. We previously identified a cryptic t(7;12) with ETV6 involvement in two cases of infant leukemia. The finding of a third case of t(7;12), also in an infant, prompted a more focussed search based on the common features found in these patients and those reported in the literature. The selection criteria were age at diagnosis < 20 months and the presence of +19 and/or +8 in the karyotype; cases with abnormalities of 7q and/or 12p were also considered. FISH studies using whole chromosome paints and probes for the ETV6 gene revealed a t(7;12) in 10 out of 23 cases studied. Seven of these had evidence of ETV6 rearrangement. Of those with ETV6 involvement, six had a 7q36 and one a 7q22 breakpoint. Importantly, in three cases the 7q36 breakpoint was within the same PAC, suggesting the existence of a new nonrandom translocation. However, in at least one patient the 7q36 breakpoint was different. The identification of the 7q partner genes will determine whether it is the disruption of ETV6 alone, or the formation of fusion genes, that is important for leukemogenesis in these patients. As both 7q36 and 7q22 are critical regions of gene loss in del(7q) leukemias, the identification of partner genes from these regions may also be important in understanding the pathogenesis of these diseases.