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International audience; Pediatric acute myeloid leukemia is a rare and heterogeneous disease in relation to morphology, immunophenotyping, germline and somatic cytogenetic and genetic abnormalities. Over recent decades, outcomes have greatly improved, although survival rates remain around 70% and the relapse rate is high, at around 30%. Cytogenetics is an important factor for diagnosis and indication of prognosis. The main cytogenetic abnormalities are referenced in the current WHO classification of acute myeloid leukemia, where there is an indication for risk-adapted therapy. The aim of this article is to provide an updated review of cytogenetics in pediatric AML, describing well-known WHO entities, as well as new subgroups and germline mutations with therapeutic implications. We describe the main chromosomal abnormalities, their frequency according to age and AML subtypes, and their prognostic relevance within current therapeutic protocols. We focus on de novo AML and on cytogenetic diagnosis, including the practical difficulties encountered, based on the most recent hematological and cytogenetic recommendations.

Children with B-cell non-Hodgkin lymphoma (B-NHL) have an excellent chance of survival, however, current clinical risk stratification places as many as half of patients in a high-risk group receiving very intensive chemo-immunotherapy. TP53 alterations are associated with adverse outcome in many malignancies; however, whilst common in paediatric B-NHL, their utility as a risk classifier is unknown. We evaluated the clinical significance of TP53 abnormalities (mutations, deletion and/or copy number neutral loss of heterozygosity) in a large UK paediatric B-NHL cohort and determined their impact on survival. TP53 abnormalities were present in 54.7% of cases and were independently associated with a significantly inferior survival compared to those without a TP53 abnormality (PFS 70.0% vs 100%, p p = 0.002). Moreover, amongst patients clinically defined as high-risk (stage III with high LDH or stage IV), those without a TP53 abnormality have superior survival compared to those with TP53 abnormalities (PFS 100% vs 55.6%, p = 0.005, OS 100% vs 66.7%, p = 0.019). Biallelic TP53 abnormalities were either maintained from the presentation or acquired at progression in all paired diagnosis/progression Burkitt lymphoma cases. TP53 abnormalities thus define clinical risk groups within paediatric B-NHL and offer a novel molecular risk stratifier, allowing more personalised treatment protocols.

Acute leukemias (ALs) of ambiguous lineage are a heterogeneous group of high-risk leukemias characterized by coexpression of myeloid and lymphoid markers. In this study, we identified a distinct subgroup of immature acute leukemias characterized by a broadly variable phenotype, covering acute myeloid leukemia (AML, M0 or M1), T/myeloid mixed-phenotype acute leukemia (T/M MPAL), and early T-cell precursor acute lymphoblastic leukemia (ETP-ALL). Rearrangements at 14q32/BCL11B are the cytogenetic hallmark of this entity. In our screening of 915 hematological malignancies, there were 202 AML and 333 T-cell acute lymphoblastic leukemias (T-ALL: 58, ETP; 178, non-ETP; 8, T/M MPAL; 89, not otherwise specified). We identified 20 cases of immature leukemias (4% of AML and 3.6% of T-ALL), harboring 4 types of 14q32/BCL11B translocations: t(2,14)(q22.3;q32) (n = 7), t(6;14)(q25.3;q32) (n = 9), t(7;14)(q21.2;q32) (n = 2), and t(8;14)(q24.2;q32) (n = 2). The t(2;14) produced a ZEB2-BCL11B fusion transcript, whereas the other 3 rearrangements displaced transcriptionally active enhancer sequences close to BCL11B without producing fusion genes. All translocations resulted in the activation of BCL11B, a regulator of T-cell differentiation associated with transcriptional corepressor complexes in mammalian cells. The expression of BCL11B behaved as a disease biomarker that was present at diagnosis, but not in remission. Deregulation of BCL11B co-occurred with variants at FLT3 and at epigenetic modulators, most frequently the DNMT3A, TET2, and/or WT1 genes. Transcriptome analysis identified a specific expression signature, with significant downregulation of BCL11B targets, and clearly separating BCL11B AL from AML, T-ALL, and ETP-ALL. Remarkably, an ex vivo drug-sensitivity profile identified a panel of compounds with effective antileukemic activity.

Aneuploidy occurs within a significant proportion of childhood B-cell acute lymphoblastic leukemia (B-ALL). Some copy number variations (CNV), associated with novel subtypes of childhood B-ALL, have prognostic significance. A total of 233 childhood B-ALL patients were enrolled into this study. Focal copy number alterations of ERG, IKZF1, PAX5, ETV6, RB1, BTG1, EBF1, CDKN2A/2B, and the Xp22.33/Yp11.31 region were assessed by Multiplex Ligation-dependent Probe Amplification (MLPA). The MLPA telomere kit was used to identify aneuploidy through detection of whole chromosome loss or gain. We carried out these procedures alongside measurement of DNA index in order to identify, aneuploidy status in our cohort. MLPA telomere data and DNA index correlated well with aneuploidy status at higher sensitivity than cytogenetic analysis. Three masked hypodiploid patients, undetected by cytogenetics, and their associated copy number neutral loss of heterozygosity (CN-LOH) were identified by STR and SNP arrays. Rearrangements of TCF3, located to 19p, were frequently associated with 19p deletions. Other genetic alterations including iAMP21, IKZF1 deletions, ERG deletions, PAX5AMP, which have clinical significance or are associated with novel subtypes of ALL, were identified. In conclusion, appropriate application of MLPA aids the identifications of CNV and aneuploidy in childhood B-ALL.

Deregulated expression of the type I cytokine receptor, CRLF2, is observed in 5-15% of precursor B-cell acute lymphoblastic leukaemia (B-ALL). We have previously reported the genomic landscape of patients with CRLF2 rearrangements (CRLF2-r) using both whole genome and exome sequencing, which identified a number of potential clonal and sub-clonal genomic alterations. In this study, we aimed to assess when the CRLF2-r; IGH-CRLF2 or P2RY8-CRLF2, arose during the evolution of both Down syndrome-ALL (DS-ALL) and non-DS-ALL. Using fluorescence in situ hybridisation, we were able to track up to four structural variants in single cells from 47 CRLF2-r B-ALL patients, which in association with our multiplex single-cell analysis of a further four patients, permitted simultaneous tracking of copy number alterations, structural and single nucleotide variants within individual cells. We observed CRLF2-r arising as both early and late events in DS and non-DS-ALL patients. Parallel evolution of discrete clones was observed in the development of CRLF2-r B-ALL, either involving the CRLF2-r or one of the other tracked abnormalities. In-depth single-cell analysis identified both linear and branching evolution with early clones harbouring a multitude of abnormalities, including the CRLF2-r in DS-ALL patients.

The R98S mutation in ribosomal protein L10 (RPL10 R98S) affects 8% of pediatric T-cell acute lymphoblastic leukemia (T-ALL) cases, and was previously described to impair cellular proliferation. The current study reveals that RPL10 R98S cells accumulate reactive oxygen species which promotes mitochondrial dysfunction and reduced ATP levels, causing the proliferation defect. RPL10 R98S mutant leukemia cells can survive high oxidative stress levels via a specific increase of IRES-mediated translation of the anti-apoptotic factor B-cell lymphoma 2 (BCL-2), mediating BCL-2 protein overexpression. RPL10 R98S selective sensitivity to the clinically available Bcl-2 inhibitor Venetoclax (ABT-199) was supported by suppression of splenomegaly and the absence of human leukemia cells in the blood of T-ALL xenografted mice. These results shed new light on the oncogenic function of ribosomal mutations in cancer, provide a novel mechanism for BCL-2 upregulation in leukemia, and highlight BCL-2 inhibition as a novel therapeutic opportunity in RPL10 R98S defective T-ALL. ispartof: LEUKEMIA vol:33 issue:2 pages:319-332 ispartof: location:England status: published

The Janus kinase 3 (JAK3) tyrosine kinase is mutated in 10% to 16% of T-cell acute lymphoblastic leukemia (T-ALL) cases. JAK3 mutants induce constitutive JAK/STAT signaling and cause leukemia when expressed in the bone marrow cells of mice. Surprisingly, we observed that one third of JAK3-mutant T-ALL cases harbor 2 JAK3 mutations, some of which are monoallelic and others that are biallelic. Our data suggest that wild-type JAK3 competes with mutant JAK3 (M511I) for binding to the common γ chain and thereby suppresses its oncogenic potential. We demonstrate that JAK3 (M511I) can increase its limited oncogenic potential through the acquisition of an additional mutation in the mutant JAK3 allele. These double JAK3 mutants show increased STAT5 activation and increased potential to transform primary mouse pro-T cells to interleukin-7-independent growth and were not affected by wild-type JAK3 expression. These data extend our insight into the oncogenic properties of JAK3 mutations and provide an explanation of why progression of JAK3-mutant T-ALL cases can be associated with the accumulation of additional JAK3 mutations.

IKZF1 deletion (ΔIKZF1) is an important predictor of relapse in both childhood and adult B-cell precursor acute lymphoblastic leukemia (B-ALL). Previously, we revealed that COBL is a hotspot for breakpoints in leukemia and could promote IKZF1 deletions. Through an international collaboration, we provide a detailed genetic and clinical picture of B-ALL with COBL rearrangements (COBL-r). Patients with B-ALL and IKZF1 deletion (n = 133) were included. IKZF1 ∆1-8 were associated with large alterations within chromosome 7: monosomy 7 (18%), isochromosome 7q (10%), 7p loss (19%), and interstitial deletions (53%). The latter included COBL-r, which were found in 12% of the IKZF1 ∆1-8 cohort. Patients with COBL-r are mostly classified as intermediate cytogenetic risk and frequently harbor ETV6, PAX5, CDKN2A/B deletions. Overall, 56% of breakpoints were located within COBL intron 5. Cryptic recombination signal sequence motifs were broadly distributed within the sequence of COBL, and no enrichment for the breakpoint cluster region was found. In summary, a diverse spectrum of alterations characterizes ΔIKZF1 and they also include deletion breakpoints within COBL. We confirmed that COBL is a hotspot associated with ΔIKZF1, but these rearrangements are not driven by RAG-mediated recombination.

Contains fulltext : 204641.pdf (Publisher’s version ) (Open Access) Genetic abnormalities provide vital diagnostic and prognostic information in pediatric acute lymphoblastic leukemia (ALL) and are increasingly used to assign patients to risk groups. We recently proposed a novel classifier based on the copy-number alteration (CNA) profile of the 8 most commonly deleted genes in B-cell precursor ALL. This classifier defined 3 CNA subgroups in consecutive UK trials and was able to discriminate patients with intermediate-risk cytogenetics. In this study, we sought to validate the United Kingdom ALL (UKALL)-CNA classifier and reevaluate the interaction with cytogenetic risk groups using individual patient data from 3239 cases collected from 12 groups within the International BFM Study Group. The classifier was validated and defined 3 risk groups with distinct event-free survival (EFS) rates: good (88%), intermediate (76%), and poor (68%) (P < .001). There was no evidence of heterogeneity, even within trials that used minimal residual disease to guide therapy. By integrating CNA and cytogenetic data, we replicated our original key observation that patients with intermediate-risk cytogenetics can be stratified into 2 prognostic subgroups. Group A had an EFS rate of 86% (similar to patients with good-risk cytogenetics), while group B patients had a significantly inferior rate (73%, P < .001). Finally, we revised the overall genetic classification by defining 4 risk groups with distinct EFS rates: very good (91%), good (81%), intermediate (73%), and poor (54%), P < .001. In conclusion, the UKALL-CNA classifier is a robust prognostic tool that can be deployed in different trial settings and used to refine established cytogenetic risk groups.

The EBF1-PDGFRB gene fusion accounts for 0.5%) at week 14. Two UKALL 2011 patients, positive for EBF1-PDGFRB, received imatinib; 1 died 6 months after a matched unrelated bone marrow transplant as a result of undefined encephalopathy, and the other remained in remission 10 months after diagnosis.

© 2016 Ferrata Storti Foundation.With the deaths of Janet Rowley and John Goldman in December 2013, the world lost two pioneers in the field of chronic myeloid leukemia. In 1973, Janet Rowley, unraveled the cytogenetic anatomy of the Philadelphia chromosome, which subsequently led to the identification of the BCR-ABL1 fusion gene and its principal pathogenetic role in the development of chronic myeloid leukemia. This work was also of major importance to support the idea that cytogenetic changes were drivers of leukemogenesis. John Goldman originally made seminal contributions to the use of autologous and allogeneic stem cell transplantation from the late 1970s onwards. Then, in collaboration with Brian Druker, he led efforts to develop ABL1 tyrosine kinase inhibitors for the treatment of patients with chronic myeloid leukemia in the late 1990s. He also led the global efforts to develop and harmonize methodology for molecular monitoring, and was an indefatigable organizer of international conferences. These conferences brought together clinicians and scientists, and accelerated the adoption of new therapies. The abundance of praise, tributes and testimonies expressed by many serve to illustrate the indelible impressions these two passionate and affable scholars made on so many people’s lives. This tribute provides an outline of the remarkable story of chronic myeloid leukemia, and in writing it, it is clear that the historical triumph of biomedical science over this leukemia cannot be considered without appreciating the work of both Janet Rowley and John Goldman.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the Western World and it is characterized by a marked degree of clinical heterogeneity. An impaired balance between pro- and anti-apoptotic stimuli determines chemorefractoriness and outcome. The low proliferation rate of CLL cells indicates that one of the primary mechanisms involved in disease development may be an apoptotic failure. Here, we study the clinical and functional significance of DRAK2, a novel stress response kinase that plays a critical role in apoptosis, T-cell biology, and B-cell activation in CLL. We have analyzed CLL patient samples and showed that low expression levels of DRAK2 were significantly associated with unfavorable outcome in our CLL cohort. DRAK2 expression levels showed a positive correlation with the expression of DAPK1, and TGFBR1. Consistent with clinical data, the downregulation of DRAK2 in MEC-1 CLL cells strongly increased cell viability and proliferation. Further, our transcriptome data from MEC-1 cells highlighted MAPK, NF-&kappa, B, and Akt and as critical signaling hubs upon DRAK2 knockdown. Taken together, our results indicate DRAK2 as a novel marker of CLL survival that plays key regulatory roles in CLL prognosis.

In childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL), cytogenetic abnormalities remain important diagnostic and prognostic tools. A number of well-established abnormalities are routinely used in risk stratification for treatment. These include high hyperdiploidy and ETV6-RUNX1 fusion, classified as good risk, while Philadelphia chromosome (Ph) positive ALL and rearrangements of the KMT2A (MLL) gene define poor risk. A poor risk subgroup of intrachromosomal amplification of chromosome 21 (iAMP21-ALL) has been described, in which intensification of therapy has greatly improved outcome. Until recently, no consistent molecular features were defined in around 30% of BCP-ALL (known as B-other-ALL). Recent studies are classifying them into distinct subgroups, some with clear potential for novel therapeutic approaches. For example, in 1 poor risk subtype, known as Ph-like/BCR-ABL1-like ALL, approximately 10% have rearrangements of ABL-class tyrosine kinases: including ABL1, ABL2, PDGFRB, PDGFRA, and CSF1R. Notably, they show a poor response to standard chemotherapy, while they respond to treatment with tyrosine kinase inhibitors, such as imatinib. In other Ph-like-ALL patients, deregulation of the cytokine receptor, CRLF2, and JAK2 rearrangements lead to activation of the JAK-STAT signaling pathway, implicating a specific role for JAK inhibitors in their treatment. Other novel subgroups within B-other-ALL are defined by the IGH-DUX4 translocation, related to deletions of the ERG gene and a good outcome, while fusions involving ZNF384, MEF2D, and intragenic PAX5 amplification (PAX5AMP) are linked to a poor outcome. Continued genetic screening will eventually lead to complete genomic classification of BCP-ALL and define more molecular targets for less toxic therapies.

Several somatic ribosome defects have recently been discovered in cancer, yet their oncogenic mechanisms remain poorly understood. Here we investigated the pathogenic role of the recurrent R98S mutation in ribosomal protein L10 (RPL10-R98S) found in T-ALL. The JAK-STAT signaling pathway is a critical controller of cellular proliferation and survival. A proteome screen revealed overexpression of several Jak-Stat signaling proteins in engineered RPL10-R98S mouse lymphoid cells, which we confirmed in hematopoietic cells from transgenic Rpl10-R98S mice and T-ALL xenograft samples. RPL10-R98S expressing cells displayed JAK-STAT pathway hyper-activation upon cytokine stimulation, as well as increased sensitivity to clinically used JAK-STAT inhibitors like pimozide. A mutually exclusive mutation pattern between RPL10-R98S and JAK-STAT mutations in T-ALL patients further suggests that RPL10-R98S functionally mimics JAK-STAT activation. Mechanistically, besides transcriptional changes, RPL10-R98S caused reduction of apparent programmed ribosomal frameshifting at several ribosomal frameshift signals in mouse and human Jak-Stat genes, as well as decreased Jak1 degradation. Of further medical interest, RPL10-R98S cells showed reduced proteasome activity and enhanced sensitivity to clinical proteasome inhibitors. Collectively, we describe modulation of the JAK-STAT cascade as a novel cancer-promoting activity of a ribosomal mutation, and expand the relevance of this cascade in leukemia.Leukemia accepted article preview online, 24 July 2017. doi:10.1038/leu.2017.225. ispartof: Leukemia vol:32 issue:3 pages:809-819 ispartof: location:England status: published

Refinement of IKZF1 status in pediatric Philadelphia-positive acute lymphoblastic leukemia

Acute myeloid leukemia (AML) develops when there is a block in differentiation and uncontrolled proliferation of myeloid precursors, resulting in bone marrow failure. AML is a clinically, morphologically, and genetically heterogeneous disease, and biological differences between adult and childhood AML have been identified. AML comprises 15%-20% of all children15 years of age diagnosed with acute leukemia. Relapse occurs in up to 40% of children with AML and is the most common cause of death. Relapse arises from leukemic stem cells (LSCs) that persist after conventional chemotherapy. The treatment of AML is challenging, and new strategies to target LSCs are required. The cell surface marker CD33 has been identified as a therapeutic target, and novel anti-CD33 immunotherapies are promising new agents in the treatment of AML. This review summarizes recent developments emphasizing the genetic differences in adult and childhood AML and highlights the rationale for CD33 as a target for therapy in all age groups.

In childhood acute lymphoblastic leukaemia (ALL), chromosomal abnormalities are important diagnostic and prognostic biomarkers, which are used in risk stratification. For example, t(12;21)/ETV6-RUNX1 and high hyperdiploidy are associated with a favourable outcome, whereas KMT2A (MLL) rearrangements, intrachromosomal amplification of chromosome 21 (iAMP21), t(17;19)/TCF3-HLF, near-haploidy and low hypodiploidy classify high-risk disease. Patients with Philadelphia chromosome (Ph) positive, t(9;22)(q34;q11)/BCR-ABL1 ALL are now treated with tyrosine kinase inhibitors, whilst patients with iAMP21-ALL have improved outcome when treated intensively. Genomic technologies have identified copy number alterations and mutations affecting genes involving in leukaemogenesis and disease resistance. These newly described abnormalities are helping to re-define and extend genetic risk stratification in this disease. Transcriptome sequencing has identified a network of kinase-activating aberrations and emerging experimental and clinical data indicate that patients with these abnormalities respond to treatment with tyrosine kinase or other small molecule inhibitors. In conclusion, genetic biomarkers are playing an increasingly important role in the management of patients with ALL.

Aurora kinases regulate mitosis and are commonly overexpressed in leukemia. This phase I/IIa study of AT9283, a multikinase inhibitor, was designed to identify maximal tolerated doses, safety, pharmacokinetics, and pharmacodynamic activity in children with relapsed/refractory acute leukemia. The trial suffered from poor recruitment and terminated early, therefore failing to identify its primary endpoints. AT9283 caused tolerable toxicity, but failed to show clinical responses. Future trials should be based on robust preclinical data that provide an indication of which patients may benefit from the experimental agent, and recruitment should be improved through international collaborations and early combination with established treatment strategies. Refereed/Peer-reviewed

Childhood BCR-ABL1-positive B-cell precursor acute lymphoblastic leukemia (BCP-ALL) has an unfavorable outcome and is characterized by a high frequency of IKZF1 deletions. The prognostic value of IKZF1 deletions was evaluated in two cohorts of children with BCR-ABL1-positive BCP-ALL, before (pre-TKI) and after introduction of Imatinib (EsPhALL). IKZF1 deletions were found in 126/191 (66%) of the patients. In the pre-TKI cohort, IKZF1-deleted patients had an unfavorable outcome compared to wild-type patients (4-yr DFS 30.0±6.8% versus 57.5±9.4%, p=0.01). In the EsPhALL-cohort, the IKZF1 deletions were associated with an unfavorable prognosis in patients who were stratified by early clinical response in the good-risk arm (4-yr DFS 51.9±8.8% for IKZF1-deleted versus 78.6±13.9% for IKZF1 wild-type; p=0.03), even when treated with Imatinib (4-yr DFS 55.5±9.5% for IKZF1-deleted versus 75.0±21.7% for IKZF1 wild-type; p=0.05). In conclusion, IKZF1 deletions are predictive for a highly unfavorable outcome in children with BCR-ABL1-positive BCP-ALL irrespective the introduction of Imatinib. These results underscore the urgent need for alternative therapy for IKZF1-deleted BCR-ABL1-positive patients. In contrast, good-risk patients with IKZF1 wild-type responded remarkably well to Imatinib-containing regimens, thus providing a rationale to potentially avoid the use of hematopoietic stem cell transplantation in this subset of BCR-ABL1-positive children.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a heterogeneous disease that can be subdivided according to primary recurrent genetic abnormalities that are strongly associated with characteristic biological and clinical features. The first few cases with ZNF384-related rearrangements were described as early as 2002. The leukemic phenotype of these cases was not only BCP-ALL but also mixed phenotype acute leukemia (MPAL) and acute myeloid leukemia (AML) switched from ALL. The number of patients was small because this type of leukemia is rare and many of the fusions are cytogenetically cryptic. RNA sequencing revealed that 1% to 6% of childhood BCP-ALL cases, 5% to 15% of adult BCP-ALL cases and 48% of B/Myeloid MPAL cases harbored ZNF384 rearrangements. Of note, ZNF384 has a variety of partner genes such as TCF3, EP300 and TAF15. Their biological characteristics showed distinct expression profiles, and the cell origin might arise from primitive hematopoietic cells. The clinical features associated with ZNF384-related rearrangements have not been analyzed in a large cohort of patients. To identify the clinical characteristics of ZNF384-related rearrangements in childhood BCP-ALL (MPAL was excluded), we studied a total with 226 cases of ZNF384-related rearrangements from 15 international consortia who participate in the Ponte di Legno study group registered between 1987 and 2018. We analyzed the impact of outcome in association with clinical and biological characteristics. ZNF384-related rearrangements were detected by fluorescence in situ hybridization (FISH), reverse transcription polymerase chain reaction (RT-PCR) and/or next generation sequencing (NGS), according to local selection policies, or because of poor response to therapy. Additional genetic abnormalities were detected by multiplex ligation-dependent probe amplification (MLPA), single nucleotide polymorphism array (SNP array) and/or NGS. The median age of presentation was 9 years old (range, 1 - 25 years old). The female and male ratio was 1:1. Immunophenotypic characteristics were classified as BCP-ALL exclusively In addition, 33% were CD10 negative cases (cutoff 20%); 71% were CD13 positive; and 86% were CD33 positive. Complete hematological remission was achieved in 99% of cases. One third (31%) of patients were treated as high risk and one quarter (23%) of patients received a stem cell transplant in first remission. After a median follow-up of 5.3 years, the 5-year event free survival (EFS) rate was 84% (95%CI, 77 to 89%), and the 5-year overall survival (OS) rate was 91% (95% CI, 85 to 94%). There was no difference in survival rate by treatment era or by country or region of origin. The proportion of partner genes with ZNF384 was as follows: EP300 (37%, n=84), TCF3 (27%, n=60), TAF15 (8%, n=17), CREBBP (7%, n=16), others (8%, n=18) of identifiable partners, and unknown (14%, n=31), although a prospective unselected analysis is needed for an appropriate estimate of the partners distribution. Patients with an EP300-ZNF384 fusion had a significantly lower relapse rate at 5 years compared with the remaining patients: 5% (95% CI 2-14) versus 20% (12-32), hazard ratio 4.58 (1.56-13.45), p=0.006), respectively. The corresponding EFS and OS rates were 91% (81-96) vs. 76% (64-85), p=0.024 and 92% (81-96) vs. 90% (80-95), p=0.3, suggesting that the non-EP300 relapses were salvageable (Figure). Multivariate analysis adjusting for sex, age, WBC and treatment era did not alter these results. Of note, in cases of TCF3 and TAF15, relapse occurred very late even after 5 years from diagnosis. Additional genetic abnormalities such as IKZF1, PAX5, CDKN2A/2B deletions were also analyzed. The distribution of deletions by partner genes was different between fusion partners but were not significant as prognostic factors. We confirm that ZNF384 rearrangement is a biologically and clinically distinct subtype of BCP-ALL. Immunophenotype abnormalities imply that ZNF384 rearrangements arise from primitive hematopoietic cells. Even considering a potential selection bias for the retrospective nature of the study, the OS was excellent in this subtype, although, relapse events did not reach a plateau among patients with TCF3-ZNF384 and TAF15-ZNF384. On the other hand, EP300-ZNF384 showed good prognosis with a low relapse rate. The biological background in each fusion partner warrants further investigation. Disclosures Loh: Medisix Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees.

Acute lymphoblastic leukemia (ALL) is characterised by a number of recurrent chromosomal abnormalities which inform prognosis. Low hypodiploidy (HoTr) and high hyperdiploidy (HeH) are genetic subgroups associated with large non-random ploidy shifts, specifically 30-39 chromosomes and 51-65 chromosomes respectively. HoTr ALL often presents with a near triploid karyotype of 60-78 chromosomes through chromosomal endoreduplication without cytokinesis. This presents a diagnostic challenge in distinguishing this poor risk entity from good risk HeH ALL. To date, classification of such challenging cases has been based on the modal chromosome number, the pattern of specific gains, and identification of loss of heterozygosity (LOH) using single nucleotide polymorphism (SNP) arrays where possible. However, loss of cellular context and mixed cell populations (normal diploid, low hypodiploid and near-triploid) when analysing SNP arrays can pose additional analytical difficulty. The aim of this SNP array study was to (1) determine the level of inaccurate genetic subgrouping when cytogenetics only was used to distinguish HeH from doubled up HoTr and (2) develop a diagnostic algorithm to reliably call HeH and HoTr without supporting cytogenetic analysis. SNP arrays were performed on diagnostic ALL samples of 85 patients (46 HoTr, 39 HeH) using Illumina CytoSNP 850k (n=80) or Affymetrix Cytoscan HD (n=5) arrays. Probe level data were uploaded to Nexus Copy Number 10 (BioDiscovery) and manual segments spanning the length of each chromosome were created. No further data pre-processing was carried out and log2 ratio of 0 was automatically assigned to the median log2 ratio of the sample. Chromosomal log2 ratios were normalized within each sample and a variety of machine-learning techniques used to cluster the samples independently of assigned diagnosis. Cases residing in the incorrect cluster based on initial diagnosis were examined in detail using information from cytogenetics and SNP array interpretation. SNP arrays were analysed from 46 HoTr (median age 50.5 years (range 7-87), 43% male) and 39 HeH (median age 7 years (range 1-58), 56% male) patients. Unsupervised clustering of log2 ratios showed a clear distinction between HeH and HoTr patients (figure (A)). Six cases clustered incorrectly based on cytogenetic diagnosis. After detailed interpretation of all cases, including identifying LOH affecting chromosomes 3, 7, 15, 16 and 17, 3/6 cases initially classified as HeH were highly suggestive of HoTr ALL despite having Using whole chromosome log2 ratios, HoTr and HeH ALL have distinct profiles. SNP array analysis highlighted at least 4 patients whose ploidy subgroups appeared incorrectly called by cytogenetics, which can affect risk stratification. Crucially, these data call into question the accepted modal chromosome numbers for HeH and HoTr in the near triploid phase and suggest this alone cannot be used to classify patients into these ploidy groups. All samples incorrectly classified as HeH ALL were from adults aged >40 years, suggesting this good risk subgroup is even rarer than previously thought in older adults. After re-classification, our cohort only contained 5/41 adults >40 years with HeH ALL, signifying that HoTr ALL is the commonest genetic ploidy group in older adults with ALL and must still be considered in cases with 50-60 chromosomes. Copy number analysis from SNP arrays is challenging in samples with marked ploidy shift and mixed cell populations. Importantly, a number of our samples had significant contaminating normal DNA and LOH could not be confirmed visually (figure (B)), underlying the need for additional factors to aid classification. Our analysis only takes into account log2 ratio of entire chromosomes, thus permitting a measure of the relative over and under-representation of specific chromosomes within the sample. This method accurately clusters patients even when LOH cannot be clearly visualized from B-allele frequency due to contaminating non-leukemic DNA and supports the development of a diagnostic classifier based on chromosomal log2 ratios. Disclosures Fielding: Amgen: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Incyte: Consultancy.

The survival rate of childhood acute lymphoblastic leukemia (ALL) has reached >80-90% in developed countries, which is a triumph of modern medicine. This success is due to implementation of contemporary treatment protocols, optimal application of risk stratification, risk-directed multi-agent chemotherapy regimens, and improved supportive care. Unfortunately, such improvements have not translated to Low Middle Income Countries (LMICs), where 90% of the world's children live. The estimated 5 year survival rates in Asia range widely between 44.3% and 80%. The Intercontinental-BFM2002 study, conducted in 15 upper-middle and high-income countries reported a 5 year event-free survival (EFS) and overall survival (OS) rates of 74% and 82%, respectively. Factors that may contribute to the lower survival in LMICs are highly complex, including delays in presentation, diagnostic inaccuracy, restricted budget for risk-stratification and appropriate treatment, treatment abandonment and socio economic status. In Malaysia, different protocols are used by different leukemia treatment centers for treating children with ALL. In UKM Medical Centre (UKMMC), the UKALL protocols (modified UK X, XI, XII, 97(99) and 2003) have been used in the Pediatric Hemato-Oncology Unit since the 1990s. Herein, we report the adopted protocol, the stratification profile and outcome of children with ALL, treated with modified UKALL 97(99) and UKALL 2003 in our institution from 2006 to 2014. Clinical data from children with ALL, who received these modified therapies, were retrospectively reviewed. Prednisolone was used in modified UKALL97(99) and Dexamethasone in modified UKALL 2003, while 6-mercaptopurine was used in both modified protocols. Otherwise, chemotherapy and duration of treatment were identical to the original protocols of Regimens A, B and C. ALL was diagnosed based on standard morphology and immunophenotyping criteria. At diagnosis, patients were stratified according to the National Cancer Institute (NCI) risk criteria and using FISH for detection of cytogenetic abnormalities. EFS and OS were determined using the Kaplan-Meier methods. Newly diagnosed ALL in 156 children were included in the study; 103 (66.0%) were standard risk, 49 (31.4%) were high risk and 4 (2.5%) were infants. There were 2 children with Down syndrome. The success rate of FISH was 76.4% (94/123). Patients were stratified as standard risk, based on ETV6-RUNX1, and high risk based on unfavorable cytogenetics, BCR-ABL and MLL rearrangements. Half of the patients with unfavorable cytogenetics were classified in the NCI high risk group, with WCC >100x109/L. A total of 151 patients were treated as per risk stratification, 2 patients transferred care, while 3 patients refused treatment. Mortality from sepsis during treatment was approximately 10%, including 2 deaths during induction remission and induction at relapse. The majority of disease progression was relapse-related, however, treatment abandonment also contributed to relapse. Approximately 5% of patients abandoned their treatment (3 patients abandoned and 3 patients refused treatment). The 5-year OS for the standard risk group was 86.6%, with 3-year and 5-year EFS of 88.1% and 83.4%, respectively. The 5-year OS for the high risk group was 65.7%, while 3-year and 5-year EFS were 64.7% and 58.2%, respectively (Figure 1). The MRC UKALL97 stratification by NCI risk reported a 5-year EFS of 83.1% for the standard risk group and 66.9% for the high risk group, while the UKALL2003 interim analysis reported a 5-year EFS of 87.7%. The MRC UKALL97/99 reported a 5-year OS of 88%. The cure rate of children with standard risk ALL at UKMMC, using modified UKALL 97(99) and UKALL 2003 protocols, was comparable to MRC UKALL97. However, the cure rate for high risk ALL was comparatively lower. This single center study from UKMMC has highlighted some critical factors that improved the outcome of children with ALL and suggests further improvements that are necessary to reduce the relapse rate, especially in the high risk ALL patients. Disclosures No relevant conflicts of interest to declare.

The genetics of acute lymphoblastic leukemia are becoming well understood and the incidence of individual chromosomal abnormalities varies considerably with age. Cytogenetics provide reliable risk stratification for treatment: high hyperdiploidy and ETV6-RUNX1 are good risk, whereas BCR-ABL1, MLL rearrangements, and hypodiploidy are poor risk. Nevertheless, some patients within the good- and intermediate-risk groups will unpredictably relapse. With advancing technologies in array-based approaches (single nucleotide polymorphism arrays) and next-generation sequencing to study the genome, increasing numbers of new genetic changes are being discovered. These include deletions of B-cell differentiation and cell cycle control genes, as well as mutations of genes in key signaling pathways. Their associations and interactions with established cytogenetic subgroups and with each other are becoming elucidated. Whether they have a link to outcome is the most important factor for refinement of risk factors in relation to clinical trials. For several newly identified abnormalities, including intrachromosomal amplification of chromosome 21 (iAMP21), that are associated with a poor prognosis with standard therapy, appropriately modified treatment has significantly improved outcome. After the successful use of tyrosine kinase inhibitors in the treatment of BCR-ABL1–positive acute lymphoblastic leukemia, patients with alternative ABL1 translocations and rearrangements involving PDGFRB may benefit from treatment with tyrosine kinase inhibitors. Other aberrations, for example, CRLF2 overexpression and JAK2 mutations, are also providing potential novel therapeutic targets with the prospect of reduced toxicity.

Ruxolitinib is an oral Janus-activated kinase 1 (JAK1)/JAK2 inhibitor approved for the treatment of patients with myelofibrosis based on the results of two randomized clinical trials. However, discordant indications were provided by regulatory agencies and scientific societies for selecting the most appropriate candidates to this drug. The European LeukemiaNet and the Italian Society of Hematology shared the aim of building evidence-based recommendations for the use of ruxolitinib according to the GRADE methodology. Eighteen patient-intervention-comparator-outcome profiles were listed, each of them comparing ruxolitinib to other therapies with the aim of improving one of the three clinical outcomes: (a) splenomegaly, (b) disease-related symptoms, and (c) survival. Ruxolitinib was strongly recommended for improving symptomatic or severe (>15 cm below the costal margin) splenomegaly in patients with an International Prognostic Scoring System (IPSS)/dynamic IPSS risk intermediate 2 or high. Ruxolitinib was also strongly recommended for improving systemic symptoms in patients with an MPN10 score >44, refractory severe itching, unintended weight loss not attributable to other causes or unexplained fever. Because of weak evidence, the panel does not recommend ruxolitinib therapy for improving survival. Also, the recommendations given above do not necessarily apply to patients who are candidates for allogeneic stem cell transplant.

Abstract 88 Chromosomal abnormalities in childhood ALL are important disease markers and predictors of prognosis. Virtually all modern protocols recommend that patients with t(9;22), MLL translocations and haploidy ( We investigated the prognostic relevance of cytogenetics among 1,934 children treated on MRC ALL97. Patients with t(9;22), haploidy, low hypodiploidy and those aged The 5yr RFS of the whole cohort was 81% (95% CI 79–82%) with a median follow-up of 8.2yrs. Univariate analysis revealed 5 abnormalities that were significantly associated with relapse: t(12;21) Hazard ratio (HR)=0.50 (95% CI 0.36, 0.68); high hyperdiploidy (51–65 chromosomes): 0.58 (0.45, 0.74); iAMP21: 5.51 (3.57, 8.50); t(9;22): 3.31 (2.06, 5.32); other MLL translocations [not t(4;11)]: 2.70 (1.39, 5.25); and 17p loss [del(17p)]: 2.13 (1.35, 3.34) (all p Further analysis of high hyperdiploid patients revealed that there was no difference in RFS for those with (n=218) and without (n=200) triple trisomy (+4, +10 and +17) [HR=0.81 (0.49, 1.34), p=0.4]. However, high hyperdiploid patients with a +18 (n=396) had a lower risk of relapse [HR=0.44 (0.26, 0.74) (p=0.002)] than other patients (n=86). Among 369 t(12;21) patients 47 (13%) suffered a relapse. The timing of these relapses was different to the rest of the cohort with fewer early relapses (within 6 months of the end of treatment) in the t(12;21) cohort: 9/47 (19%) versus 170/285 (59%), p A total of 54/1596 (3.4%) patients had del(17p) by cytogenetics: i(17q) (n=18), del(17)(p) (n=7), monosomy 17 (n=8) and unbalanced translocations (n=21). It is a secondary abnormality co-existing with high hyperdiploidy (n=21), t(12;21) (n=6), MLL translocations (n=2) and t(9;22) (n=1). Overall, these patients had an inferior RFS (64%, (49%–75%), p=0.004). However, the presence of del(17p) did not abrogate the good outcome of patients with t(12;21) and high hyperdiploidy [HR=1.70 (0.75, 3.87) p=0.206] but did represent a strong marker of relapse among other patients [HR=2.96 (1.68, 5.20) p We classified 1,733 patients into three cytogenetic risk groups: good (GRG) [t(12;21), high hyperdiploidy] n=928 (54%); poor (PRG) [t(9;22), t(4;11), other MLL, t(17;19), haploidy, low hypodiploidy, iAMP21, del(17p)] n=153 (9%); standard (SRG) [all other cases] n=652 (38%). The 5 year RFS were GRG 88% (85–90%), SRG 79% (75–82%) and PRG 49% (40–57%). In multivariate analysis, patients in the GRG or PRG were less or more likely to relapse compared to patients in the SRG: HR=0.65 (0.50–0.83), p=0.001 and HR=2.67 (2.01–3.53), p These data clarify the prognostic relevance of several chromosomal abnormalities in the context of a modern childhood ALL therapy and provide further evidence that cytogenetics is a powerful and independent indicator of relapse risk. Disclosures: No relevant conflicts of interest to declare.

Del (9q) is a recurrent cytogenetic abnormality in acute myeloid leukaemia (AML). We report an analysis of 81 patients with del(9q) as a diagnostic karyotypic abnormality entered into the Medical Research Council AML trials 10, 11 and 12. Patients were divided into three groups: (i) Sole del (9q), 21 patients ; (ii) Del(9q) in association with t(8 ; 21), 29 patients ; (iii) Del(9q) in association with other cytogenetic abnormalities, 31 patients. Sole del(9q) was associated with a characteristic bone marrow phenotype at diagnosis: a single Auer rod was found in all cases examined. There was also an association with erythroid dysplasia (74%) and granylocytic lineage vacuolation (90%). The incidence of all three of these features was significantly higher (P < 0.05) in the sole del(9q) group compared with control cases lacking del(9q). The overall survival (OS) of all 81 patients was compared with a control group of 1738 patients with normal cytogenetics entered in the same trials over the period of investigation. The 5-year OS for patients with del(9q) was 45%, compared with 35% for the control group (P = 0.09). Patients with del(9q) in association with t(8 ; 21) had a 5-year OS of 75%, which was significantly better than the groups with either sole del(9q) (40%) and del(9q) with other abnormalities (26% ; P = 0.008). Karyotyping indicated a common area of deletion in the region 9q21-22, which was present in 94% of cases. It is likely that the deletion of single or multiple tumour suppressor genes located in this region may underlie the pathogenesis of del (9q) AML.

Abstract 290 Multiplex Ligation-dependent Probe Amplification (MLPA) provides an accurate and reliable high throughput method to screen for copy number abnormalities (CNA) of the significant genes in BCP-ALL. We have screened a consecutive cohort of 1427 childhood BCP-ALL patients treated on UKALL97 and UKALL2003 using the P335 SALSA MLPA kit IKZF1 (MRC Holland, The Netherlands). We here report the association of CNA involving these genes with clinical features. The overall breakdown of CNA was CDKN2A/B 28% (395), ETV6 22% (312), PAX5 19% (272), IKZF1 14% (196), RB1 6% (92), BTG1 6% (89) and EBF1 2% (30), as well as the rearrangement, P2RY8-CRLF2 4% (63). The cohort comprised 762 (54%) males and 665 (46%) females. There was no shift in the gender balance according to CNA. The median age of the entire cohort was 5 years (range 1–23) with 24% of patients being ≥10 years. There was some correlation between the distribution of CNA and patient age. Patients with IKZF1 and CDKN2A/B deletions were significantly older at 7 years (p50×109/L (each p There is particular interest in IKZF1 and CRLF2 in relation to outcome in BCP-ALL. We have previously shown an intermediate outcome for patients with CRLF2 rearrangements. We here examined the prognostic effect of IKZF1 deletions in this patient cohort; but excluded Down syndrome and BCR-ABL1 positive patients from survival analyses. Among 469 ALL97 patients, 13% (63) had IKZF1 deletions and after a median follow-up time of 9.5 years, 49% (31) had relapsed and 40% (25) had died. In the NCI standard risk (SR) group, patients with IKZF1 deletions had a significantly inferior event free survival (EFS) at 5 years (61% v 85%, p=0.0006). However, the effect appeared to be largely confined to patients with intermediate risk cytogenetics (63% v 85%, p=0.025) rather than those with good risk cytogenetics (75% v 87%, p=0.2915). As expected the NCI high risk (HR) patients with IKZF1 deletions had an inferior EFS: 50% v 68%, p=0.0112. In ALL2003, 776 patients with a median follow-up time of 3.9 years were available for analysis. Among 11% (87) patients with IKZF1 deletions, 13% (11) had relapsed and 5% (4) died in remission, resulting in a significantly inferior 5 year EFS: 78% v 90% p=0.0025. Of note, 8/11 IKZF1 deleted patients who relapsed were MRD positive (>0.01%) at day 28 and only 1/22 patients with good risk cytogenetics and IKZF1 deletion relapsed. There was no evidence that the type of deletion (deletion of exons 4–7 vs others) was linked to outcome in either the ALL97 or ALL2003 cohort. In conclusion, we have shown that IKZF1, PAX5 and/or CDKN2A/B aberrations are significantly associated with NCI HR, which may point to an associated poor outcome. Indeed, we showed that patients with IKZF1 deletions had an inferior outcome overall. However, there was evidence to suggest that the effect was pleiotropic and, importantly, IKZF1 deleted patients treated on ALL2003 in general achieved a respectable EFS rate of 75% at 5 years. Although in most studies IKZF1 deletions have been associated with a poor prognosis in BCP-ALL, while the risk relating to CRLF2 has been variable, thus far these findings, including those from this study, do not support changes to treatment for patients with these abnormalities. Disclosures: No relevant conflicts of interest to declare.

Abstract 292 Children with ETV6-RUNX1 or high hyperdiploid (HeH, 51–65 chromosomes) acute lymphoblastic leukaemia (ALL) have an excellent prognosis when treated on modern protocols and achieve survival rates in excess of 90% at 5 years. They are prevalent aberrations and together account for nearly 60% cases at diagnosis. Approximately 10–15% patients with these aberrations will suffer a relapse; usually off-treatment. Despite their good outcome their high prevalence means that they account for roughly 40% childhood ALL relapse. Therefore predicting those that will suffer a relapse remains a significant clinical issue. Numerous studies have postulated that certain secondary genetic abnormalities or cytogenetic characteristics correlate with outcome. However, the results have been inconsistent and few abnormalities or characteristics have been studied or confirmed in independent cohorts. In this study, we have investigated the prognostic impact of the major secondary abnormalities and characteristics among ETV6-RUNX1 and HeH patients treated on MRC ALL97/99 between 1997 and 2002. The cohort had a median follow-up time of 8.5 years and survival rates are quoted at 7 years. Additional genetic abnormalities were detected either by fluorescence in situ hybridization (FISH) using probes for ETV6 and RUNX1 or Multiplex Ligation-Dependent Probe Amplification (MLPA) using the SALSA P335 ALL IKZF1 kit. Patients were also analysed by National Cancer Institute (NCI) risk status: standard risk (SR) patient were less than 10 years old with a white cell count (WCC) lower than 50×109/L; high risk (HR) included all other patients. Among 368 ETV6-RUNX1 patients, 47 (13%) relapsed and 20 died (5%) resulting in event free (EFS) and overall survival (OS) rates of 88% and 96%, respectively. The majority of patients were SR (n=283, 77%) and although they did not have a significantly lower relapse rate (32/283 (11%) v 15/85 (18%), p=0.14) they did have a lower death rate (8/283 (3%) v 12/85 (14%), p Among 553 HeH patients, 81 (15%) relapsed and 48 (9%) died resulting in EFS and OS rates of 83% and 91%, respectively. The majority of patients were SR (79%) with a significantly lower relapse and death rate: 57/439 (13%) v 24/114 (21%), p=0.033 and 30/439 (7%) v 18/114 (16%), p=0.002). HR patients had significantly inferior EFS and OS rates compared to SR patients: 76% v 85% (p=0.03) and 85% v 94% (p=0.002). HeH karyotypes were classified according to the presence of triple trisomy (+4,+10,+17) (47%), double trisomy (+4,+10) (60%), trisomy 18 (78%) and modal chromosome number (51–53, 21%; 54–57, 56%; 58–65, 23%). Only the incidence of trisomy 18 varied by NCI risk status: SR 80% v HR 69%, p=0.016. The incidence of micro-deletions detected among 144 patients was similar for SR and HR patients: CDKN2A/B (16%), IKZF1 (9%), ETV6 (8%), PAX5 (3%), RB1 (3%), BTG1 (1%) and EBF1 (0%). Among SR patients only trisomy 18 was significantly associated with a good outcome: EFS 88% v 73% (p=0.0013); OS 96% v 90% (p=0.016). There were no significant risk factors among the HR group; although all 4 patients with an IKZF1 deletion relapsed. In contrast to previous studies most secondary genetic abnormalities or cytogenetic characteristics were not associated with outcome in this study. Although NCI HR patients did have a significantly inferior outcome compared to their SR counterparts with the same chromosomal abnormality, the EFS and OS rates at 7 years were in excess of 75%, highlighting the excellent prognosis for patients with these abnormalities. These findings support the risk stratification of ETV6-RUNX1 and HeH patients by NCI risk status on current protocols. Disclosures: No relevant conflicts of interest to declare.

The clinical features, cytogenetics and response to treatment have been examined in 180 infants (aged

Recent genomic studies have provided a refined genetic map of acute lymphoblastic leukemia (ALL) and increased the number of potential prognostic markers. Therefore, we integrated copy-number alteration data from the 8 most commonly deleted genes, subordinately, with established chromosomal abnormalities to derive a 2-tier genetic classification. The classification was developed using 809 ALL97/99 patients and validated using 742 United Kingdom (UK)ALL2003 patients. Good-risk (GR) genetic features included ETV6-RUNX1, high hyperdiploidy, normal copy-number status for all 8 genes, isolated deletions affecting ETV6/PAX5/BTG1, and ETV6 deletions with a single additional deletion of BTG1/PAX5/CDKN2A/B. All other genetic features were classified as poor risk (PR). Three-quarters of UKALL2003 patients had a GR genetic profile and a significantly improved event-free survival (EFS) (94%) compared with patients with a PR genetic profile (79%). This difference was driven by a lower relapse rate (4% vs 17%), was seen across all patient subgroups, and was independent of other risk factors. Even genetic GR patients with minimal residual disease (>0.01%) at day 29 had an EFS in excess of 90%. In conclusion, the integration of genomic and cytogenetic data defines 2 subgroups with distinct responses to treatment and identifies a large subset of children suitable for treatment deintensification.

This study identifies multiple copies of the AML1 gene on a duplicated chromosome 21, dup(21), as a recurrent abnormality in acute lymphoblastic leukemia (ALL). Clusters of AML1 signals were visible at interphase by fluorescence in situ hybridization (FISH). In metaphase, they appeared tandemly duplicated on marker chromosomes of five distinct morphological types: large or small acrocentrics, metacentrics, submetacentrics or rings. The markers comprised only chromosome 21 material. Karyotypes were near-diploid and, besides dup(21), no other established chromosomal changes were observed. A total of 20 patients, 1.5 and

Contains fulltext : 172831.pdf (Publisher’s version ) (Closed access) Somatic genetic abnormalities are initiators and drivers of disease and have proven clinical utility at initial diagnosis. However, the genetic landscape and its clinical utility at relapse are less well understood and have not been studied comprehensively. We analyzed cytogenetic data from 427 children with relapsed B-cell precursor ALL treated on the international trial, ALLR3. Also we screened 238 patients with a marrow relapse for selected copy number alterations (CNAs) and mutations. Cytogenetic risk groups were predictive of outcome postrelapse and survival rates at 5 years for patients with good, intermediate-, and high-risk cytogenetics were 68%, 47%, and 26%, respectively (P < .001). TP53 alterations and NR3C1/BTG1 deletions were associated with a higher risk of progression: hazard ratio 2.36 (95% confidence interval, 1.51-3.70, P < .001) and 2.15 (1.32-3.48, P = .002). NRAS mutations were associated with an increased risk of progression among standard-risk patients with high hyperdiploidy: 3.17 (1.15-8.71, P = .026). Patients classified clinically as standard and high risk had distinct genetic profiles. The outcome of clinical standard-risk patients with high-risk cytogenetics was equivalent to clinical high-risk patients. Screening patients at relapse for key genetic abnormalities will enable the integration of genetic and clinical risk factors to improve patient stratification and outcome. This study is registered at www.clinicaltrials.org as #ISCRTN45724312.

Recurrent deletions of the long arm of chromosome 5 were detected in 23/200 cases of T-cell acute lymphoblastic leukemia. Genomic studies identified two types of deletions: interstitial and terminal. Interstitial 5q deletions, found in five cases, were present in both adults and children with a female predominance (chi-square, P=0.012). Interestingly, these cases resembled immature/early T-cell precursor acute lymphoblastic leukemia showing significant down-regulation of five out of the ten top differentially expressed genes in this leukemia group, including TCF7 which maps within the 5q31 common deleted region. Mutations of genes known to be associated with immature/early T-cell precursor acute lymphoblastic leukemia, i.e. WT1, ETV6, JAK1, JAK3, and RUNX1, were present, while CDKN2A/B deletions/mutations were never detected. All patients had relapsed/resistant disease and blasts showed an early differentiation arrest with expression of myeloid markers. Terminal 5q deletions, found in 18 of patients, were more prevalent in adults (chi-square, P=0.010) and defined a subgroup of HOXA-positive T-cell acute lymphoblastic leukemia characterized by 130 up- and 197 down-regulated genes. Down-regulated genes included TRIM41, ZFP62, MAPK9, MGAT1, and CNOT6, all mapping within the 1.4 Mb common deleted region at 5q35.3. Of interest, besides CNOT6 down-regulation, these cases also showed low BTG1 expression and a high incidence of CNOT3 mutations, suggesting that the CCR4-NOT complex plays a crucial role in the pathogenesis of HOXA-positive T-cell acute lymphoblastic leukemia with terminal 5q deletions. In conclusion, interstitial and terminal 5q deletions are recurrent genomic losses identifying distinct subtypes of T-cell acute lymphoblastic leukemia.

Intrachromosomal amplification of chromosome 21 (iAMP21) defines a distinct subgroup of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) that has a dismal outcome when treated with standard therapy. For improved diagnosis and risk stratification, the initiating genetic events need to be elucidated. To investigate the genetic basis of BCP-ALL, genomes of 94 iAMP21 patients were interrogated by arrays, FISH, and multiplex ligation-dependent probe amplification. Most copy number alterations targeted chromosome 21, reinforcing the complexity of this chromosome. The common region of amplification on chromosome 21 was refined to a 5.1-mb region that included RUNX1, miR-802, and genes mapping to the Down syndrome critical region. Recurrent abnormalities affecting genes in key pathways were identified: IKZF1 (22%), CDKN2A/B (17%), PAX5 (8%), ETV6 (19%), and RB1 (37%). Investigation of clonal architecture provided evidence that these abnormalities, and P2RY8-CRLF2, were secondary to chromosome 21 rearrangements. Patient outcome was uniformly poor with standard therapy irrespective of the presence or absence of these changes. This study has provided evidence that chromosome 21 instability is the only anomaly among those so far investigated that is common to all iAMP21 patients, and therefore the initiating event is likely to be found among the complex structural rearrangements of this abnormal chromosome.

Deregulated expression of CRLF2 (CRLF2-d) arises via its juxtaposition to the IGH@ enhancer or P2RY8 promoter. Among 865 BCP-ALL children treated on MRC ALL97, 52 (6%) had CRLF2-d, but it was more prevalent among Down syndrome patients (54%). P2RY8-CRLF2 (n = 43) was more frequent than IGH@-CRLF2 (n = 9). CRLF2-d was not associated with age, sex, or white cell count, but IGH@-CRLF2 patients were older than P2RY8-CRLF2 patients (median 8 vs 4 years, P = .0017). Patients with CRLF2-d were more likely to present with enlarged livers and spleens (38% vs 18%, P < .001). CRLF2-d was not seen in conjunction with established chromosomal translocations but 6 (12%) cases had high hyperdiploidy, and 5 (10%) had iAMP21. Univariate analysis suggested that CRLF2-d was associated with an inferior outcome: (event-free survival [EFS] hazard ratio 2.27 [95% confidence interval 1.48-3.47], P < .001; OS 3.69 [2.34-5.84], P < .001). However, multivariate analysis indicated that its effect was mediated by other risk factors such as cytogenetics and DS status (EFS 1.45 [0.88-2.39], P = .140; OS 1.90 [1.08-3.36], P = .027). Although the outcome of IGH@-CRLF2 patients appeared inferior compared with P2RY8-CRLF2 patients, the result was not significant (EFS 2.69 [1.15-6.31], P = .023; OS 2.86 [1.15-6.79], P = .021). Therefore, we concluded that patients with CRLF2-d should be classified into the intermediate cytogenetic risk group.

Diagnostic karyotype provides the framework for risk-stratification schemes in acute myeloid leukemia (AML); however, the prognostic significance of many rare recurring cytogenetic abnormalities remains uncertain. We studied the outcomes of 5876 patients (16-59 years of age) who were classified into 54 cytogenetic subgroups and treated in the Medical Research Council trials. In multivariable analysis, t(15;17)(q22;q21), t(8;21)(q22;q22), and inv(16)(p13q22)/t(16;16)(p13;q22) were the only abnormalities found to predict a relatively favorable prognosis (P < .001). In patients with t(15;17) treated with extended all-trans retinoic acid and anthracycline-based chemotherapy, additional cytogenetic changes did not have an impact on prognosis. Similarly, additional abnormalities did not have a significant adverse effect in t(8;21) AML; whereas in patients with inv(16), the presence of additional changes, particularly +22, predicted a better outcome (P = .004). In multivariable analyses, various abnormalities predicted a significantly poorer outcome, namely abn(3q) (excluding t(3;5)(q25;q34)), inv(3)(q21q26)/t(3;3)(q21;q26), add(5q)/del(5q), −5, −7, add(7q)/del(7q), t(6;11)(q27;q23), t(10;11)(p11∼13;q23), other t(11q23) (excluding t(9;11)(p21∼22;q23) and t(11;19)(q23;p13)), t(9;22)(q34;q11), −17, and abn(17p). Patients lacking the aforementioned favorable or adverse aberrations but with 4 or more unrelated abnormalities also exhibited a significantly poorer prognosis (designated “complex” karyotype group). These data allow more reliable prediction of outcome for patients with rarer abnormalities and may facilitate the development of consensus in reporting of karyotypic information in clinical trials involving younger adults with AML. This study is registered at http://www.isrctn.org as ISRCTN55678797 and ISRCTN17161961.

BACKGROUND: Chromosomal abnormalities in childhood acute lymphoblastic leukaemia are well established disease markers and indicators of outcomes. However, the long-term prognosis and independent prognostic effect of some abnormalities has been questioned. Also, little is known about the association between cytogenetics and the characteristics of relapse (eg, time and site of relapse) that are known to predict outcome after relapse. METHODS: We analysed cytogenetic data from 1725 children with B-cell precursor acute lymphoblastic leukaemia who were included in the UK Medical Research Council ALL97/99 study and followed up for a median time of 8.2 years. Univariate and multivariate analysis were done to examine risk of relapse, event-free survival, and overall survival associated with 21 chromosomal abnormalities and three cytogenetic risk groups constructed from these data. FINDINGS: Two chromosomal abnormalities were associated with a significantly better outcome (ETV6-RUNX1, hazard ratio [HR] 0.51, 95% CI 0.38-0.70 and high hyperdiploidy, 0.60, 0.47-0.78), whereas five abnormalities were associated with an increased risk of relapse (intrachromosomal amplification of chromosome 21 [iAMP21], 6.04, 3.90-9.35; t(9;22), 3.55, 2.21-5.72; MLL translocations, 2.98, 1.71-5.20; abnormal 17p, 2.09, 1.30-3.37; and loss of 13q, 1.87, 1.09-3.20). Multivariate analysis incorporating age, white-cell count, and treatment parameters showed that six cytogenetic abnormalities (ETV6-RUNX1, high hyperdiploidy, iAMP21, t(9;22), loss of 13q, and abnormal 17p) retained their significance for effect on relapse risk. Based on these data, patients were classified into good, intermediate, and poor cytogenetic risk groups. Slow early treatment response correlated with cytogenetic risk group: 34 of 460 (7%) in the good-risk group, 22 of 211 (10%) in the intermediate-risk group, and 27 of 95 (28%) in the poor-risk group had a slow response (p

A large series of plasma cell dyscrasias (n=2207) was examined for translocations which deregulate the MAF genes, t(14;20)(q32;q12) and t(14;16)(q32;q23), and their disease behavior was compared to a group characterized by the t(4;14)(p16;q32) where CCND2 is also up-regulated. The t(14;20) showed low prevalence in myeloma (27/1830, 1.5%) and smoldering myeloma (1/148

Between 1980 and 2001, the United Kingdom Medical Research Council Childhood Leukemia Working Party conducted four clinical trials in acute lymphoblastic leukaemia (ALL), which recruited a total of 6516 patients. UKALL VIII examined the role of daunorubicin in induction chemotherapy, and UKALL X examined the role of post-induction intensification. Both resulted in major improvement in the outcomes. UKALL XI examined the efficacy of different methods of central nervous system-directed therapy and the effects of an additional intensification. ALL97, which was initially based on the UKALL XD template (two intensification phases), examined the role of different steroids in induction and of different thiopurines through continuing chemotherapy. A reappraisal of results from UKALL XI compared with other cooperative group results led to a redesign in 1999, which subsequently resulted in a major improvement in outcomes. In addition, ALL97 and ALL97/99 showed a significant advantage for the use of dexamethasone rather than prednisolone; although the use of 6-thioguanine resulted in fewer relapses, this advantage was offset by an increased incidence of deaths in remission. Over the era encompassed by these four trials, there has been a major improvement in both event-free and overall survival for children in the United Kingdom with ALL.

Background Multiple myeloma, monoclonal gammopathy of undetermined significance and smoldering multiple myeloma harbor common chromosomal abnormalities but the prevalence and relative association of aberrations in these diagnostic groups remains controversial. We investigated these aspects in a large series of patients.Design and Methods Chromosome 13 deletion (Δ13), deletion of TP53, ploidy status and immunoglobulin heavy chain (IgH) translocations were evaluated by fluorescence in situ hybridization in patients with monoclonal gammopathy of undetermined significance (n=189), smoldering multiple myeloma (n=127) and multiple myeloma (n=400).Results Overall, Δ13 (25%, 34% and 47%), 16q23 deletions (6%, 8% and 21%) and 17p13 deletions (3%, 1% and 10%) were less frequent in patients with monoclonal gammopathy of undetermined significance and smoldering multiple myeloma than in those with multiple myeloma. When distinct genetic groups were considered, no differences in the prevalence of Δ13 were found with t(4;14)(p16;q32) and t(14;16)(q32;q23) among the three diagnostic groups; in contrast Δ13 was rarer in t(11;14)(q13;q32) in patients with monoclonal gammopathy (1/28) and smoldering myeloma (2/13) than in those with multiple myeloma (40%). Similar results were seen for the few t(6;14)(p21;q32) cases: 0/3 patients with monoclonal gammopathy or smoldering myeloma had the Δ13, whereas 4/6 (67%) patients with multiple myeloma and this translocation also had the deletion. In multiple myeloma patients with both an IgH translocation and Δ13, the proportions of cells affected by the two abnormalities were similar, as was the case for t(4;14) and t(14;16) monoclonal gammopathy patients positive for Δ13. In contrast, in monoclonal gammopathy patients with t(14;20)(q32;q11), the translocation was present in almost all cells, while the Δ13 was present in only a sub-population.Conclusions These results indicate that the presence and time of occurrence of Δ13 depends on the presence of specific concurrent abnormalities. The observation that Δ13 was extremely rare in monoclonal gammopathy of undetermined significance and smoldering multiple myeloma with translocations directly involving cyclin D genes (CCND1 and CCND3) suggest a possible role of Δ13 in the progression of the disease specifically in these genetic sub-groups. (clinicaltrials.gov identifier: ISRCTN 68454111; UKCRN ID 1176).

We report 2 novel, cryptic chromosomal abnormalities in precursor B-cell acute lymphoblastic leukemia (BCP-ALL): a translocation, either t(X;14)(p22;q32) or t(Y;14)(p11;q32), in 33 patients and an interstitial deletion, either del(X)(p22.33p22.33) or del(Y)(p11.32p11.32), in 64 patients, involving the pseudoautosomal region (PAR1) of the sex chromosomes. The incidence of these abnormalities was 5% in childhood ALL (0.8% with the translocation, 4.2% with the deletion). Patients with the translocation were older (median age, 16 years), whereas the patients with the deletion were younger (median age, 4 years). The 2 abnormalities result in deregulated expression of the cytokine receptor, cytokine receptor-like factor 2, CRLF2 (also known as thymic stromal-derived lymphopoietin receptor, TSLPR). Overexpression of CRLF2 was associated with activation of the JAK-STAT pathway in cell lines and transduced primary B-cell progenitors, sustaining their proliferation and indicating a causal role of CRLF2 overexpression in lymphoid transformation. In Down syndrome (DS) ALL and 2 non-DS BCP-ALL cell lines, CRLF2 deregulation was associated with mutations of the JAK2 pseudokinase domain, suggesting oncogenic cooperation as well as highlighting a link between non-DS ALL and JAK2 mutations.

Translocations involving chromosome 11q23 frequently occur in pediatric acute myeloid leukemia (AML) and are associated with poor prognosis. In most cases, the MLL gene is involved, and more than 50 translocation partners have been described. Clinical outcome data of the 11q23-rearranged subgroups are scarce because most 11q23 series are too small for meaningful analysis of subgroups, although some studies suggest that patients with t(9;11)(p22;q23) have a more favorable prognosis. We retrospectively collected outcome data of 756 children with 11q23- or MLL-rearranged AML from 11 collaborative groups to identify differences in outcome based on translocation partners. All karyotypes were centrally reviewed before assigning patients to subgroups. The event-free survival of 11q23/MLL-rearranged pediatric AML at 5 years from diagnosis was 44% (± 5%), with large differences across subgroups (11% ± 5% to 92% ± 5%). Multivariate analysis identified the following subgroups as independent prognostic predictors: t(1;11)(q21;q23) (hazard ratio [HR] = 0.1, P = .004); t(6;11)(q27;q23) (HR = 2.2, P < .001); t(10;11)(p12;q23) (HR = 1.5, P = .005); and t(10;11)(p11.2;q23) (HR = 2.5, P = .005). We could not confirm the favorable prognosis of the t(9;11)(p22;q23) subgroup. We identified large differences in outcome within 11q23/MLL-rearranged pediatric AML and novel subgroups based on translocation partners that independently predict clinical outcome. Screening for these translocation partners is needed for accurate treatment stratification at diagnosis.

Chromosomal rearrangements are recurrent findings in human cancer and result in aberrant restructuring of the genome. The majority of known fusion genes are the consequence of reciprocal (balanced) translocations. However, most translocations described in human cancer are unbalanced, suggesting that other cancer genes remain to be identified. Historically, it was assumed that these unbalanced rearrangements affected gene function through the loss or gain of chromosomal material. However, emerging data supports direct disruption of genes located at or close to the unbalanced translocation breakpoints. New approaches are required for the identification of those gene loci underlying unbalanced translocations in cancer, as traditional methods have had limited success. This review focuses on one such strategy, using traditional and innovative molecular technologies to characterize breakpoint heterogeneity within a series of acute lymphoblastic leukemia (ALL) patients with dicentric chromosomes. This approach has shown that in ALL, specific gene loci can be targeted by heterogeneous translocation breakpoints involving multiple partner chromosomes. Carcinomas have a high proportion of unbalanced rearrangements and relatively few significant genes have been identified. The application of the same strategy to their analysis will lead to the discovery of novel cancer genes and improve our understanding of the genetic basis of tumorigenesis.

We previously reported that susceptibility to childhood B cell precursor ALL (BCP ALL) is associated with HLA-DPB1 alleles having glutamic acid (E) rather than lysine (K) in the P4 antigenic peptide-binding pocket. Clustering approximately 90% of DPB1 alleles into DPB69E (DP2, 6, 8) and DPB69K (DP1, 3, 4) supertypes revealed that DP2 and DP8 are associated with BCP ALL, but DP6 is also associated with non-BCP leukaemia. Here, we report that only one of seven alleles with the DP6 supertype (DPB1(*)0601) is associated with childhood leukaemia (leukaemia vs controls: odds ratio, 95% confidence interval [OR, CI]: 4.6, 2.0-10.4; corrected P=0.019), but not with childhood solid tumours or lymphomas. DPB1(*)0601 is also significantly associated with leukaemia subtypes, including BCP ALL, Pro-B ALL, T-ALL and AML. DPB1(*)0601 is significantly over-transmitted (76.9%) from parents to children with BCP ALL (OR; CI: 4.7; 1.01-22.2). Sequencing the coding region of DPB1(*)0601 revealed an exon 1-4 haplotype [T-DEAV-KIL-RVI] shared with DPB1(*)0301 and 0901, but no evidence of germline mutations in childhood leukaemia. These results suggest that the DPbeta0601 molecule may be functionally involved in childhood leukaemia. Analysis of peptide binding and T-cell activation by DPbeta0601-peptide complexes should help determine its role in childhood leukaemia causation.

Two children presenting with sporadic unilateral retinoblastoma and exhibiting a high degree of chromosome breakage were noted to have unusual facies, microcephaly and abnormal skin pigmentation. In the first child the pattern of both spontaneous and mitomycin-C-induced chromosome breakage was characteristic of Fanconi's anaemia although the degree of breakage was extreme. She also exhibited a striking increase in X-ray-induced chromosomal damage in G0 lymphocytes as measured by dicentric formation and increase in chromatid-type aberrations. She had a number of typical clinical features, including cafe-au-lait patches and abnormalities involving the kidney; however, she demonstrated neither the hypoplasia of radius and thumb nor the typical aplastic phase of this disorder. At age 22 months the child became anaemic with trilineage myelodysplasia, which was rapidly followed by the development of acute myeloblastic leukaemia. The early onset (at age 4 months) of retinoblastoma may have been associated with the underlying genomic instability. The second child exhibited a pattern of chromosome breakage characteristic of Bloom's syndrome, in addition to a moderate increase in damage induced by mytomycin-C. She had the typical stunted growth and malar hypoplasia of Bloom's syndrome although she did not demonstrate the frequently described erythematous 'butterfly rash' Although patients with Fanconi's anaemia and Bloom's syndrome are recognised to be at an increased risk of cancer, retinoblastoma has not previously been described in patients with either condition. We suggest that underlying recessive chromosome breakage syndromes may be underdiagnosed in paediatric cancer patients, with important implications for prognosis and genetic counselling.

Children with Down syndrome (DS) have a markedly increased risk of acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). To identify chromosomal changes cooperating with +21 that may provide information on the pathogenesis of these leukemias, we analyzed 215 DS-ALLs and 189 DS-AMLs. Unlike previous smaller series, a significant proportion of DS-ALLs had the typical B-cell precursor ALL abnormalities high hyperdiploidy (HeH; 11%) and t(12;21)(p13;q22) (10%). The HeH DS-ALLs were characterized by gains of the same chromosomes as non–DS-HeH, suggesting the same etiology/pathogenesis. In addition, specific genetic subtypes of DS-ALL were suggested by the significant overrepresentation of cases with +X, t(8;14)(q11;q32), and del(9p). Unlike DS-ALL, the common translocations associated with non–DS-AML were rare in DS-AML, which instead were characterized by the frequent presence of dup(1q), del(6q), del(7p), dup(7q), +8, +11, del(16q), and +21. This series of DS leukemias—the largest to date—reveals that DS-ALL is a heterogeneous disorder that comprises both t(12;21) and HeH as well as DS-related abnormalities. Furthermore, this analysis confirms that DS-AML is a distinct entity, originating through other genetic pathways than do non–DS-AMLs, and suggests that unbalanced changes such as dup(1q), +8, and +21 are involved in the leukemogenic process.