A genome-wide association study identifies risk loci for childhood acute lymphoblastic leukemia at 10q26.13 and 12q23.1.

Genome-wide association studies (GWASs) have shown that common genetic variation contributes to the heritable risk of childhood acute lymphoblastic leukemia (ALL). To identify new susceptibility loci for the largest subtype of ALL, B-cell precursor ALL (BCP-ALL), we conducted a meta-analysis of two GWASs with imputation using 1000 Genomes and UK10K Project data as reference (totaling 1658 cases and 7224 controls). After genotyping an additional 2525 cases and 3575 controls, we identify new susceptibility loci for BCP-ALL mapping to 10q26.13 (rs35837782, LHPP, P=1.38 × 10-11) and 12q23.1 (rs4762284, ELK3, P=8.41 × 10-9). We also provide confirmatory evidence for the existence of independent risk loci at 9p21.3, but show that the association marked by rs77728904 can be accounted for by linkage disequilibrium with the rare high-impact CDKN2A p.Ala148Thr variant rs3731249. Our data provide further insights into genetic susceptibility to ALL and its biology.

The role of the RAS pathway in iAMP21-ALL.

Intrachromosomal amplification of chromosome 21 (iAMP21) identifies a high-risk subtype of acute lymphoblastic leukaemia (ALL), requiring intensive treatment to reduce their relapse risk. Improved understanding of the genomic landscape of iAMP21-ALL will ascertain whether these patients may benefit from targeted therapy. We performed whole-exome sequencing of eight iAMP21-ALL samples. The mutation rate was dramatically disparate between cases (average 24.9, range 5-51) and a large number of novel variants were identified, including frequent mutation of the RAS/MEK/ERK pathway. Targeted sequencing of a larger cohort revealed that 60% (25/42) of diagnostic iAMP21-ALL samples harboured 42 distinct RAS pathway mutations. High sequencing coverage demonstrated heterogeneity in the form of multiple RAS pathway mutations within the same sample and diverse variant allele frequencies (VAFs) (2-52%), similar to other subtypes of ALL. Constitutive RAS pathway activation was observed in iAMP21 samples that harboured mutations in the predominant clone (⩾35% VAF). Viable iAMP21 cells from primary xenografts showed reduced viability in response to the MEK1/2 inhibitor, selumetinib, in vitro. As clonal (⩾35% VAF) mutations were detected in 26% (11/42) of iAMP21-ALL, this evidence of response to RAS pathway inhibitors may offer the possibility to introduce targeted therapy to improve therapeutic efficacy in these high-risk patients.

Prognostic value of rare IKZF1 deletion in childhood B-cell precursor acute lymphoblastic leukemia: an international collaborative study.

Deletions in IKZF1 are found in ~15% of children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). There is strong evidence for the poor prognosis of IKZF1 deletions affecting exons 4-7 and exons 1-8, but evidence for the remaining 33% of cases harboring other variants of IKZF1 deletions is lacking. In an international multicenter study we analyzed the prognostic value of these rare variants in a case-control design. Each IKZF1-deleted case was matched to three IKZF1 wild-type controls based on cytogenetic subtype, treatment protocol, risk stratification arm, white blood cell count and age. Hazard ratios for the prognostic impact of rare IKZF1 deletions on event-free survival were calculated by matched pair Cox regression. Matched pair analysis for all 134 cases with rare IKZF1 deletions together revealed a poor prognosis (P<0.001) that was evident in each risk stratification arm. Rare variant types with the most unfavorable event-free survival were DEL 2-7 (P=0.03), DEL 2-8 (P=0.002) and DEL-Other (P<0.001). The prognosis of each type of rare variant was equal or worse compared with the well-known major DEL 4-7 and DEL 1-8 IKZF1 deletion variants. We therefore conclude that all variants of rare IKZF1 deletions are associated with an unfavorable prognosis in pediatric BCP-ALL.

An international study of intrachromosomal amplification of chromosome 21 (iAMP21): cytogenetic characterization and outcome.

Intrachromosomal amplification of chromosome 21 (iAMP21) defines a distinct cytogenetic subgroup of childhood B-cell precursor acute lymphoblastic leukaemia (BCP-ALL). To date, fluorescence in situ hybridisation (FISH), with probes specific for the RUNX1 gene, provides the only reliable detection method (five or more RUNX1 signals per cell). Patients with iAMP21 are older (median age 9 years) with a low white cell count. Previously, we demonstrated a high relapse risk when these patients were treated as standard risk. Recent studies have shown improved outcome on intensive therapy. In view of these treatment implications, accurate identification is essential. Here we have studied the cytogenetics and outcome of 530 iAMP21 patients that highlighted the association of specific secondary chromosomal and genetic changes with iAMP21 to assist in diagnosis, including the gain of chromosome X, loss or deletion of chromosome 7, ETV6 and RB1 deletions. These iAMP21 patients when treated as high risk showed the same improved outcome as those in trial-based studies regardless of the backbone chemotherapy regimen given. This study reinforces the importance of intensified treatment to reduce the risk of relapse in iAMP21 patients. This now well-defined patient subgroup should be recognised by World Health Organisation (WHO) as a distinct entity of BCP-ALL.

Hyperdiploidy with 49-65 chromosomes represents a heterogeneous cytogenetic subgroup of acute myeloid leukemia with differential outcome.

Chromosome gain is frequent in acute myeloid leukemia (AML) and is counted alongside structural abnormalities when determining karyotype complexity. However, there are few studies investigating the cytogenetic profile and outcome of patients with a hyperdiploid karyotype (49-65 chromosomes, HK). We identified 221 (14%) patients with HK out of 1563 patients with three or more chromosomal abnormalities. HK was not associated with sex, white cell count and secondary disease status, but was more prevalent among children (22% vs 13%). The pattern of chromosomal gain and loss was non-random and chromosomes 8, 13 and 21 were the most frequently gained. Three distinct subgroups (numerical, structural and adverse) were identified with differential outcome: 5-year cumulative incidence of relapse of 52%, 68% and 76%, respectively (P=0.008). Patients in the adverse subgroup had poorer survival compared with patients with only numerical abnormalities (adjusted hazard ratio: 2.01 (95% confidence interval: 1.43-2.83), P=0.0002). This outcome heterogeneity was similar among children and adults. In conclusion, AML patients with a HK should not automatically be assigned to the adverse cytogenetic risk group on the basis of complexity. Instead they should be assessed for the presence of specific chromosomal abnormalities, which are known to harbour an adverse effect.

High hyperdiploidy among adolescents and adults with acute lymphoblastic leukaemia (ALL): cytogenetic features, clinical characteristics and outcome.

High hyperdiploidy (HeH, 51-65 chromosomes) is an established genetic subtype of acute lymphoblastic leukaemia (ALL). The clinical and cytogenetic features as well as outcome of HeH among adolescents and adults have not been thoroughly investigated. Among 1232 B-cell precursor ALL patients (15-65 years) treated in the UKALLXII/ECOG2993 trial, 160 (13%) had a HeH karyotype, including 80 patients aged >24 years. The frequency of HeH was the same in Philadelphia chromosome (Ph)-positive and -negative cases, but Ph-positive patients were older. The cytogenetic profiles of Ph-positive and Ph-negative HeH cases were similar, although trisomy 2 was strongly associated with Ph-positive HeH. Overall, Ph-positive HeH patients did not have an inferior overall survival compared with Ph-negative patients (P=0.2: 50 vs 57% at 5 years). Trisomy of chromosome 4 was associated with a superior outcome in Ph-negative patients, whereas +5 and +20 were associated with an inferior outcome in Ph-positive and Ph-negative patients, respectively. All three markers retained significance in multivariate analysis adjusting for age and white cell count: hazard ratio for risk of death 0.47 (95% CI: 0.27-0.84) (P=0.01), 3.73 (1.51-9.21) (P=0.004) and 2.63 (1.25-5.54) (P=0.01), respectively. In conclusion, HeH is an important subtype of ALL at all ages and displays outcome heterogeneity according to chromosomal gain.

Impact of NOTCH1/FBXW7 mutations on outcome in pediatric T-cell acute lymphoblastic leukemia patients treated on the MRC UKALL 2003 trial.

Activating mutations in the NOTCH1 pathway are frequent in pediatric T-cell acute lymphoblastic leukemia (T-ALL) but their role in refining risk stratification is unclear. We screened 162 pediatric T-ALL patients treated on the MRC UKALL2003 trial for NOTCH1/FBXW7 gene mutations and related genotype to response to therapy and long-term outcome. Overall, 35% were wild-type (WT) for both genes (NOTCH1(WT)FBXW7(WT)), 38% single NOTCH1 mutant (NOTCH1(Single)FBXW7(WT)), 3% just FBXW7 mutant (NOTCH1(WT)FBXW7(MUT)) and 24% either double NOTCH1 mutant (NOTCH1(Double)FBXW7(WT)) or mutant in both genes (NOTCH1(MUT)FBXW7(MUT)), hereafter called as NOTCH1±FBXW7(Double). There was no difference between groups in early response to therapy, but NOTCH1±FBXW7(Double) patients were more likely to be associated with negative minimal residual disease (MRD) post-induction than NOTCH1(WT)FBXW7(WT) patients (71% versus 40%, P=0.004). Outcome improved according to the number of mutations, overall survival at 5 years 82%, 88% and 100% for NOTCH1(WT)FBXW7(WT), NOTCH1(Single)FBXW7(WT) and NOTCH1±FBXW7(Double) patients, respectively (log-rank P for trend=0.005). Although 14 NOTCH1±FBXW7(Double) patients were classified as high risk (slow response and/or MRD positive), only two had disease progression and all remain alive. Patients with double NOTCH1 and/or FBXW7 mutations have a very good outcome and should not be considered for more intensive therapy in first remission, even if slow early responders or MRD positive after induction therapy.

Outcome in children with Down's syndrome and acute lymphoblastic leukemia: role of IKZF1 deletions and CRLF2 aberrations.

Children with Down's syndrome (DS) have an increased risk of developing acute lymphoblastic leukemia (ALL) and have a low frequency of established genetic aberrations. We aimed to determine which genetic abnormalities are involved in DS ALL. We studied the frequency and prognostic value of deletions in B-cell development genes and aberrations of janus kinase 2 (JAK2) and cytokine receptor-like factor 2 (CRLF2) using array-comparative genomic hybridization, and multiplex ligation-dependent probe amplification in a population-based cohort of 34 Dutch Childhood Oncology Group DS ALL samples. A population-based cohort of 88 DS samples from the UK trials was used to validate survival estimates for IKZF1 and CRLF2 abnormalities. In total, 50% of DS ALL patients had ≥1 deletion in the B-cell development genes: PAX5 (12%), VPREB1 (18%) and IKZF1 (35%). JAK2 was mutated in 15% of patients, genomic CRLF2 rearrangements in 62%. Outcome was significantly worse in patients with IKZF1 deletions (6-year event-free survival (EFS) 45 ± 16% vs 95 ± 4%; P=0.002), which was confirmed in the validation cohort (6-year EFS 21 ± 12% vs 58 ± 11%; P=0.002). This IKZF1 deletion was a strong independent predictor for outcome (hazard ratio EFS 3.05; P=0.001). Neither CRLF2 nor JAK2 were predictors for worse prognosis. If confirmed in prospective series, IKZF1 deletions may be used for risk-group stratification in DS ALL.

Temporal changes in the incidence and pattern of central nervous system relapses in children with acute lymphoblastic leukaemia treated on four consecutive Medical Research Council trials, 1985-2001.

Despite the success of contemporary treatment protocols in childhood acute lymphoblastic leukaemia (ALL), relapse within the central nervous system (CNS) remains a challenge. To better understand this phenomenon, we have analysed the changes in incidence and pattern of CNS relapses in 5564 children enrolled in four successive Medical Research Council-ALL trials between 1985 and 2001. Changes in the incidence and pattern of CNS relapses were examined and the relationship with patient characteristics was assessed. The factors affecting outcome after relapse were determined. Overall, relapses declined by 49%. Decreases occurred primarily in non-CNS and combined relapses with a progressive shift towards later (> or =30 months from diagnosis) relapses (P<0.0001). Although isolated CNS relapses declined, the proportional incidence and timing of relapse remained unchanged. Age and presenting white blood cell (WBC) count were risk factors for CNS relapse. On multivariate analysis, the time to relapse and the trial period influenced outcomes after relapse. Relapse trends differed within biological subtypes. In ETV6-RUNX1 ALL, relapse patterns mirrored overall trends whereas in high hyperdiploidy (HH) ALL, these seem to have plateaued over the latter two trial periods. Intensive systemic and intrathecal chemotherapy have decreased the overall CNS relapse rates and changed the patterns of recurrence. The heterogeneity of therapeutic response in the biological subtypes suggests room for further optimization using currently available chemotherapy.

Heterogeneous patterns of amplification of the NUP214-ABL1 fusion gene in T-cell acute lymphoblastic leukemia.

Episomes with the NUP214-ABL1 fusion gene have been observed in 6% of T-ALL. In this multicentric study we collected 27 cases of NUP214-ABL1-positive T-ALL. Median age was 15 years with male predominance. Outcome was poor in 12 patients. An associated abnormality involving TLX1 or TLX3 was found in all investigated cases. Fluorescent in situ hybridization revealed a heterogeneous pattern of NUP214-ABL1 amplification. Multiple episomes carrying the fusion were detected in 24 patients. Episomes were observed in a significant number of nuclei in 18 cases, but in only 1-5% of nuclei in 6. In addition, intrachromosomal amplification (small hsr) was identified either as the only change or in association with episomes in four cases and two T-ALL cell lines (PEER and ALL-SIL). One case showed insertion of apparently non-amplified NUP214-ABL1 sequences at 14q12. The amplified sequences were analyzed using array-based CGH.These findings confirm that the NUP214-ABL1 gene requires amplification for oncogenicity; it is part of a multistep process of leukemogenesis; and it can be a late event present only in subpopulations. Data also provide in vivo evidence for a model of episome formation, amplification and optional reintegration into the genome. Implications for the use of kinase inhibitors are discussed.

Genome complexity in acute lymphoblastic leukemia is revealed by array-based comparative genomic hybridization.

Chromosomal abnormalities are important for the classification and risk stratification of patients with acute lymphoblastic leukemia (ALL). However, approximately 30% of childhood and 50% of adult patients lack abnormalities with clinical relevance. Here, we describe the use of array-based comparative genomic hybridization (aCGH) to identify copy number alterations (CNA) in 58 ALL patients. CNA were identified in 83% of cases, and most frequently involved chromosomes 21 (n=42), 9 (n=21), 6 (n=16), 12 (n=11), 15 (n=11), 8 (n=10) and 17 (n=10). Deletions of 6q (del(6q)) were heterogeneous in size, in agreement with previous data, demonstrating the sensitivity of aCGH to measure CNA. Although 9p deletions showed considerable variability in both the extent and location, all encompassed the CDKN2A locus. Six patients showed del(12p), with a common region encompassing the ETV6 gene. Complex CNA were observed involving chromosomes 6 (n=2), 15 (n=2) and 21 (n=11) with multiple regions of loss and gain along each chromosome. Chromosome 21 CNA shared a common region of gain, with associated subtelomeric deletions. Other recurrent findings included dim(13q), dim(16q) and enh(17q). This is the first report of genome-wide detection of CNA in ALL patients using aCGH, and it has demonstrated a higher level of karyotype complexity than anticipated from conventional cytogenetic analysis.

Population-based demographic study of karyotypes in 1709 patients with adult acute myeloid leukemia.

Few large demographic studies of acute myeloid leukemia (AML) are derived from population-based registries. Demographic and karyotypic data were provided for AML cases from two regional leukemia registry databases in Scotland and the Northern Region of England. A population-based dataset was compiled, comprising 1709 patients aged >16 years (1235 North England/474 Scotland patients). The most common cytogenetic abnormalities involved chromosomes 5 and/or 7 (17%). Patients with the following abnormal chromosome 5/7 combinations: -5, del(5q), -5/-7 and del(5q)/-7 represented a significantly older population (P < 0.01, ANOVA). t(8;21) was the only 'favourable' karyotype found in older age. Karyotypic complexity varied within chromosome 5/7 combination groups; those containing -5, -5/-7, -5/del(7q), del(5q)/-7 or del(5q)/del(7q) combinations were significantly more frequently complex than those containing -7 and del(7q) (P < 0.01, chi2 test). Additional recurring cytogenetic abnormalities within complex karyotypes containing chromosome 5/7 combinations included (in order of frequency), abnormalities of chromosomes 17, 12, 3 and 18. Complex karyotypes not involving chromosomes 5 or 7 represented 30% of all complex karyotypes, occurred in younger patients than those involving chromosomes 5 and 7, and frequently included additional trisomy 8 (26%). In conclusion, we describe subgroups within adverse karyotypes, with different demographics, degree of complexity and additional chromosome abnormalities.

No prognostic effect of additional chromosomal abnormalities in children with acute lymphoblastic leukemia and 11q23 abnormalities.

This study characterized the additional chromosomal abnormalities (ACA) associated with 11q23 rearrangements in 450 infants and children with acute lymphoblastic leukemia (ALL) and examined the impact of these ACA on survival. Overall, 213 (47%) cases had ACA but the incidence varied according to patient age and 11q23 subgroup. Infants and patients with t(4;11)(q21;q23) had the lowest incidence of ACA (50/182 (27%) and 57/216 (26%) respectively), whereas patients with del(11)(q23) had the highest incidence (66/93 (71%)). Del(11)(q23) abnormalities were heterogeneous and occasionally secondary to t(9;22)(q34;q11.2). Thus, patients with del(11)(q23) comprised a separate biological entity, which was clearly distinct from those with an 11q23 translocation. The most frequent specific ACA were trisomy X (n = 38), abnormal 12p (n = 32), abnormal 9p (n = 28) and del(6q) (n = 19). The presence of ACA did not change the 5 year event-free survival estimates among children (56% (95% Cl 46-65%) vs 62% (54-69%)) or infants (22% (15-29%) vs 18% (9-29%)), nor when the different 11q23 subgroups were analyzed separately. This study has conclusively demonstrated that there is no prognostic effect of secondary chromosomal changes in association with 11q23 abnormalities in childhood ALL. However, characterization of these ACA is important to determine their potential role in initiation of MLL driven leukemogenesis.