Sarah L. Wright

A comprehensive analysis of the CDKN2A gene in childhood acute lymphoblastic leukemia reveals genomic deletion, copy number neutral loss of heterozygosity, and association with specific cytogenetic subgroups.

Inactivation of the tumor suppressor gene, CDKN2A, can occur by deletion, methylation, or mutation. We assessed the principal mode of inactivation in childhood acute lymphoblastic leukemia (ALL) and frequency in biologically relevant subgroups. Mutation or methylation was rare, whereas genomic deletion occurred in 21% of B-cell precursor ALL and 50% of T-ALL patients. Single nucleotide polymorphism arrays revealed copy number neutral (CNN) loss of heterozygosity (LOH) in 8% of patients. Array-based comparative genomic hybridization demonstrated that the mean size of deletions was 14.8 Mb and biallelic deletions composed a large and small deletion (mean sizes, 23.3 Mb and 1.4 Mb). Among 86 patients, only 2 small deletions were below the resolution of detection by fluorescence in situ hybridization. Patients with high hyperdiploidy, ETV6-RUNX1, or 11q23/MLL rearrangements had low rates of deletion (11%, 15%, 13%), whereas patients with t(9;22), t(1;19), TLX3, or TLX1 rearrangements had higher frequencies (61%, 42%, 78%, and 89%). In conclusion, CDKN2A deletion is a significant secondary abnormality in childhood ALL strongly correlated with phenotype and genotype. The variation in the incidence of CDKN2A deletions by cytogenetic subgroup may explain its inconsistent association with outcome. CNN LOH without apparent CDKN2A inactivation suggests the presence of other relevant genes in this region.

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.

Variable Breakpoints Target PAX5 in Patients with Dicentric Chromosomes: A Model for the Basis of Unbalanced Translocations in Cancer

The search for target genes involved in unbalanced acquired chromosomal abnormalities has been largely unsuccessful, because the breakpoints of these rearrangements are too variable. Here, we use the example of dicentric chromosomes in B cell precursor acute lymphoblastic leukemia to show that, despite this heterogeneity, single genes are targeted through a variety of mechanisms. FISH showed that, although they were heterogeneous, breakpoints on 9p resulted in the partial or complete deletion of PAX5. Molecular copy number counting further delineated the breakpoints and facilitated cloning with long-distance inverse PCR. This approach identified 5 fusion gene partners with PAX5: LOC392027 (7p12.1), SLCO1B3 (12p12), ASXL1 (20q11.1), KIF3B (20q11.21), and C20orf112 (20q11.1). In each predicted fusion protein, the DNA-binding paired domain of PAX5 was present. Using quantitative PCR, we demonstrated that both the deletion and gene fusion events resulted in the same underexpression of PAX5, which extended to the differential expression of the PAX5 target genes, EBF1, ALDH1A1, ATP9A, and FLT3. Further molecular analysis showed deletion and mutation of the homologous PAX5 allele, providing further support for the key role of PAX5. Here, we show that specific gene loci may be the target of heterogeneous translocation breakpoints in human cancer, acting through a variety of mechanisms. This approach indicates an application for the identification of cancer genes in solid tumours, where unbalanced chromosomal rearrangements are particularly prevalent and few genes have been identified. It can be extrapolated that this strategy will reveal that the same mechanisms operate in cancer pathogenesis in general.

The complex genomic profile of ETV6-RUNX1 positive acute lymphoblastic leukemia highlights a recurrent deletion of TBL1XR1.

The ETV6‐RUNX1 fusion is the molecular consequence of the t(12;21)(p13;q22) seen in ∼25% of children with acute lymphoblastic leukemia (ALL). Studies have shown that the fusion alone is insufficient for the initiation of leukemia; additional genetic changes are required. Genomic profiling identified copy number alterations at high frequencies in these patients. Focal deletions of TBL1XR1 were observed in 15% of cases; 3 patients exhibited deletions distal to the gene. Fluorescence in situ hybridization confirmed these deletions and quantitative RT‐PCR showed that the TBL1XR1 gene was significantly under‐expressed. TBL1XR1 is a key component of the SMRT and N‐CoR compressor complexes, which control hormone–receptor mediated gene expression. Differential expression of the retinoic acid target genes, RARB, CRABP1, and CRABP2, indicated that deletion of TBL1XR1 compromised the function of SMRT/N‐CoR in the appropriate control of gene expression. This study identifies deletions of TBL1XR1 as a recurrent abnormality in ETV6‐RUNX1 positive ALL. We provide evidence that implicates this deletion in the inappropriate control of gene expression in these patients. The target of the interaction between TBL1XR1 and the signaling pathways described here may be exploited in cancer therapy.

Molecular cytogenetic characterization of TCF3 (E2A)/19p13.3 rearrangements in B-cell precursor acute lymphoblastic leukemia

The t(1;19)(q23;p13.3) is one of the most common chromosomal abnormalities in B‐cell precursor acute lymphoblastic leukemia (BCP‐ALL) and usually gives rise to the TCF3‐PBX1 fusion gene. Additional rare, and sometimes cytogenetically cryptic, translocations involving the TCF3 gene have also been described. Using a dual color split‐signal fluorescence in situ hybridization (FISH) probe, we have investigated the involvement of this gene in a series of BCP‐ALLs harboring 19p13 translocations, as well as an unselected patient cohort. The TCF3 gene was shown to be involved in the majority of cases with a cytogenetically visible t(1;19) translocation, while the remaining TCF3‐negative ALLs demonstrated breakpoint heterogeneity. Although most “other” 19p13 translocations did not produce a split‐signal FISH pattern, a novel t(13;19)(q14;p13) involving TCF3 was discovered. A prospective screen of 161 children with BCP‐ALL revealed a cryptic t(12;19)(p13;p13), another novel TCF3 rearrangement, and a series of patients with submicroscopic deletions of TCF3. These results demonstrate the utility of a split‐signal FISH strategy in confirming the involvement of the TCF3 gene in 19p13 rearrangements and in identifying novel and cryptic TCF3 translocations. In addition to its role as a fusion partner gene, we propose that TCF3 can also act as a tumor suppressor gene in BCP‐ALL.

Heterogeneous breakpoints in patients with acute lymphoblastic leukemia and the dic(9;20)(p11~13;q11) show recurrent involvement of genes at 20q11.21

Dicentric chromosomes are rare in acute lymphoblastic leukemia, dic(9;20) being a recurrent aberration. This study provides insight into the breakpoint complexity underlying dicentric chromosomal formation in acute lymphoblastic leukemia and highlights putative target gene loci. The dic(9;20)(p11~13;q11) is a recurrent chromosomal abnormality in patients with acute lymphoblastic leukemia. Although it results in loss of material from 9p and 20q, the molecular targets on both chromosomes have not been fully elucidated. From an initial cohort of 58 with acute lymphoblastic leukemia patients with this translocation, breakpoint mapping with fluorescence in situ hybridization on 26 of them revealed breakpoint heterogeneity of both chromosomes. PAX5 has been proposed to be the target gene on 9p, while for 20q, FISH analysis implicated the involvement of the ASXL1 gene, either by a breakpoint within (n=4) or centromeric (deletion, n=12) of the gene. Molecular copy-number counting, long-distance inverse PCR and direct sequence analysis identified six dic(9;20) breakpoint sequences. In addition to the three previously reported: PAX5-ASXL1, PAX5-C20ORF112 and PAX5-KIF3B; we identified three new ones in this study: sequences 3’ of PAX5 disrupting ASXL1, and ZCCHC7 disrupted by sequences 3’ of FRG1B and LOC1499503. This study provides insight into the breakpoint complexity underlying dicentric chromosomal formation in acute lymphoblastic leukemia and highlights putative target gene loci.

Disruption of ETV6 in intron 2 results in upregulatory and insertional events in childhood acute lymphoblastic leukaemia

We describe four cases of childhood B-cell progenitor acute lymphoblastic leukaemia (BCP-ALL) and one of T-cell (T-ALL) with unexpected numbers of interphase signals for ETV6 with an ETV6–RUNX1 fusion probe. Three fusion negative cases each had a telomeric part of 12p terminating within intron 2 of ETV6, attached to sequences from 5q, 7p and 7q, respectively. Two fusion positive cases, with partial insertions of ETV6 into chromosome 21, also had a breakpoint in intron 2. Fluorescence in situ hybridisation (FISH), array comparative genomic hybridization (aCGH) and Molecular Copy-Number Counting (MCC) results were concordant for the T-cell case. Sequences downstream of TLX3 on chromosome 5 were deleted, leaving the intact gene closely apposed to the first two exons of ETV6 and its upstream promoter. qRT-PCR showed a significant upregulation of TLX3. In this study we provide the first incontrovertible evidence that the upstream promoter of ETV6 attached to the first two exons of the gene was responsible for the ectopic expression of a proto-oncogene that became abnormally close as the result of deletion and translocation. We have also shown breakpoints in intron 2 of ETV6 in two cases of insertion with ETV6–RUNX1 fusion.

Cytogenetics of long-term survivors of ETV6-RUNX1 fusion positive acute lymphoblastic leukemia

This study describes the cytogenetics of 33 children with ETV6‐RUNX1 positive acute lymphoblastic leukemia (ALL) who had been in continuous complete remission for a minimum of 8.8 years [median event‐free survival (EFS) 10.9 years]. The results were compared with a published series of 16 fusion positive patients treated on the same childhood ALL trial, who had relapsed (median EFS, 2.3 years). Interphase fluorescence in situ hybridization (FISH) at diagnosis showed deletion of the second ETV6 signal from all fusion positive cells in 45% of the long‐term survivors but in none of the relapsed patients, whereas patients with mixed populations with retained or lost second signals were more frequent among those who had relapsed (69%) than the long‐term survivors (21%). Interphase populations with two fusion signals in 18% of the long‐term survivors and 31% of relapsed patients were smaller in the long‐term survivors (median, 4% of total cells) than in the relapsed patients (median, 84%). The additional copy of chromosome 21 in 30% of long‐term survivors and in 69% of relapsed patients was a derived chromosome 21 in 20% and 55% of patients, respectively. Metaphase FISH for 26 long‐term survivors and 15 relapsed patients revealed complex karyotypes in both groups. Variant translocations involved different chromosome arms between the long‐term survivors and relapsed patients. It appears that the two groups have some distinguishing cytogenetic features at the time of diagnosis, which may provide pointers to relapse that are worthy of more detailed study.

Fluorescence In Situ Hybridization (FISH) as a Tool for the Detection of Significant Chromosomal Abnormalities in Childhood Leukaemia

Cytogenetics is integral to the diagnosis of childhood leukaemia, particularly in relation to the risk stratification of patients for treatment. Fluorescence in situ hybridization (FISH) has become an important complementary technique, expanding chromosomal analysis into the molecular arena. It has greatly improved the accuracy and applicability of cytogenetics and led to the discovery of novel chromosomal changes of prognostic significance. Many probes are now commercially available, providing robust and reliable detection of chromosomal abnormalities. Since the cloning of the human genome, it is now possible to access detailed genomic information and develop FISH probes for virtually any known DNA sequence. The range of procedures necessary for the successful application of FISH in the accurate detection of significant chromosomal abnormalities in childhood acute leukaemia is described here.

Heterogeneous breakpoints target PAX5 in acute lymphoblastic leukaemia patients with dicentric chromosomes

Although both MRD and karyotype are powerful determinants of outcome in childhood ALL, few studies have examined the kinetics of MRD clearance by cytogenetics. In ALL2003, patients are stratified by NCI criteria to a three or four drug induction. MRD is assessed at day 29 and week 11 using a standardised and quality controlled RQPCR of 21patient specific immunoglobulin or T-cell receptor rearrangements. MRD risk groups were defined as: (1) High risk MRD410-4 at day 29 (HR); (2) Low risk MRD negative or o10-4 at day 29 and negative at week 11 (LR); or (3) MRD indeterminate risk. Among 1000 patients entered into the trial, 98% were eligible for these analyses, 94% had a successful cytogenetics and 57% were assigned to a clinically relevant MRD groups. Among these latter 555 patients, 54% were MRD-HR whereas 45% were MRD-LR. Collectively, patients with high-risk cytogenetics ‘t(9;22), o40 chromosomes, 11q23/MLL, t(17;19) and iAMP21’ were more likely to be MRDHR ‘83% vs 52%, P50.003’. Patients with ETV6-RUNX1 fusion were less likely to be MRD-HR ‘28% vs 63%, Po0.001’ whereas high hyperdiploid patients were more likely ‘64% vs 49%, P50.002’. However, excluding ETV6-RUNX1 patients from the latter analysis revealed that high hyperdiploid patients were as likely to be MRDHR as other ETV6-RUNX1 negative patients. T-ALL patients were also more likely to be MRD-HR compared to BCP-ALL patients ‘70% vs 52%, P50.022’. In particular, 9/10 patients with t(5;14)/TLX3- BCL11B fusion and 6/6 patients with SIL-TAL1 fusion were MRD-HR. In conclusion, we have clearly demonstrated that MRD status varies by cytogenetic subgroup with ETV6-RUNX1 patients having the fastest MRD clearance rate. Despite the good prognosis associated with high hyperdiploidy, these patients were as likely to be MRD-HR as other standard risk patients. Longer follow-up is required to determine the clinical significance of this finding.Genetic alterations play a key role in the leukemogenesis of childhood ALL. Inactivation of CDKN2A (p16), a tumour suppressor gene located at 9p21, can occur by deletion, methylation or mutation. Published reports are inconsistent in terms of incidence and mode of inactivation. We report a comprehensive analysis of CDKN2A inactivation in 1230 diagnostic and 101 relapse samples, including 46 matched diagnostic and relapse pairs, from 1285 children with ALL. Using data from cytogenetics (CC) (n=1088), FISH (n=1209), SNP arrays (SNPA) (n=106), CGH arrays (aCGH) (n=106), dHPLC (n=48) and methylation specific-PCR (MSP) (n=96) we have assessed the mode and frequency of CDKN2A inactivation. Mutation or methylation of CDKN2A was rare occurring in 1 patient each (2% and 1% respectively). In contrast, CDKN2A deletion was highly prevalent. The frequencies of deletion detected by the different methodologies were: CC 166 (15%), FISH 335 (28%), SNPA 17 (16%) and aCGH 35 (33%). The proportion of biallelic deletions also varied: CC 15 (9%), FISH 174 (52%) and aCGH 15 (65%). This variation was directly related to the resolution of each technique with a high degree of concordance across samples investigated by >1 method. Analysis of 50 deletions by aCGH showed that the size of the deletion ranged from 0.03Mb to 39.1Mb with a mean of 14.8Mb. Furthermore, analysis of 15 biallelic deletions demonstrated that they comprised one large deletion (mean size 23.3Mb) and a second much smaller deletion (mean size 1.4Mb). In addition, SNPA revealed copy number neutral LOH in 8 (8%) cases, but only once in association with a CDKN2A mutation. At diagnosis CDKN2A inactivation by any method was noted in 329 (27%) patients which was not different from that observed at relapse [25 (25%)]. However, the frequencies of CDKN2A inactivation and biallelic deletion were significantly greater in T-ALL compared with B cell precursor (BCP) ALL: 135/269 (50%) v 190/918 (21%) (p1 Prognostic impact of minimal residual disease in childhood acute lymphoblastic leukaemia depends on immunological subtype: results of trial AIEOP-BFM ALL 2000 M Schrappe , C Rizzari, G Mannz, A Moericke , MG Valsecchi, M Zimmermann, CR Bartram, R PanzerGrümayerz, A Schrauder , G Cazzaniga, T Flohr , R Parasole, A Reiter , WD Ludwig, L Lo Nigrozz, G Basso, G de Rossi, E Barisone, F Niggli , H Gadnerz and V Conter Department of Paediatrics and University Medical Center Schleswig-Holstein, Kiel, Germany, Paediatric Clinic, Ospedale Nuovo S Gerardo, Monza, Italy, zDepartment of Paediatric Haemato-Oncology, St Anna Children’s Hospital, Vienna, Austria, Department of Paediatric Haemato-Oncology, Medical School Hannover, Germany, Institute of Human Genetics, Ruprecht-Karls University, Heidelberg, Germany, Department of Paediatric Haemato-Oncology, Pausilipon Hospital, Napoli, Italy, Department of Paediatric Haemato-Oncology, University Hospital, Giessen, Germany, Department of Haemato-Oncology, Robert-Roessle-Klinik at the HELIOS Klinikum Charité, Berlin, Germany, zzDepartment of Paediatric Haematology/Oncology, University Hospital, Catania, Italy, Department of Paediatrics, University Hospital, Padova, Italy, Department of Paediatric Haematology/Oncology, Hospital Bambino Gesù, Rome, Italy, Ospedale Infantile Regina Margherita, Torino, Italy, Department of Paediatric Haematology/Oncology, University Hospital, Zurich, Switzerland