Abnormalities of the der(12)t(12;21) in ETV6-RUNX1 acute lymphoblastic leukemia.

ETV6-RUNX1 fusion [t(12;21)(p13;q22)] occurs in 25% of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and is associated with a favorable outcome. Additional abnormalities involving der(21)t(12;21) and nonrearranged chromosome 12 are well characterized but aberrations involving the der(12)t(12;21) have rarely been described. Herein, we describe two novel abnormalities affecting the der(12)t(12;21): a deletion (20/247, 8%) and duplication (10/247, 4%). All 30 patients were under 10 years of age, had a median white blood count of 12.4 × 10(9)/L and 19.2 × 10(9)/L, respectively, with a good outcome. Deletions of der(12)t(12;21) on both sides of the breakpoint were confirmed and mapped: centromeric (12p11.21-12p13.2) and telomeric (21q22.12-21q22.3). The size of these deletions extended from 0.4-13.4 to 0.8-2.5 Mb, respectively. The centromeric deletion encompassed the following genes: LRP6, BCL2L14, DUSP16, CREBL2, and CDKN1B. We postulate that this deletion occurs at the same time as the translocation because it was present in all ETV6-RUNX1-positive cells. A second abnormality representing duplication of the reciprocal RUNX1-ETV6 fusion gene was a secondary event, which we hypothesize arose through mitotic recombination errors. This led to the formation of the following chromosome: der(12)(21qter→21q22.12::12p13.2-12p12.3::12p12.3→12qter). Both abnormalities affect the reciprocal RUNX1-ETV6 fusion product which could either eliminate or amplify its expression and thus contribute to leukemogenesis. However, other consequences such as haploinsufficiency of tumor suppressor genes and amplification of oncogenes could also be driving forces behind these aberrations. In conclusion, this study has defined novel abnormalities in ETV6-RUNX1 BCP-ALL, which implicate new genes involved in leukemogenesis.

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.

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.

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.

ETV6/RUNX1 fusion at diagnosis and relapse: some prognostic indications.

This study was undertaken in order to compare the interphase and metaphase cytogenetics of 28 patients with ETV6/RUNX1 positive acute lymphoblastic leukemia, at diagnosis and relapse. The median time to relapse was 26 months. The significant fusion positive population heterogeneity revealed at interphase by a commercial probe for ETV6/RUNX1 fusion has not been described before. Six diagnostic samples had a single abnormal population; others had up to five each, which differed in the numbers of RUNX1 signals, and in the retention or loss of the second ETV6 signal. In contrast, the number of fusion signals was more constant. At relapse, there were fewer populations; the largest or unique clone was sometimes a re-emergence of a minor, diagnostic one, with a retained copy of ETV6 and the most RUNX1 signals. Abnormal, fusion negative clones were identified in bone marrow samples at extra-medullary relapse. Variant three or four-way translocations, which involved chromosomes 12 and 21, were prominent among the complex rearrangements revealed by metaphase FISH. The frequency of their occurrence at diagnosis and reappearance at relapse, sometimes accompanied by minor clonal evolution, was another new observation. Other recurrent cytogenetic features included a second copy of the fusion signal in six cases, partial duplication of the long arm of the X chromosome in two cases, and trisomy 10 in three cases. In comparing our data with previously reported cases, a picture is beginning to emerge of certain diagnostic features, which may provide circumstantial evidence of an increased risk of relapse.

Interphase molecular cytogenetic screening for chromosomal abnormalities of prognostic significance in childhood acute lymphoblastic leukaemia: a UK Cancer Cytogenetics Group Study.

Summary Interphase fluorescence in situ hybridization (iFISH) was used independently to reveal chromosomal abnormalities of prognostic importance in a large, consecutive series of children (n = 2367) with acute lymphoblastic leukaemia (ALL). The fusions, TEL/AML1 and BCR/ABL, and rearrangements of the MLL gene occurred at frequencies of 22% (n = 447/2027) (25% in B-lineage ALL), 2% (n = 43/2027) and 2% (n = 47/2016) respectively. There was considerable variation in iFISH signal patterns both between and within patient samples. The TEL/AML1 probe showed the highest incidence of variation (59%, n = 524/884), which included 38 (2%) patients with clustered, multiple copies of AML1. We were thus able to define amplification of AML1 as a new recurrent abnormality in ALL, associated with a poor prognosis. Amplification involving the ABL gene, a rare recurrent abnormality confined to T ALL patients, was identified for the first time. The use of centromeric probes revealed significant hidden high hyperdiploidy of 33% and 59%, respectively, in patients with normal (n = 21/64) or failed (n = 32/54) cytogenetic results. The iFISH contributed significantly to the high success rate of 91% (n = 2114/2323) and the remarkable abnormality detection rate of 89% (n = 1879/2114). This study highlights the importance of iFISH as a complementary tool to cytogenetics in routine screening for significant chromosomal abnormalities in ALL.

MLL translocations with concurrent 3' deletions: interpretation of FISH results.

Rearrangements involving the MLL gene at 11q23 occur in a clinically relevant subgroup of patients with acute lymphoblastic leukemia (ALL) at all ages, and therefore their accurate identification at diagnosis is important. It has become commonplace to screen ALL patients for rearrangements of MLL using a dual-color fluorescence in situ hybridization (FISH) assay. We report on 12 ALL patients with an unusual FISH result consisting of the following signal pattern: one 5' green, no 3' red, and one/two fusion signals. This configuration is consistent with a MLL translocation and simultaneous deletion of 3' MLL-a well-established phenomenon-which has been interpreted as a positive result. G-banded and complementary metaphase FISH analyses confirmed an 11q23/MLL translocation in 8 of the 12 cases, whereas in one case, the identification of a del(11)(q23) was restricted to G-banded analysis only. In three cases, an MLL rearrangement was excluded by extensive FISH analysis and/or Southern blotting. In conclusion, the loss of the 3' MLL signal should not be assumed to be the result of a concurrent translocation and deletion event, and such aberrant FISH signal patterns should be investigated further by alternative methods for determining their MLL status.

Three distinct subgroups of hypodiploidy in acute lymphoblastic leukaemia.

This study of children and adults with acute lymphoblastic leukaemia (ALL) is the largest series of patients with hypodiploidy (<46 chromosomes) yet reported. The incidence of 5% was independent of age. Patients were subdivided by the number of chromosomes; near-haploidy (23-29 chromosomes), low hypodiploidy (33-39 chromosomes) and high hypodiploidy (42-45 chromosomes). The near-haploid and low hypodiploid groups were characterized by their chromosomal gains and a doubled hyperdiploid population. Structural abnormalities were more frequent in the low hypodiploid group. Near-haploidy was restricted to children of median age 7 years (range 2-15) whereas low hypodiploidy occurred in an older group of median age 15 years (range 9-54). Patients with 42-45 chromosomes were characterized by complex karyotypes involving chromosomes 7, 9 and 12. The features shared by the few patients with 42-44 chromosomes and the large number with 45 justified their inclusion in the same group. Survival analysis showed a poor outcome for the near-haploid and low hypodiploid groups compared to those with 42-45 chromosomes. Thus cytogenetics, or at least a clear definition of the modal chromosome number, is essential at diagnosis in order to stratify patients with hypodiploidy into the appropriate risk group for treatment.