Zoë J. Broadfield

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.

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

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.

Amplification of AML1 in acute lymphoblastic leukemia is associated with a poor outcome

A total of 28 children and nine adults with relapsed T-ALL were analyzed for the configuration of their T-cell receptor (TCR) and TAL1 genes at diagnosis and relapse to evaluate their stability throughout the disease course. A total of 150 clonal TCR and TAL1 gene rearrangements were identified in the 37 patients at diagnosis. In 65% of cases all rearrangements and in 27% of cases most rearrangements found at diagnosis were preserved at relapse. Two children with unusually late T-ALL recurrences displayed completely different TCR gene rearrangement sequences between diagnosis and relapse. This indicates that a proportion of very late T-ALL recurrences might represent second T-ALL. Specifically, 88% of clonal rearrangements identified at diagnosis in truly relapsed T-ALL were preserved at relapse. This is significantly higher as compared to previously studied precursor-B-ALL (~70%). Thus, from biological point of view, immunogenotype of T-ALL is more stable as compared with precursor-B-ALL. The overall stability of TCR gene rearrangements was higher in adult T-ALL (97%) than in childhood T-ALL (86%). Based on the stability of TCR gene rearrangements, we propose a strategy for PCR target selection (TCRD+TAL1 TCRB TCRG), which probably allows reliable minimal residual disease detection in all T-ALL patients.

Amplification of the ABL gene in T-cell acute lymphoblastic leukemia.

Expression of CD86 in acute myelogenous leukemia is a marker of dendritic/monocytic lineage. Exp Hematol 2002; 30: 126–134. 6 Houtenbos I, Westers TM, Ossenkoppele GJ, Van De Loosdrecht AA. Identification of CD14 as a predictor for leukemic dendritic cell differentiation in acute myeloid leukemia. Leukemia 2003; 17: 1683–1684. 7 Brockhaus M, Schoenfeld HJ, Schlaeger EJ, Hunziker W, Lesslauer W, Loetscher H. Identification of two types of tumor necrosis factor receptors on human cell lines by monoclonal antibodies. Proc Natl Acad Sci USA 1990; 87: 3127–3131. 8 Santini V, Gozzini A, Scappini B, Rossi FP. Maturation and apoptosis of primary human acute myeloblastic leukemia cells are determined by TNF-alpha exclusively through CD120A stimulation. Haematologica 1999; 84: 291–297.

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.

Derivative chromosome 9 deletions are a significant feature of childhood Philadelphia chromosome positive acute lymphoblastic leukaemia.

Deletions from the derivative chromosome 9, der(9), of the translocation, t(9;22)(q34;q11), at the site of the ABL/BCR fusion gene, have been demonstrated by fluorescence in situ hybridisation (FISH), in both Philadelphia chromosome (Ph)-positive chronic myeloid leukaemia (CML) and acute lymphoblastic leukaemia (ALL). In CML they occur in 10–15% of cases and appear to indicate a worse prognosis, whereas in ALL, the situation is unclear. This study presents the findings of dual fusion FISH used to detect such deletions in a series of 27 BCR/ABL-positive childhood ALL patients. Metaphase FISH was essential for the accurate interpretation of interphase FISH signal patterns. Three cases (11%) had a single fusion signal, resulting from deletions of the der(9). Three other patients with variant translocations and one with an insertion, also had a single fusion, but with no evidence of deletions. Gain of a fusion in approximately one-third of patients indicated a second Ph, which appears to be a diagnostic marker of Ph-positive ALL. This study shows that the incidence of deletions from the der(9) in childhood ALL is at least as high as that reported for CML.

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.

Complex chromosomal abnormalities in utero, 5 years before leukaemia.

Prenatal acquisition of leukaemia‐associated gene rearrangements is a well‐established phenomenon. This is the first report of a complex cytogenetic clone, in association with an ETV6/AML1 fusion, developing in utero. Identical twin girls, aged 4 years, developed ETV6/AML1‐positive acute lymphoblastic leukaemia (ALL) within 3 months of one another. Both demonstrated an identical four way, variant t(12;21). There was gain of an AML1 signal in twin 1 and loss of an ETV6 one in twin 2 at interphase. This unique case study demonstrates that ETV6/AML1 fusion and the associated complex chromosomal rearrangements occurred in utero. Clonal expansion of the abnormal cell in one twin was followed by metastasis to the other. There was a prolonged preleukaemic phase, which lasted well into childhood. The short time between the two diagnoses of ALL suggests a common precipitating event. The significance of the different secondary markers remains unclear.

Cytogenetic classification of T lineage acute lymphoblastic leukaemia

Chromosomal translocations lead to oncogene activation in a significant number of haematological malignancies. Those involving the immunoglobulin heavy chain locus, IGH, at chromosome band 14q32 are frequently observed in B-cell malignant proliferation. A small number have been described in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL). However, their biological and clinical significance is currently unknown. Detailed fluorescence in situ hybridisation (FISH) and molecular studies were carried out on a series of BCP-ALL patients with chromosomal abnormalities involving 14q32. Novel and recurrent translocations affecting different chromosomes were highlighted. Refined FISH mapping identified putative IGH partner genes at, or flanking, the translocation breakpoints. Four translocations: two previously reported, t(14;19)(q32;q13), t(8;14)(q11;q32), and two novel, t(14;14)(q11;q32)/ inv(14)(q11q32) and t(14;20)(q32;q13), were identified. Molecular analyses showed that four different members of the CAATT enhancer binding protein (CEBP) gene family were involved: CEBPA (19q13, n59), CEBPD (8q11, n58), CEBPE (14q11, n53) and CEBPB (20q13, n52). One patient with a t(14;19)(q32;q13) was observed to involve the fifth family member CEBPG (19q13, n51). Breakpoints were located within the 30 untranslated region (UTR) of CEBPA and either 30 UTR or 50 of CEBPE, whereas breakpoints in 8q11 were B30 kb centromeric of CEBPD. Where material was available, over-expression of target genes was shown by quantitative real-time PCR. Overall, this study has demonstrated for the first time the involvement of five members of the same gene family in a single subtype of haematological disease. It has indicated that transcriptional upregulation of CEBP gene family members, by juxtaposition to IGH, is important in BCP-ALL: a mechanism in complete contrast to that involving CEPBA in acute myeloid leukaemia.