Christine J. Harrison

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.

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.

Treatment of infants with lymphoblastic leukaemia: results of the UK Infant Protocols 1987-1999

Summary.  One hundred and twenty‐six infants with acute lymphoblastic leukaemia (ALL) were treated on two consecutive protocols, Infant 87 (n = 40) and Infant 92 (n = 86), in an attempt to improve the poor prognosis of this disease. Both included intensive induction and consolidation with intrathecal and high‐dose systemic therapy for central nervous system (CNS) protection. Intensification therapy was modified and high‐dose chemotherapy with bone marrow transplantation in first remission was permitted in Infant 92. Four‐year event‐free survival was superior in Infant 92 (33%; 95% CI 23–44%) compared with Infant 87 (22·5%; 95% CI 12–37%) (P = 0·04) and survival at 4 years was also superior, 46% (95% CI 35–57%) c.f. 32·5% (95% CI 20–48%) (P = 0·01), largely as a result of a significant reduction in remission deaths. Twelve patients in Infant 92 underwent bone marrow transplantation (BMT) in first remission, but their survival was no better than that of patients receiving chemotherapy. Multivariate analysis of prognostic factors showed the adverse influence of younger age, CNS involvement at diagnosis and a high initial leucocyte count, but not of CD10 expression. Cytogenetic analysis, available in 93% of patients in Infant 92, showed that 67% had chromosomal rearrangements involving 11q23 of which 39% had the translocation t(4;11) (q21;q23). There was no significant difference in event‐free survival between cytogenetic subgroups, although no children under 6 months of age with 11q23 abnormalities, other than the t(4;11), survived. In conclusion, infants with lymphoblastic leukaemia remain a high‐risk group, but it is unclear whether their adverse prognosis can be attributed to unfavourable cytogenetics alone. The role of high‐dose therapy and BMT in first remission remains uncertain.

Intrachromosomal amplification of chromosome 21 (iAMP21) may arise from a breakage-fusion-bridge cycle.

Intrachromosomal amplification of chromosome 21 (iAMP21), involving amplification of the RUNX1 gene and duplication of chromosome 21, dup(21q), defines a new cytogenetic subgroup in B‐lineage acute lymphoblastic leukemia (ALL) with a poor prognosis. Characterization of this abnormality has become vital to ensure that the most accurate detection method is used. We have previously defined common regions of amplification and deletion of chromosome 21 in these patients, although the level and extent of amplification within the amplicon was highly variable. This study, using interphase fluorescence in situ hybridization (FISH) with chromosome 21 locus specific probes, substantiated these findings in a large series of patients and confirmed that the amplicon always included RUNX1. Thus, FISH with probes directed to the RUNX1 gene remains the most reliable detection method. Metaphase FISH, supported by G‐ and multiple color chromosomal banding (mBAND) revealed the patient specific morphology and genetic profile of the dup(21q) chromosomes, as well as the complexity of the intrachromosomal changes giving rise to them. These findings suggested that iAMP21 had arisen from a breakage–fusion–bridge cycle: a mechanism previously described in tumors, which we report for the first time in ALL.

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.

Del (9q) AML: clinical and cytological characteristics and prognostic implications.

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.

t(14;19)(q32;q13): a recurrent translocation in B-cell precursor acute lymphoblastic leukemia

The recurrent t(14;19)(q32;q13) translocation associated with chronic B‐cell lymphoproliferative disorders, such as atypical chronic lymphocytic leukemia, results in the juxtaposition of the IGH@ and BCL3 genes and subsequent overexpression of BCL3. We report six patients with B‐cell precursor acute lymphoblastic leukemia who have a cytogenetically identical translocation with different breakpoints at the molecular level. Fluorescence in situ hybridization with locus‐specific probes confirmed the involvement of the IGH@ gene but showed that the breakpoint on 19q13 lay outside the region documented in t(14;19)(q32;q13)‐positive chronic lymphocytic leukemia. This newly described translocation constitutes a distinct cytogenetic subgroup that is confined to older children and younger adults with B‐cell precursor acute lymphoblastic leukemia.

Comparative expressed sequence hybridization studies of high-hyperdiploid childhood acute lymphoblastic leukemia

The functional consequences of a high‐hyperdiploid karyotype, found in up to one‐third of cases of acute lymphoblastic leukemia (ALL), are unknown. Using the technique of comparative expressed sequence hybridization (CESH), we sought to address the question of whether increased chromosome copies in hyperdiploid ALL lead to increased gene expression. Relative expression of hyperdiploid ALL blasts versus peripheral blood mononuclear cells was analyzed in 18 patients. Common regions of overexpression corresponding to the presence of tri‐/tetrasomies included: Xp22.1–22.2, 4q28, 6q14–15, 6q24, 10p13, 14q23–24, 17q21, 18q12, and 21q21, identified in 28–89% of cases. However, increased expression without underlying trisomy occurred at 3p21.3, 7q11.2, 8p21, and 8q24.1 in 39–90% of cases. High expression at 7q11.2, the most consistent change detected, was confirmed by quantitative PCR. Poor correlation between the presence of tri‐/tetrasomy and overexpression was observed for chromosomes 14 and 17. Two cases were reanalyzed versus (i) B cells, (ii) transformed B cells, and (iii) CD34+19+ cells (the putative counterpart of the leukemic cell). A reduction in the number of relatively overexpressed regions was observed with CD34+19+ cells. In particular, the peak at 7q11.2 disappeared, suggesting up‐regulation of genes from this region in the early ontology of normal B‐cell development. In conclusion, we have shown that tri‐/tetrasomies in hyperdiploid ALL lead to an increase in the expression of associated sequences. The choice of a biologically relevant reference is crucial for data interpretation.

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.