Mary Martineau

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.

Amplification of AML1 on a duplicated chromosome 21 in acute lymphoblastic leukemia: a study of 20 cases.

This study identifies multiple copies of the AML1 gene on a duplicated chromosome 21, dup(21), as a recurrent abnormality in acute lymphoblastic leukemia (ALL). Clusters of AML1 signals were visible at interphase by fluorescence in situ hybridization (FISH). In metaphase, they appeared tandemly duplicated on marker chromosomes of five distinct morphological types: large or small acrocentrics, metacentrics, submetacentrics or rings. The markers comprised only chromosome 21 material. Karyotypes were near-diploid and, besides dup(21), no other established chromosomal changes were observed. A total of 20 patients, 1.5 and <0.5% among consecutive series of childhood and adult ALL respectively, showed this phenomenon. Their median age was 9 years, white cell counts were low and all had a pre-B/common immunophenotype. Although this series is not the first report of this abnormality, it is the largest, permitting a detailed description of the variety of morphological forms that duplicated chromosome 21 can assume.

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.

The t(6;11)(q27;q23) translocation in acute leukemia: a laboratory and clinical study of 30 cases

Thirty patients representing 5.5% of those collected by the 11q23 workshop had a t(6;11)(q27;q23). They included 27 cases of acute myeloid leukemia (AML) (M1, three cases; M2, two cases; M4, nine cases; M4/M5, one case; M5, 12 cases) of age range 3–72 years and three cases of acute lymphoblastic leukemia (ALL) (B-lineage ALL, two cases; T-ALL, one case) of age range 0.5–13 years. In 20 cases the t(6;11) was the sole abnormality. In 10 cases the recurrent additional abnormalities were extra copies of chromosomes 8, 19, 21, or the der(6). Translocation t(6;11) was identified by cytogenetics alone in 13 cases. In three cases it was confirmed by fluorescence in situ hybridization (FISH) using whole chromosome paints (wcps) 6 and 11. In a further 14 cases involvement of MLL was demonstrated by FISH, by reverse transcriptase polymerase chain reaction (RT-PCR), by Southern blotting (SB) or by a combination of these methods. One case had a direct insertion of 11 into 6-dir ins(6;11)(q27;q13q23). Molecular investigations showed that one case had a 3′ deletion of MLL. The median overall survival for the patients was 12 months, indicating a poor prognosis for patients with a t(6;11) translocation.

The t(10;11)(p12;q23) translocation in acute leukaemia: a cytogenetic and clinical study of 20 patients

The clinical, haematological and cytogenetic data for 20 patients with an acquired abnormality of 11q23 and 10p have been reviewed at this workshop. Patients predominantly presented with de novo AML M5a and the most common cytogenetic finding was an inversion of part of the long arm of chromosome 11 followed by a translocation between 11q and 10p. Band p12 represented the most common breakpoint on chromosome 10. The t(10;11) subgroup defined a subset of younger 11q23 patients, the majority of whom achieve a first complete remission despite the differing treatment regimens.

ETV6/AML1 fusion by FISH in adult acute lymphoblastic leukemia

Dual-color interphase fluorescence in situ hybridization (FISH) with ETV6 and AML1 probes was used for the first time on a series of 159 adult patients with acute lymphoblastic leukemia (ALL), for detection of the t(12;21)(p13;q22) translocation. Seven patients (4.4%) were found, with 50–100% of positive cells, of whom one of two tested, proved negative for the fusion product by RT-PCR. Two of them, aged 43 and 50 years, are the oldest patients so far confirmed to have the translocation. Three who relapsed at 10, 11 and 24 months, suggest that adults may not enjoy the good short-term prognosis reported for t(12;21)-positive children. Thirty-one-negative cases had signal numbers differing from the two expected for each gene. In 15 cases these results were consistent with the karyotype. In nine cases with uninformative cytogenetics, the numbers were consistent with those for centromeres and indicated a hidden aneuploidy. Loss of ETV6 genes in two cases and AML1 amplification in three others were not suspected from the cytogenetics. In conclusion, FISH proved to be reliable in defining ETV6/AML1 positivity in this group of patients as well as providing valuable insights into negative cases.

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.

A Fluorescence in Situ Hybridization Map of 6q Deletions in Acute Lymphocytic Leukemia: Identification and Analysis of a Candidate Tumor Suppressor Gene

With the objective of identifying candidate tumor suppressor genes, we used fluorescence in situ hybridization to map leukemia-related deletions of the long arm of chromosome 6 (6q). Twenty of 24 deletions overlapped to define a 4.8-Mb region of minimal deletion between markers D6S1510 and D6S1692 within chromosome 6 band q16. Using reverse transcription-PCR, we found evidence of expression in hematopoietic cells for 3 of 15 genes in the region (GRIK2, C6orf111, and CCNC). Comparison between our own and published deletion data singled out GRIK2 as the gene most frequently affected by deletions of 6q in acute lymphocytic leukemia (ALL). Sequence analysis of GRIK2 in 14 ALL cases carrying heterozygous 6q deletions revealed a constitutional and paternally inherited C to G substitution in exon 6 encoding for an amino acid change in one patient. The substitution was absent among 232 normal alleles tested, leaving open the possibility that heterozygous carriers of such mutations may be susceptible to ALL. Although low in all normal hematopoietic tissues, quantitative reverse transcription-PCR showed higher baseline GRIK2 expression in thymus and T cells than other lineages. Among T-cell ALL patients, 6q deletion was associated with a statistically significant reduction in GRIK2 expression (P = 0.0001). By contrast, elevated GRIK2 expression was measured in the myelomonocytic line THP-1 and in one patient with common ALL. Finally, we detected significant levels of GRIK2 expression in prostate, kidney, trachea, and lung, raising the possibility that this gene may be protective against multiple tumor types.

Monosomy 20 as a pointer to dicentric (9;20) in acute lymphoblastic leukemia

Twenty new cases of acute lymphoblastic leukemia (ALL) with the dicentric chromosome dic(9;20)(p1113;q11) are presented. This chromosomal abnormality is difficult to identify from G-banding alone. It masquerades as monosomy 20 and is only accurately identified by fluorescence in situ hybridization (FISH). Monosomy 20 was found in 59/2790 patients with successful karyotypes entered to the Leukaemia Research Fund/UK Cancer Cytogenetics Group Karyotype Database in ALL (LRF/UKCCG Karyotype Database). FISH revealed dic(9;20) in 20/25 cases with available material. Extra copies of chromosome 21 were found in 8 of the 20 cases. Patients were 14 females and six males, aged 1–32 years (median 4 years), with leukocyte counts of 2–536 (median 23) × 109/l and immunophenotypes of common or pre-B ALL (17 cases), T-ALL (one case) or unknown (two cases). Four patients relapsed at 2, 22, 28 and 47 months and two died at 49 and 63 months (median follow-up 37 months). FISH studies on the remaining five patients showed one with monosomy 20 and four with other rearrangements of the chromosome. This study has increased the number of reported cases of dic(9;20) from 17 to 37. It has identified dic(9;20) in one case of T-ALL and shows an association of this translocation with trisomy 21.