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Pediatric acute myeloid leukemia (AML) is a rare disease whose prognosis is highly variable according to factors such as chromosomal abnormalities. Recurrent genomic rearrangements are detected in half of pediatric AML by karyotype. NUcleoPorin 98 (NUP98) gene is rearranged with 31 different fusion partner genes. These rearrangements are frequently undetected by conventional cytogenetics, as the NUP98 gene is located at the end of the chromosome 11 short arm (11p15). By screening a series of 574 pediatric AML, we detected a NUP98 rearrangement in 22 cases (3.8%), a frequency similar to CBFB-MYH11 fusion gene (4.0%). The most frequent NUP98 fusion gene partner is NSD1. These cases are homogeneous regarding their biological and clinical characteristics, and associated with bad prognosis only improved by bone marrow transplantation. We detailed the biological characteristics of these AML by exome sequencing which demonstrated few recurrent mutations (FLT3 ITD, WT1, CEBPA, NBPF14, BCR and ODF1). The analysis of the clonal structure in these cases suggests that the mutation order in the NUP98-rearranged pediatric AML begins with the NUP98 rearrangement leading to epigenetic dysregulations then followed by mutations of critical hematopoietic transcription factors and finally, activation of the FLT3 signaling pathway.

Episomes with the NUP214-ABL1 fusion gene have been observed in 6% of T-ALL. In this multicentric study we collected 27 cases of NUP214-ABL1-positive T-ALL. Median age was 15 years with male predominance. Outcome was poor in 12 patients. An associated abnormality involving TLX1 or TLX3 was found in all investigated cases. Fluorescent in situ hybridization revealed a heterogeneous pattern of NUP214-ABL1 amplification. Multiple episomes carrying the fusion were detected in 24 patients. Episomes were observed in a significant number of nuclei in 18 cases, but in only 1-5% of nuclei in 6. In addition, intrachromosomal amplification (small hsr) was identified either as the only change or in association with episomes in four cases and two T-ALL cell lines (PEER and ALL-SIL). One case showed insertion of apparently non-amplified NUP214-ABL1 sequences at 14q12. The amplified sequences were analyzed using array-based CGH.These findings confirm that the NUP214-ABL1 gene requires amplification for oncogenicity; it is part of a multistep process of leukemogenesis; and it can be a late event present only in subpopulations. Data also provide in vivo evidence for a model of episome formation, amplification and optional reintegration into the genome. Implications for the use of kinase inhibitors are discussed.

The t(8;16)(p11;p13) is a rare translocation involved in de novo and therapy-related myelomonocytic and monocytic acute leukemia. It fuses two genes encoding histone acetyltransferases (HATs), MYST3 located at 8p11 to CREBBP located at 16p13. Variant translocations involve other HAT-encoding genes such as EP300, MYST4, NCOA2 or NCOA3. MYST3-linked acute myeloid leukemias (AMLs) share specific clinical and biological features and a poor prognosis. Because of its rarity, the molecular biology of MYST3-linked AMLs remains poorly understood. We have established the genome and gene expression profiles of a multicentric series of 61 M4/M5 AMLs including 18 MYST3-linked AMLs by using array comparative genome hybridization (aCGH) (n=52) and DNA microarrays (n=44), respectively. We show that M4/5 AMLs have a variety of rare genomic alterations. One alteration, a gain of the MYB locus, was found recurrently and only in the MYST3-linked AMLs (7/18 vs 0/34). MYST3-AMLs have also a specific a gene expression profile, which includes overexpression of MYB, CD4 and HOXA genes. These features, reminiscent of T-cell acute lymphoid leukemia (ALL), suggest the targeting of a common T-myeloid progenitor.

Thirty cases of acute myeloid leukaemia (AML) with MYST histone acetyltransferase 3 (MYST3) rearrangement were collected in a retrospective study from 14 centres in France and Belgium. The mean age at diagnosis was 59.4 years and 67% of the patients were females. Most cases (77%) were secondary to solid cancer (57%), haematological malignancy (35%) or both (8%), and appeared 25 months after the primary disease. Clinically, cutaneous localization and disseminated intravascular coagulation were present in 30 and 40% of the cases, respectively. AMLs were myelomonocytic (7%) or monocytic (93%), with erythrophagocytosis (75%) and cytoplasmic vacuoles (75%). Immunophenotype showed no particularity compared with monocytic leukaemia without MYST3 abnormality. Twenty-eight cases carried t(8;16)(p11;p13) with MYST3-CREBBP fusion, one case carried a variant t(8;22)(p11;q13) and one case carried a t(8;19)(p11;q13). Type I (MYST3 exon 16-CREBBP exon 3) was the most frequent MYST3-CREBBP fusion transcript (65%). MYST3 rearrangement was associated with a poor prognosis, as 50% of patients deceased during the first 10 months. All those particular clinical, cytologic, cytogenetic, molecular and prognostic characteristics of AML with MYST3 rearrangement may have allowed an individualization into the World Health Organization classification.

A series of 38 patients with acute myeloblastic leukemia (AML) with 49 or more chromosomes and without structural abnormalities was selected within the Groupe Francophone de Cytogénétique Hématologique (GFCH) to better define their characteristics. The median age of the patients was 65 years, and all FAB subtypes were represented. Although all chromosomes were gained, some seems to prevail: chromosome 8 (68%), 21 (47%), 19 (37%), and 13 and 14 (34% each). Since MLL rearrangement leads patients in a group with an unfavorable prognosis, search for cryptic rearrangements of MLL was performed in 34 patients and showed abnormalities in 5 (15%). When we applied the most frequent definition of complex karyotypes (three or more abnormalities), all patients with high hyperdiploid AML fall in the unfavorable category. Among the 18 patients without MLL rearrangement receiving an induction therapy, 16 (89%) reached CR and 6 (33%) were still alive after a 31-month median follow-up (14-61 months). Although this study was retrospective, these results suggest that high hyperdiploid AML without chromosome rearrangement seems to be a subgroup of uncommon AML (less than 1%), and may be better classified in the intermediate prognostic group.

Recently, we and others described a new chromosomal rearrangement, that is, inv(7)(p15q34) and t(7;7)(p15;q34) involving the T-cell receptor beta (TCRbeta) (7q34) and the HOXA gene locus (7p15) in 5% of T-cell acute lymphoblastic leukemia (T-ALL) patients leading to transcriptional activation of especially HOXA10. To further address the clinical, immunophenotypical and molecular genetic findings of this chromosomal aberration, we studied 330 additional T-ALLs. This revealed TCRbeta-HOXA rearrangements in five additional patients, which brings the total to 14 cases in 424 patients (3.3%). Real-time quantitative PCR analysis for HOXA10 gene expression was performed in 170 T-ALL patients and detected HOXA10 overexpression in 25.2% of cases including all the cases with a TCRbeta-HOXA rearrangement (8.2%). In contrast, expression of the short HOXA10 transcript, HOXA10b, was almost exclusively found in the TCRbeta-HOXA rearranged cases, suggesting a specific role for the HOXA10b short transcript in TCRbeta-HOXA-mediated oncogenesis. Other molecular and/or cytogenetic aberrations frequently found in subtypes of T-ALL (SIL-TAL1, CALM-AF10, HOX11, HOX11L2) were not detected in the TCRbeta-HOXA rearranged cases except for deletion 9p21 and NOTCH1 activating mutations, which were present in 64 and 67%, respectively. In conclusion, this study defines TCRbeta-HOXA rearranged T-ALLs as a distinct cytogenetic subgroup by clinical, immunophenotypical and molecular genetic characteristics.

The NUP98 gene is fused with 19 different partner genes in various human hematopoietic malignancies. In order to gain additional clinico-hematological data and to identify new partners of NUP98, the Groupe Francophone de Cytogénétique Hématologique (GFCH) collected cases of hematological malignancies where a 11p15 rearrangement was detected. Fluorescence in situ hybridization (FISH) analysis showed that 35% of these patients (23/66) carried a rearrangement of the NUP98 locus. Genes of the HOXA cluster and the nuclear-receptor set domain (NSD) genes were frequently fused to NUP98, mainly in de novo myeloid malignancies whereas the DDX10 and TOP1 genes were equally rearranged in de novo and in therapy-related myeloid proliferations. Involvement of ADD3 and C6ORF80 genes were detected, respectively, in myeloid disorders and in T-cell acute lymphoblastic leukemia (T-ALL), whereas the RAP1GDS1 gene was fused to NUP98 in T-ALL. Three new chromosomal breakpoints: 3q22.1, 7p15 (in a localization distinct from the HOXA locus) and Xq28 were detected in rearrangements with the NUP98 gene locus. The present study as well as a review of the 73 cases previously reported in the literature allowed us to delineate some chromosomal, clinical and molecular features of patients carrying a NUP98 gene rearrangements.

Imatinib mesylate (Gleevec), an inhibitor of the BCR-ABL tyrosine kinase, was introduced recently into the therapy of chronic myeloid leukemia (CML). Several cases of emergence of clonal chromosomal abnormalities after therapy with imatinib have been reported, but their incidence, etiology and prognosis remain to be clarified. We report here a large series of 34 CML patients treated with imatinib who developed Philadelphia (Ph)-negative clones. Among 1001 patients with Ph-positive CML treated with imatinib, 34 (3.4%) developed clonal chromosomal abnormalities in Ph-negative cells. Three patients were treated with imatinib up-front. The most common cytogenetic abnormalities were trisomy 8 and monosomy 7 in twelve and seven patients, respectively. In 15 patients, fluorescent in situ hybridization with specific probes was performed in materials archived before the initiation of imatinib. The Ph-negative clone was related to previous therapy in three patients, and represented a minor pre-existing clone that expanded after the eradication of Ph-positive cells with imatinib in two others. However, in 11 patients, the new clonal chromosomal abnormalities were not detected and imatinib may have had a direct effect. No myelodysplasia was found in our cohort. With a median follow-up of 24 months, one patient showed CML acceleration and two relapsed.

To accurately estimate the incidence of HOX11L2 expression, and determine the associated cytogenetic features, in T-cell acute lymphoblastic leukemia (T-ALL), the Groupe Français de Cytogénétique Hématologique (GFCH) carried out a retrospective study of both childhood and adult patients. In total, 364 patients were included (211 children

The MLL gene, located at 11q23, is frequently rearranged in acute leukaemia as either chimaeric fusion genes or partial tandem duplications. We report a series of 12 acute leukaemia cases with apparent amplification of the MLL gene ascertained using fluorescence in situ hybridisation (FISH). Seven cases showed intrachromosomal amplification of MLL, four cases showed extrachromosomal amplification as double minute chromosomes (dmin) and one case had separate subclones with dmin and homogenously staining region (hsr). Southern blot analysis of the MLL gene showed MLL gene rearrangement in three of the 10 successful cases. These cases do not naturally fall into either of the two recognised categories of MLL rearrangement and may represent a third variety of MLL gene abnormalities.

Therapy-related leukemia associated with chemotherapy, particularly alkylating agents and topoisomerase II inhibitors, are being reported with increasing frequency in the literature mainly after breast cancer. We also observed an increasing number of such leukemias in the data base of the specialized registry of hematological malignancies of the Côte d'Or department. Between 1980 and 1998, 156 AML and RAEB-t were registered in women in Côte d'Or. Among them, 12 occurred in women with breast cancer history (7.7%). Analysis by period of time shows a significant increase in the proportion of therapy-related leukemia secondary to breast cancer (P < 0.02). Chemotherapy including topoisomerase II inhibitors was used in 10 cases in which mitoxantrone was used in eight cases. In these eight cases, leukemia had clinical and biological characteristics usually described with topoisomerase II inhibitors but 44% were promyelocytic sub-type with the t(15;17) specific karyotypic abnormality. These data on a well-defined population demonstrate the increased proportion of therapy-related leukemia secondary to breast cancer, probably due to the use of mitoxantrone.

The MLL gene located at 11q23 has been described as a 'promiscuous' gene due its involvement with a large number of genetic partners. The EU Concerted Action Workshop on 11q23 provided 550 cases for study of which 82 showed abnormalities which did not involve the established translocations or deletion of 11q23. In these 'other' cases, which included inversions and duplications, 11q23 was found to be involved with 25 chromosome partners of which 10 had not been previously reported. These were 1q31, 4p11, 6q13, 8q21, 10q22, 10q25, 11q11, 11q21, 13q34 and 18q23. This study demonstrated the value of the Workshop, in confirming the diversity of chromosomal partner sites involved with 11q23 and in the identification of new partners.

This study prospectively analysed the relationships between immunophenotypic and cytogenetic features of blast cells in 432 acute non-lymphoblastic leukemias (ANLL) at presentation. An abnormal karyotype was detected in 232 cases (54%). These abnormalities were related to immunophenotypic markers as detected using a consensual panel of monoclonal antibodies allowing lineage assignment and investigation of myeloid marker expression on blast cells. In univariate analysis, CD9, CD10, CD15, CD34 and TdT expression appeared significantly associated with chromosomal anomalies. Multivariate analysis identified CD34 and CD9 expression as independently predictive of the presence of at least one cytogenetic abnormality (P < 10(-4) and P < 0.03, respectively). Significant associations between immunophenotypic and karyotypic features were observed both within individual FAB subgroups and independently from morphological criteria. Specific features were seen in five ANLL entities: M0 or M1/B lineage antigen positivity/t(9;22) or del(11)(q23); M2/CD13-/t(8;21); M4/CD13+, CD34+, CD36+/inv(16); M4 or M5/lack of B lineage antigen/del(11)(q23) or t(9;11). More practically, and although the relationships demonstrated only represent a fraction of homogeneous immunophenotypic subgroups, identification of such immunophenotypic features should prompt careful karyotypic examination, eventually using molecular biology analysis on non-growing cells.

Aneuploidy is a frequent feature in multiple myeloma. Cytogenetic analyses have shown that a 14q+ chromosome resulting from either a t(8;14)(q24;q32) or a t(11;14)(q13;q32) was the most consistent abnormality but no specific chromosomal aberration has been identified in this disease. Bone marrow cells from 121 consecutive patients with multiple myeloma were analyzed cytogenetically by standard banding techniques including RHG, GTG and CBG banding. Cells were cultured for 24-96 h in the presence or in the absence of interleukin-6. Clonal abnormalities were detected in 41 of the 121 patients (34%). A der(16)t(1;16)(q10;p10) abnormality was identified in nine of these 41 patients (22%). Der(16) was identified at diagnosis in five patients, during disease progression in two additional patients, and at the time of a relapse in the two last cases. The t(1;15)(q10;p10) translocation was always unbalanced, resulting in a monosomy 16q in all cases. The CBG banding did not demonstrate dicentric chromosomes and the whole chromosome painting confirmed the der(16). A large number of other chromosomal abnormalities were associated with der(16), including chromosomal rearrangements involving the 8q24 band in five cases. Four of these five cases were Burkitt's-type translocations. This observation suggests that der(16)t(1;16)(q10;p10) could be one of the most frequent chromosomal abnormalities that can be identified in multiple myeloma cells.

The polymerase chain reaction (PCR) was used to amplify cDNA in order to characterize normal and hybrid T-cell receptor (TCR) gene rearrangements derived from a T-cell acute lymphoblastic leukemia (T-ALL) bearing a chromosome 7 inversion. The nucleotide sequence analysis of the amplified product showed the presence of an out-of-frame V beta/J gamma/C gamma transcript and an in-frame V gamma/J gamma/C beta transcript which result from an interlocus recombination between the TCR-beta and gamma loci and the transcription of the reciprocal hybrid TCR gene. The sequence analysis of the reciprocal DNA segment directly involved in the breakpoint of the inversion showed a recombination between a J gamma-sequence heptamer signal and a coding J beta gene segment. The exonuclease nibbling of each DNA extremity and the non-templated nucleotide insertion observed at both coding and reciprocal joints demonstrate that the inversion is mediated by the lymphocyte recombinase complex. During T-cell differentiation, TCR-beta genes are rearranged prior to TCR-alpha genes. In the present case of T-ALL, we have shown that TCR-delta genes are rearranged at both loci, excluding productive rearrangements of TCR-alpha genes. The molecular analysis of the normal TCR genes derived from the leukemic cells revealed the presence of productively rearranged TCR-beta, gamma, and delta genes. Cell surface staining of these cells showed the presence of CD3 molecules and of a TCR-beta chain corresponding to the normal beta allele expressed in a disulfide-linked complex with a protein which is not TCR-alpha, gamma, or delta. This could represent the cell surface expression of the hybrid TCR-V gamma/C beta protein or the expression of a TCR-beta homodimer.

The expression of the P-glycoprotein which is associated with the development of multidrug resistance in various cell lines was investigated in 87 fresh acute leukaemia and multiple myeloma samples using the specific mouse monoclonal antibody MRK16 in an indirect immunofluorescence assay. Considering a 10% positive cell cut-off value, a heterogeneous expression of P-glycoprotein was observed in 5/22 (22.7%) de novo acute leukaemias, 7/22 (31.8%) relapse or secondary acute leukaemias, 14/27 (51.8%) acute transformation of myeloproliferative or myelodysplastic syndromes and 5/16 (31.2%) multiple myelomas. This expression was not associated with specific cytogenetic abnormalities, especially alterations of chromosome 7q. Verapamil, a calcium channel blocker, has been demonstrated to circumvent the multidrug resistance in cell lines, possibly by interfering with P-glycoprotein function. Using the microculture tetrazolium assay, verapamil was demonstrated to increase the sensitivity of fresh leukaemic or myeloma cells to doxorubicin in 19/43 (43.1%) samples. The doxorubicin IC50 level and the capacity of verapamil to increase the sensitivity of blast cells to doxorubicin in vitro did not correlate with the expression of P-glycoprotein. We conclude that high non-cytotoxic concentrations of verapamil were able to increase the in vitro doxorubicin sensitivity of fresh acute leukaemia and myeloma cells without detectable expression of the P-glycoprotein.