Clinical, cytogenetic and molecular characteristics of 14 T-ALL patients carrying the TCRbeta-HOXA rearrangement: a study of the Groupe Francophone de Cytogénétique Hématologique.

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.

Fluorescence in situ hybridization analysis of 110 hematopoietic disorders with chromosome 5 abnormalities: do de novo and therapy-related myelodysplastic syndrome-acute myeloid leukemia actually differ?

A retrospective cytogenetic study of acute myeloid leukemias (AML) and myelodysplastic syndromes (MDS) was conducted by the Groupe Francophone de Cytogénétique Hématologique (GFCH) to evaluate the structural abnormalities of chromosome 5 associated with other chromosomal abnormalities, in particular of chromosome 7, in these pathologies. In all, 110 cases of AML/MDS were recruited based on the presence of chromosome 5 abnormalities under conventional cytogenetics and supplemented by a systematic fluorescence in situ hybridization study of chromosomes 5 and 7. The abnormalities of the long arm of chromosome 5 (5q) were deletions of various sizes and sometimes cryptic. The 5q abnormalities were associated with translocations in 54% of cases and were simple deletions in 46%. In 68% of cases, 5q deletions were associated with chromosome 7 abnormalities, and 90% of these presented a complex karyotype. Of the 110 patients, 28 had a hematopoietic disorder secondary to chemotherapy, radiotherapy, or both. Among 82 patients with de novo AML/MDS, 63 were older than 60 years. Chromosomal abnormalities often associated hypodiploidy and chromosome 5 and 7 abnormalities in complex karyotypes, features resembling those of secondary hemopathies. Systematic investigation of the exposure to mutagens and oncogenes is thus essential to specify the factors potentially involved in MDS/AML with 5q abnormalities.

Loss of the NPM1 gene in myeloid disorders with chromosome 5 rearrangements.

The assignment with chromosome banding techniques of the breakpoints of the recurrent translocation t(3;5) which leads to NPM1/MLF1 gene fusion in myeloid malignancies has not been unequivocal. In order to assess whether this is due to uncertainty in interpretation of the observed banding pattern or whether it reflects true genomic heterogeneity, we decided to analyze the breakpoint positions using fluorescence in situ (FISH) techniques in eight patients with myeloid malignancies and rearrangements of chromosomes 3 and 5. In three patients, colocalization of the NPM1 and MLF1 spanning BACs was demonstrated and NPM1/MLF1 fusion shown by PCR in one while in the remaining cases breakpoints were located outside the NPM1 and MLF1 loci. Interestingly, loss of a copy of the NPM1 gene was found in three of these latter patients. This findings suggest that haploinsufficiency of NPM1 may play a role in subtypes of myelodysplasias and leukemias.

Molecular cytogenetic study of 126 unselected T-ALL cases reveals high incidence of TCRbeta locus rearrangements and putative new T-cell oncogenes.

Chromosomal aberrations of T-cell receptor (TCR) gene loci often involve the TCRalphadelta (14q11) locus and affect various known T-cell oncogenes. A systematic fluorescent in situ hybridization (FISH) screening for the detection of chromosomal aberrations involving the TCR loci, TCRalphadelta (14q11), TCRbeta (7q34) and TCRgamma (7p14), has not been conducted so far. Therefore, we initiated a screening of 126 T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma cases and 19 T-ALL cell lines using FISH break-apart assays for the different TCR loci. Genomic rearrangements of the TCRbeta locus were detected in 24/126 cases (19%), most of which (58.3%) were not detected upon banding analysis. Breakpoints in the TCRalphadelta locus were detected in 22/126 cases (17.4%), whereas standard cytogenetics only detected 14 of these 22 cases. Cryptic TCRalphadelta/TCRbeta chromosome aberrations were thus observed in 22 of 126 cases (17.4%). Some of these chromosome aberrations target new putative T-cell oncogenes at chromosome 11q24, 20p12 and 6q22. Five patients and one cell line carried chromosomal rearrangements affecting both TCRbeta and TCRalphadelta loci. In conclusion, this study presents the first inventory of chromosomal rearrangements of TCR loci in T-ALL, revealing an unexpected high number of cryptic chromosomal rearrangements of the TCRbeta locus and further broadening the spectrum of genes putatively implicated in T-cell oncogenesis.

Allogeneic stem cell transplantation improves the outcome of adults with t(1;19)/E2A-PBX1 and t(4;11)/MLL-AF4 positive B-cell acute lymphoblastic leukemia: results of the prospective multicenter LALA-94 study.

Adult patients with acute lymphoblastic leukemia (ALL) and t(1;19)/E2A-PBX1 or t(4;11)/MLL-AF4 have a poor outcome. We have evaluated the impact of an intensified post-remission therapy using a high-dose chemotherapy course followed by allogeneic or autologous SCT on the outcome of 58 patients with t(1;19)/E2A-PBX1 (E2A group, n=24) or t(4;11)/MLL-AF4 (MLL group, n=34) treated in the LALA-94 multicenter prospective study. Patients in the MLL group had higher WBC counts and more frequent DIC. CR rates achieved by MLL and E2A groups were similar to other B-cell ALL (87, 82 and 86% respectively). While in CR, patients with a donor were assigned to alloSCT (n=22), the remaining patients with were randomized between autoSCT (n=15) or chemotherapy (n=8). Five-year overall survival was 31 and 45% for E2A and MLL groups, respectively. In both groups, DFS was higher in the alloSCT arm as compared to autoSCT and chemotherapy arms. The results of this study show that chemotherapy intensification did not overcome the poor prognosis of adults with t(1;19)/E2A-PBX1. Allogeneic SCT should thus be offered in first CR to patients with t(1;19)/E2A-PBX1 or t(4;11)/MLL-AF4. New therapeutic approaches are needed for patients without donor.

NUP98 rearrangements in hematopoietic malignancies: a study of the Groupe Francophone de Cytogénétique Hématologique.

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.

Cytogenetics and outcome of allogeneic transplantation in first remission of acute myeloid leukemia: the French pediatric experience.

We analyzed the impact of cytogenetics on 193 children enrolled in two successive French trials (LAME89/91 and ELAM02), who received hematopoietic stem cell transplantation during CR1. Detailed karyotype was available for 66/74 (89%) in LAME89/91 and 118/119 (99%) in ELAM02. Several karyotype and transplant characteristics differed according to therapeutic protocol: unfavorable karyotypes were more frequent in ELAM02 (36% vs 18%), pretransplant chemotherapy included high-dose cytarabine in ELAM02 and not in LAME89/91, IV replaced oral busulfan in the conditioning regimen, methotrexate was removed from post-transplant immunosuppression, and matched unrelated donor and cord blood transplantation were introduced. Five-year overall survival (OS) was 78.2% in LAME89 and 81.4% in ELAM02. OS was significantly lower for the unfavorable cytogenetic risk group in LAME89/91 when compared with intermediate and favorable groups (50% vs 90.6 and 86.4%, P=0.001). This difference was no longer apparent in ELAM02 (80.9% vs 71.3% and 5/5, respectively). Survival improvement for children with unfavorable karyotype was statistically significant (P=0.026) and was due to decrease in relapse risk. Five-year transplantation-related mortality was 6.75% in LAME89/91. In ELAM02, it was 3.2% for patients with a sibling donor and 10.9% with an unrelated donor or cord blood. We conclude that the outcome of children with unfavorable karyotype transplanted in CR1 has improved.

CALM-AF10+ T-ALL expression profiles are characterized by overexpression of HOXA and BMI1 oncogenes.

The t(10;11)(p13;q14-21) is found in T-ALL and acute myeloid leukemia and fuses CALM (Clathrin-Assembly protein-like Lymphoid-Myeloid leukaemia gene) to AF10. In order to gain insight into the transcriptional consequences of this fusion, microarray-based comparison of CALM-AF10+ vs CALM-AF10- T-ALL was performed. This analysis showed upregulation of HOXA5, HOXA9, HOXA10 and BMI1 in the CALM-AF10+ cases. Microarray results were validated by quantitative RT-PCR on an independent group of T-ALL and compared to mixed lineage leukemia-translocated acute leukemias (MLL-t AL). The overexpression of HOXA genes was associated with overexpression of its cofactor MEIS1 in CALM-AF10+ T-ALL, reaching levels of expression similar to those observed in MLL-t AL. Consequently, CALM-AF10+ T-ALL and MLL-t AL share a specific HOXA overexpression, indicating they activate common oncogenic pathways. In addition, BMI1, located close to AF10 breakpoint, was overexpressed only in CALM-AF10+ T-ALL and not in MLL-t AL. BMI1 controls cellular proliferation through suppression of the tumor suppressors encoded by the CDKN2A locus. This locus, often deleted in T-ALL, was conserved in CALM-AF10+ T-ALL. This suggests that decreased CDKN2A activity, as a result of BMI1 overexpression, contributes to leukemogenesis in CALM-AF10+ T-ALL. We propose to define a HOXA+ leukemia group composed of at least MLL-t, CALM-AF10 and HOXA-t AL, which may benefit from adapted management.

A new recurrent inversion, inv(7)(p15q34), leads to transcriptional activation of HOXA10 and HOXA11 in a subset of T-cell acute lymphoblastic leukemias.

Chromosomal translocations with breakpoints in T-cell receptor (TCR) genes are recurrent in T-cell malignancies. These translocations involve the TCRalphadelta gene (14q11), the TCRbeta gene (7q34) and to a lesser extent the TCRgamma gene at chromosomal band 7p14 and juxtapose T-cell oncogenes next to TCR regulatory sequences leading to deregulated expression of those oncogenes. Here, we describe a new recurrent chromosomal inversion of chromosome 7, inv(7)(p15q34), in a subset of patients with T-cell acute lymphoblastic leukemia characterized by CD2 negative and CD4 positive, CD8 negative blasts. This rearrangement juxtaposes the distal part of the HOXA gene cluster on 7p15 to the TCRbeta locus on 7q34. Real time quantitative PCR analysis for all HOXA genes revealed high levels of HOXA10 and HOXA11 expression in all inv(7) positive cases. This is the first report of a recurrent chromosome rearrangement targeting the HOXA gene cluster in T-cell malignancies resulting in deregulated HOXA gene expression (particularly HOXA10 and HOXA11) and is in keeping with a previous report suggesting HOXA deregulation in MLL-rearranged T- and B cell lymphoblastic leukemia as the key factor in leukaemic transformation. Finally, our observation also supports the previous suggested role of HOXA10 and HOXA11 in normal thymocyte development.

t(6;8), t(8;9) and t(8;13) translocations associated with stem cell myeloproliferative disorders have close or identical breakpoints in chromosome region 8p11-12.

A stem-cell myeloproliferative disorder involving T- and B-cell, and myeloid lineages, is associated with three different translocations with a breakpoint in region p11-12 of chromosome 8: t(6;8)(q27;p11), t(8;9)(p11;q33), and t(8;13)(p12;q12), respectively. Using fluorescence in situ hybridization (FISH), we have analysed blood cells from a series of five patients carrying these different translocations. We have identified cosmids from chromosome region 8p11-12 that span the breakpoint in all the cases. They are specific for the FCFR1 gene that encodes a receptor for members of the FGF family. The breakpoint was further detected by Southern and pulsed-field gel electrophoresis analyses with probes from the FGFR1 locus.

Value of high-dose cytarabine during interval therapy of a Berlin-Frankfurt-Munster-based protocol in increased-risk children with acute lymphoblastic leukemia and lymphoblastic lymphoma: results of the European Organization for Research and Treatment of Cancer 58881 randomized phase III trial.

PURPOSE: The European Organization for Research and Treatment of Cancer 58881 study was designed to test in a prospective multicentric randomized trial the value of high-dose (HD) intravenous (IV) cytarabine (Ara-C) added to HD IV methotrexate (MTX) to reduce the incidence of CNS and systemic relapses in children with increased-risk acute lymphoblastic leukemia (ALL) or stage III and IV lymphoblastic lymphoma treated with a Berlin-Frankfurt-Munster (BFM)-based regimen. PATIENTS AND METHODS: After completion of induction-consolidation phase, children with increased-risk (risk factor > 0.8 or T-lineage) ALL or stage III and IV lymphoblastic lymphoma were randomized to receive four courses of HD MTX (5 g/m(2) over 24 hours every 2 weeks) and four intrathecal administrations of MTX (Arm A) or the same treatment schedule with additional HD IV Ara-C (1 g/m(2) in bolus injection 12 and 24 hours after the start of each MTX infusion) (Arm B). RESULTS: Between January 1990 and January 1996, 653 patients with ALL (593 patients) or lymphoblastic lymphoma (60 patients) were randomized: 323 were assigned to Arm A (without Ara-C) and 330 to Arm B (with Ara-C). A total of 190 events (177 relapses and 13 deaths without relapse) were reported, and the median follow up was 6.5 years (range, 2 to 10 years). The incidence rates of CNS relapse were similar in both arms whether isolated (5.6% and 3.3%, respectively) or combined (5.3% and 4.6%, respectively). The estimated 6-year disease-free survival (DFS) rate was similar (log-rank P =.67) in the two treatment groups: 70.4% (SE = 2.6%) in Arm A and 71.0% (SE = 2.5%) in Arm B. The 6-year DFS rate was similar for ALL and LL patients: 70.2% (SE = 1.9%) versus 76.3% (SE = 5.6%). CONCLUSION: Prevention of CNS relapse was satisfactorily achieved with HD IV MTX and intrathecal injections of MTX in children with increased-risk ALL or stage III and IV lymphoblastic lymphoma treated with our BFM-based treatment protocol in which cranial irradiation was omitted. Disappointingly, with the dose schedule used in this protocol, HD Ara-C added to HD MTX, although well tolerated, failed to further decrease the incidence of CNS relapse or to improve the overall DFS.

Genetic abnormalities associated with the t(12;21) and their impact in the outcome of 56 patients with B-precursor acute lymphoblastic leukemia.

The ETV6/RUNX1 rearrangement is found in 20-30% of children with B-cell precursor acute lymphoblastic leukemia and is associated with a good outcome. To determine the cytogenetic and molecular abnormalities associated with the ETV6/RUNX1 rearrangement and the influence of this rearrangement in patients' evolution, we analyzed the molecular cytogenetic profiles of 56 children with this rearrangement and B-cell precursor acute lymphoblastic leukemia. Secondary changes detected with conventional cytogenetics and with fluorescence in situ hybridization were found in 71.4% of cases, the most frequent being the loss of the normal ETV6 allele, 12p aberrations, duplication of the fusion gene, and trisomy 21, as in replicating the results of previous studies. In this preliminary series, with a mean follow-up of 69.3 months, secondary abnormalities did not influence patients' outcome. It seems therefore that the prognostic value of the t(12;21) does not vary and that ETV6/RUNX1 rearrangement is an independent indicator of good prognosis.

Delineation of a 6 cM commonly deleted region in childhood acute lymphoblastic leukemia on the 6q chromosomal arm.

Deletion of the long arm of human chromosome 6 in acute lymphoblastic leukemia (ALL) has been shown by cytogenetic studies in 4-11% of cases. To characterize further the region of deletion and to precisely establish its frequency, we studied loss of heterozygosity (LOH) in 120 children with ALL using polymorphic markers located from the 6q14-15 chromosomal band to the telomere. LOH was detected in eight patients. A single region of LOH, flanked distally by D6S1594 and proximally by D6S301 was detected. These DNA markers are separated by 6 cM and are approximately located at the 6q21-22 band. Our present results delineate a region that is likely to contain a tumor-suppressor gene involved in a subset of childhood ALLs.

Cytogenetic abnormalities associated with the t(12;21): a collaborative study of 169 children with t(12;21)-positive acute lymphoblastic leukemia.

The t(12;21)(p13;q22) is a cryptic abnormality observed in 25% of children with B-lineage acute lymphoblastic leukemia (ALL), associated with a favorable prognosis. To determine whether specific cytogenetic abnormalities accompany the t(12;21), we analyzed the cytogenetic profiles of blast cells from 169 ALL cases positive for the t(12;21), previously identified by molecular methods. Only 13.6% of samples had normal karyotypes. Structural changes were detected in 89.7% of abnormal karyotypes, and numerical abnormalities in 47%. Rearrangements of 12p were the most frequent structural aberration (57 out of 146 patients with chromosomal abnormalities). Nonspecific deletions of chromosomes 6 and 9 were also found. The most frequent numerical abnormalities was trisomy for chromosomes 21. Blast cells were pseudodiploid (45.6%), hyperdiploid with 47 to 51 chromosomes (24.3%), hypodiploid with 44 to 45 chromosomes (10%), near-triploid (0.6%), or near-tetraploid (5.9%). Our results show that the t(12;21) is not associated with hyperdiploidy of 52 to 68 chromosomes or with the prognostic t(1;19), t(4;11) or t(9;22). Only children with B-lineage ALL who lack these abnormalities detected by conventional cytogenetics will probably benefit from additional testing by molecular methods to detect the t(12;21).

Establishment of a novel anaplastic large-cell lymphoma-cell line (COST) from a 'small-cell variant' of ALCL.

Anaplastic large-cell lymphoma (ALCL) is a distinct biological and cytogenetic entity with a broad spectrum of morphological features (common type, small-cell variant and lymphohistiocytic variant). Few cell lines of ALCL are available and they all originate from primary tumors demonstrating the common type morphology (ie large-sized lymphoma cells). We established a new ALCL cell line (COST) from the peripheral blood of a patient with a small-cell variant of ALCL, at diagnosis. Cells growing in vitro and in SCID mice consisted of two populations, that is, small- and large-sized cells as seen in the patient's tumor. Both large and small malignant cells were positive for CD43/MT1 T-cell associated antigen, perforin, granzyme B and TIA-1, but negative for CD2, CD3, CD5, CD7, CD4 and CD8 antigens. Standard cytogenetic studies as well as multiplex FISH confirmed the presence of the canonical t(2;5)(p23;q35) translocation, but also revealed additional numerical and structural abnormalities. The COST cell line is the first ALCL small-cell variant cell line, and thus provides a potentially useful tool for further functional and molecular studies that should improve our understanding of the small-cell variant of ALCL, which is more frequently complicated by a leukemic phase.

Effects of H-7 and staurosporine on proliferation and self-renewal of acute myeloid leukemia progenitors.

In this study, we compared the impact of two protein kinase (PK) inhibitors, H-7 and staurosporine, on the normal myeloid progenitors (CFU-GM) and acute myeloid leukemia progenitors (AML-CFU) proliferation measured by in vitro clonogenic assay. H-7 and staurosporine displayed a biphasic dose-effect on both CFU-GM and AML-CFU recovery. At the lowest concentration range (0.1 microM to 20 microM for H-7 and 0.1 nM to 1 nM for staurosporine), we observed growth stimulation whereas higher concentrations induced dose-dependent growth inhibition. Moreover, AML-CFU proved to be significantly more sensitive to the inhibitory effect of both H-7 and staurosporine than CFU-GM (3.16- and 2.12-fold, respectively). These results were further confirmed with comparable murine cell line models (FDC-P1, a hematopoietic cell line generated from normal bone marrow and WEHI, a myelomonocytic leukemia cell line). Furthermore, we report that both H-7 and staurosporine present similar inhibitory effects on proliferation (PE1) as on self-renewal (PEs) of AML-CFU. In an attempt to understand more fully the mechanism of action of H-7 and staurosporine, we investigated their impact (when used at their D50) on the human myelogenous leukemia cell line, K562. H-7 and staurosporine induced a transient decrease of cell growth, between 0 and 24 hours, and produced a transient blockade of K562 cells in the S-phase, either 24 or 48 hours after the addition of staurosporine and H-7, respectively.

B-cell lymphoma following polycythemia vera: evidence for the involvement of two different clones.

We report here two patients with polycythemia vera (PV) who developed secondary non-Hodgkin's lymphoma (NHL). Both cases were high grade B-cell NHL. Cytogenetic analysis of bone marrow and lymph node was performed in each case and showed numerous chromosomal abnormalities. Of interest, chromosomal abnormalities of the PV and of the NHL clones were different, suggesting the possible involvement of two different clones. A 11q23 breakpoint was common between the two cases and the putative role of this breakpoint in the pathogenesis of the NHLs is discussed.

Prognostic significance of karyotype in de novo adult acute myeloid leukemia. The BGMT group.

A group of 201 adult patients, 127 younger and 74 older than 55 years, with de novo acute myeloid leukemia were investigated to determine the prognostic significance of karyotype on early death (toxic or aplastic death occurring before hematopoietic recovery), drug resistance, continuous complete remission (CCR) and survival probabilities at 5 years. A good prognostic impact was found for t(8;21), t(15;17) and inv(16). The best factor proved to be t(8;21) (5-year survival probability: 50%), followed by t(15;17) (5-year survival probability: 39%) and by inv(16) (5-year survival probability: 43%). An intermediate outcome was found in patients with trisomy 8 (27% alive at 5 years) and in patients with numerical abnormalities other than -7 and +8 (33% in CCR and 62% alive at 5 years). Normal karyotypes had a different prognostic impact according to age: intermediate in young and good in older patients. A poor outcome was observed among patients with del(5q)/-5 (median survival: 1 month), with 11q23 rearrangements (median survival: 1.5 months) and with del(7q)/-7 (median survival: 10 months). The 'other structural change' group was also found to be a poor risk population (5-year survival probability: 5%) whereas complex karyotypes were predictive of short survivals only in older patients. Conversely, del(7q)/-7 and +8 as secondary changes, had no prognostic impact.

Effect of melphalan against self-renewal capacity of leukemic progenitors in acute myeloblastic leukemia.

This study aimed to evaluate the effect of melphalan on both terminal divisions and self-renewal capacity of acute myeloblastic leukemia (AML) progenitors (colony-forming units, CFU-L) grown in methylcellulose. Terminal divisions and self-renewal were assayed by primary (PE1) and secondary (PE2) colony formation, respectively. Thirteen cases of AML, were tested. Melphalan induced a negative exponential dose-effect on CFU-L survival. Moreover, melphalan was equally effective in inhibiting CFU-L growth in both PE1 and PE2 assays, with D10 values of 1.53 +/- 0.17 micrograms/ml and 1.59 +/- 0.21 micrograms/ml for PE1 and PE2, respectively (p = 0.48). Cytotoxicity of melphalan on CFU-L did not differ significantly from that observed for normal hemopoietic granulocyte-macrophage colony-forming units, erythroid burst-forming units, and granulocyte-erythroid-macrophage-megakaryocyte progenitors. Mafosfamide-lysine, a stable cyclophosphamide congener, strongly inhibited primary colony formation (PE1) with a D10 value of 14.46 +/- 1.76 micrograms/ml, but was much less efficient in the PE2 assay. Our findings suggest that the self-renewal capacity of AML progenitors can be differentially affected by alkylating agents. Moreover, since it is now considered that chemotherapy should be preferentially directed against the self-renewal of leukemic progenitors, melphalan might offer a greater potential than cyclophosphamide or cyclophosphamide derivatives in the therapy of AML.

Large deletions 5' to the ETO breakpoint are recurrent events in patients with t(8;21) acute myeloid leukemia.

Recurrent chromosomal rearrangements are observed in many leukemia subtypes. Recently, it has been shown that several of these translocations/inversions were associated with the loss of sequences located in the vicinity of the chromosomal breakpoints. So far, such deletions have not been described for the t(8;21) translocation. We have analyzed a series of 65 patients with t(8;21) using several probes specific for the ETO and AML1 regions. We have found six patients (9%) with deletion of the region 5' to ETO. In all six patients, the deletion encompassed at least 260 kb, and was even larger in two patients (up to 2 Mb). A similar analysis of the 21q22 region did not reveal any deletion of the 3'AML1 region. In conclusion, cytogenetically undetectable small deletions located immediately 5' to the ETO breakpoint were found to accompany the t(8;21) translocation in a significant percentage of cases. The clinical significance, if any, of these deletions remains to be determined.