Low frequency of the TEL/AML1 fusion gene in acute lymphoblastic leukaemia in Spain

The molecular defect of a congenitally dysfunctional form of prothrombin, prothrombin Segovia, was identified in a patient with a severe bleeding tendency, reduced prothrombin coagulant activity, and normal antigen level. Nucleotide sequencing of amplified DNA revealed a G --> A change at nucleotide 7539 of exon 9 of the prothrombin gene. This resulted in the substitution of Gly319 by Arg. The proband was homozygous for this mutation, his father and brother were heterozygous. We surmised that the substitution, which occurs near the site of cleavage of prothrombin by factor Xa (Arg320-Ile321), altered the conformation of the protein making the cleavage site inaccessible.     

AML1 gene amplification: a novel finding in childhood acute lymphoblastic leukemia

BACKGROUND AND OBJECTIVE: We previously found a high-level amplification in chromosomal region 21q22 in two children with acute lymphoblastic leukemia (ALL) using comparative genomic hybridization. The same region harbors the AML1 gene. The aim of the present study was to investigate whether AML1 is a target gene in these amplifications. DESIGN AND METHODS: Bone marrow samples were obtained from 112 childhood ALL patients. The copy number of AML1 was studied using fluorescent in situ hybridization with a dual color DNA probe specific for the AML1 and TEL genes. RESULTS: Three of the patients had 3-to-8 fold amplification of AML1 and showed a high-level amplification of 21q22 by comparative genomic hybridization. In two of them the extra copies were shown to be located tandemly in a derivative of chromosome 21. Thirty-seven of the patients (33%) had 1-to-2 extra copies of AML1, most probably reflecting the incidence of trisomy 21 and tetrasomy 21. The TEL-AML1 fusion was less frequent in the patients with extra copies of AML1 (7/40; 18%) than in the patients with no extra copy (24/72; 33%). None of the three patients with 3-to-8 fold amplification of AML1 showed the fusion or loss of TEL. INTERPRETATION AND CONCLUSIONS: Our findings suggest that the AML1 gene is a target gene in the 21q22 amplicon in childhood ALL. To understand the role, if any, of the AML1 amplification in leukemogenesis, further studies are needed.     

Translocation t(12;21) is related to in vitro cellular drug sensitivity to doxorubicin and etoposide in childhood acute lymphoblastic leukemia

The t(12;21) (p13;q22) translocation resulting in ETV6/RUNX1 (previously named TEL/AML1) gene fusion is present in about 25% of children with precursor B-lineage acute lymphoblastic leukemia (B-ALL). We successfully tested 275 precursor B-ALL samples from children aged 1 to 17 years to determine the relation between t(12;21) and in vitro cellular drug resistance, measured by the fluorometric microculture cytotoxicity assay (FMCA). Samples from 83 patients (30%) were positive for t(12;21). The ETV6/RUNX1(+) samples were significantly more sensitive than ETV6/RUNX1(-) samples to doxorubicin, etoposide, amsacrine, and dexamethasone, whereas the opposite was true for cytarabine. After matching for unevenly distributed patient characteristics, that is, excluding patients with high hyperdiploidy (> 51 chromosomes), t(9; 22), t(1;19), or 11q23 rearrangement, the ETV6/RUNX1(+) samples remained significantly more sensitive to doxorubicin (P = .001) and etoposide (P = .001). For the other drugs tested (amsacrine, cytarabine, dexamethasone, prednisolone, vincristine, 6-thioguanine, and 4-hydroperoxy-cyclophosphamide), no significant difference in cellular drug sensitivity was found. In conclusion, we found that the presence of the t(12;21) translocation in childhood precursor B-ALL is associated with a high tumor cell sensitivity to doxorubicin and etoposide. High throughput techniques should now be used to elucidate the cellular mechanisms by which ETV6/RUNX1 gene fusion is linked to increased sensitivity to these drugs.     

Interphase FISH on TEL/AML1 positive acute lymphoblastic leukemia relapses – analysis of clinical relevance of additional TEL and AML1 copy number changes

Objectives: TEL/AML1 (ETV6/RUNX1) fusion resulting from the translocation t(12;21)(p13;q22) constitutes the most common chimeric fusion gene in initial childhood B‐cell precursor (BCP) acute lymphoblastic leukemia (ALL) (19–27%) and has been associated with good prognosis. Three secondary aberrations in TEL/AML1 positive ALL have been suspected to negatively influence outcome: deletion of the second TEL allele (T), gain of the second AML1 allele (A) and duplication of the derivative chromosome 21 (der(21), TA). Many studies have explored such aberrations in initial disease, while only few reports have investigated them in relapses. Methods: In this study, bone marrow samples from 38 children with relapsed TEL/AML1 RT‐PCR positive and negative BCP‐ALL were analyzed for these mutations by interphase fluorescence in situ hybridization and results were compared with published data. Results: In children with TEL/AML1 positive ALL relapse, additional (a) TEL loss, (b) combined AML1 and der(21) gain, (c) combined TEL loss and AML1 gain as well as (d) the occurrence of a subpopulation with the signal pattern 1T/3A/1TA appear to be related to higher peripheral blast counts (PBCs) at relapse diagnosis (a and d) or a tendency towards the occurrence of a subsequent relapse (b and c) (P‐values <0.05). Conclusions: Our data together with published results on TEL/AML1 positive ALL suggest that frequencies of additional TEL and AML1 mutations are, with the exception of loss of untranslocated TEL, higher in first relapses than in initial disease. They also show that it is important to consider combined mutations in the analysis of this leukemia entity.     

Increased in vitro cellular drug resistance is related to poor outcome in high-risk childhood acute lymphoblastic leukaemia

We determined the in vitro cellular drug resistance in 370 children with newly diagnosed acute lymphoblastic leukaemia (ALL). The resistance to each of 10 drugs was measured by the fluorometric microculture cytotoxicity assay (FMCA) and was related to clinical outcome. The median follow-up time was 41 months. Risk-group stratified analyses indicated that in vitro resistance to dexamethasone, doxorubicin and amsacrine were each significantly related to the probability of disease-free survival. In the high-risk (HR) group, increased in vitro resistance to dexamethasone (P = 0.014), etoposide (P = 0.025) and doxorubicin (P = 0.05) was associated with a worse clinical outcome. Combining the results for these drugs provided a drug resistance score with an independent prognostic significance superior to that of any other factor studied, with a relative risk of relapse in the most resistant group 9.8 times that in the most sensitive group (P = 0.007). The results in the intermediate-risk (IR) and standard-risk (SR) groups were less clear cut. In conclusion, our data indicate that in vitro testing of cellular drug resistance can be used to predict the clinical outcome in HR ALL, while the final evaluation of the results in IR and SR patients must await longer follow-up.     

Cellular drug resistance in childhood acute myeloid leukemia is related to chromosomal abnormalities

Specific cytogenetic abnormalities predict prognosis in childhood acute myeloid leukemia (AML). However, it is unknown why they are predictive and whether this is related to drug resistance. We previously reported that Down syndrome (DS) AML was associated with favorable resistance profiles. Here, we successfully analyzed drug resistance and (cyto-) genetic abnormalities of 109 untreated childhood AML samples using the 4-day total cell-kill methyl-thiazolyl tetrazolium (MTT) assay. Patients were classified according to the genetic abnormalities in the leukemic cells: t(8;21), inv(16), t(15;17), t(9;11), other 11q23 translocations, abnormalities of chromosome 5/7, trisomy 8 alone, normal karyotype, single random, and multiple (defined as 2 or more) abnormalities. The DS AML samples were excluded from the subgroup analysis. Samples with chromosome 5/7 abnormalities were median 3.9-fold (P =.01) more resistant to cytarabine than other AML samples. The t(9;11) samples were more sensitive to cytarabine (median 2.9-fold, P =.002), etoposide (13.1-fold, P =.001), the anthracyclines (2.9- to 8.0-fold, P <.01), and 2-chlorodeoxyadenosine (10.0-fold, P =.002) than other AML samples. The trisomy 8 and t(15;17) groups were too small for meaningful analysis. All other genetic subgroups did not show specific resistance profiles. Overall, we found no differences in drug resistance in samples taken at diagnosis between patients remaining in continuous complete remission (CCR) versus the refractory/relapsed patients. Within several genetic subgroups, however, relapsed/refractory patients were more cytarabine resistant when compared with patients remaining in CCR, but numbers were small and the results were not significant. We conclude that some, but not all, cytogenetic subgroups in childhood AML display specific drug-resistance profiles.     

Dipyridamole enhancement of drug sensitivity in acute lymphoblastic leukemia cells

The effect of dipyridamole (DPM) on cell sensitivity to anticancer drugs was examined in acute lymphoblastic leukemia (ALL) cell lines. We established two ALL cell lines (KMO-90 and KMO-R) from bone marrow samples of a 12-year-old girl with ALL. The drug concentrations needed to reduce optical density to 50% of that of control cells (IC₅₀) showed that KMO-R was about twofold more resistant to doxorubicin (DOX), mitxantrone (MIT), vincristine (VCR), and etoposide (VP-16) than was KMO-90. Considering that both KMO-90 and KMO-R were established from a patient with ALL at the time of presentation and relapse, respectively, these two cell lines might be novel and useful models for research into the acquisition of drug resistance in ALL cells. Although cytotoxicity of DPM in KMO-90 was about 6% at 1 μg/ml, DPM enhanced cell sensitivity to DOX, MIT, VCR, and VP-16 at this concentration. Cytotoxicity of DPM in KMO-R was less than 5% at 1,5, and 10 μgg/ml. In KMO-R, DPM enhanced cell sensitivity to these four drugs in a dose-dependent manner. The plasma concentrations achieved by oral administration of DPM is about 1 μg/ml. At clinically achievable concentrations, DPM enhanced cell sensitivity to DOX, MIT, VCR, and VP-16 in both KMO-90 and KMO-R, thus showing DPM to be a useful agent for potentiating anticancer chemotherapy of hematopoietic malignancy.     

Correlation of drug sensitivity in vitro with clinical responses in childhood acute myeloid leukemia

Clonogenic cells from 41 children with newly diagnosed acute myeloid leukemia (AML) were tested in vitro for their sensitivity to cytarabine (Ara-C) and daunorubicin (DNR). The findings were then compared with the patients' responses to induction chemotherapy that uniformly included Ara-C and DNR. Light-density marrow cells were incubated with either or both drugs for one hour and cultured over leukocyte feeder layers; clusters and colonies were scored on days 7, 10, and 14. Only the percentage of cell kill in the presence of 1.8 mumol/L DNR was significantly associated with responses to induction therapy: median of 45% (range, 0% to 98%) for patients achieving complete remission v 16% (range, 4% to 23%) for nonresponders (P = .007). The relationship between clonogenic cell kill less than or equal to 23% and clinical responses was striking. Of the 11 evaluable patients with in vitro findings in this category, ten either failed induction therapy or relapsed within 1 year after attaining remission. Kaplan-Meier analysis of relapse-free survival times indicated longer durations of remission for patients whose blast cells showed increased sensitivity in vitro to Ara-C alone, DNR alone, or a combination of the two agents. Seven of 11 patients with cell kills of greater than or equal to 49% in the presence of 1.25 mumol/L Ara-C remain free of leukemia, compared with only one of 12 whose cells were less sensitive to the drug (P = .006). We conclude that the in vitro sensitivity of clonogenic leukemic progenitors to DNR and Ara-C correlates with treatment outcome in children with newly diagnosed AML.     

Biological characteristics and prognostic value of in vitro three-drug resistance to prednisolone, L-asparaginase, and vincristine in childhood acute lymphoblastic leukemia

The purpose of this study was to investigate the biological characteristics and prognostic value of in vitro three-drug resistance to prednisolone, L-asparaginase, and vincristine in childhood acute lymphoblastic leukemia (ALL). We carried out in vitro tests with a 4-day culture and a methyl-thiazol-tetrazolium assay on bone marrow samples from 209 children newly diagnosed with ALL. After testing the resistance of leukemic cells to 14 drugs, we classified the patients into two groups according to their sensitivity to three drugs (prednisolone, L-asparaginase, and vincristine) used in remission induction therapy. The three-drug resistant group (RR: sensitive to no drugs or to one drug) correlated with both short-term and long-term treatment failure. Three-year event-free survival (95% confidence interval) for the sensitive group (SS: sensitive to two or three drugs) was 0.813 (0.773-0.853) and that of the RR group was 0.616 (0.569-0.669) (P = 0.0001). Univariate analysis showed that Philadelphia-chromosome (Ph1) positivity and immunophenotype of mixed lineage were also prognostic factors in the 209 patients. The prognosis of the SS/RR drug resistance profile within 14 Ph1 patients was marginally significant (P = 0.062). Multivariate Cox regression analysis showed that Ph1 was an overwhelmingly adverse factor in event-free survival, with a relative hazard of 5.37 (2.57-11.21, P < 0.0001), followed by RR, with a relative hazard of 2.98 (1.69-5.25, P = 0.0001). Furthermore, we clarified the characteristics of the RR group by examination of the pattern of drug resistance to other drugs in comparison with the SS group. The leukemic cells of RR patients were more resistant than those of SS patients (P < 0.0001) to all the drugs tested, with resistance ratios of 1.6 to 13.1 (mean 3.4). In conclusion, in vitro three-drug resistance at the initial stage is an important independent predictor of treatment failure for both induction response and long-term outcome in childhood ALL.