Distinct gene expression profiles determine molecular treatment response in childhood acute lymphoblastic leukemia

Treatment resistance, as indicated by the presence of high levels of minimal residual disease (MRD) after induction therapy and induction consolidation, is associated with a poor prognosis in childhood acute lymphoblastic leukemia (ALL). We hypothesized that treatment resistance is an intrinsic feature of ALL cells reflected in the gene expression pattern and that resistance to chemotherapy can be predicted before treatment. To test these hypotheses, gene expression signatures of ALL samples with high MRD load were compared with those of samples without measurable MRD during treatment. We identified 54 genes that clearly distinguished resistant from sensitive ALL samples. Genes with low expression in resistant samples were predominantly associated with cell-cycle progression and apoptosis, suggesting that impaired cell proliferation and apoptosis are involved in treatment resistance. Prediction analysis using randomly selected samples as a training set and the remaining samples as a test set revealed an accuracy of 84%. We conclude that resistance to chemotherapy seems at least in part to be an intrinsic feature of ALL cells. Because treatment response could be predicted with high accuracy, gene expression profiling could become a clinically relevant tool for treatment stratification in the early course of childhood ALL.     

In vitro culture with prednisolone increases BCL-2 protein expression in adult acute lymphoblastic leukemia cells

The presence of BCL-2 gene rearrangement has been detected also in cellular populations lacking the t(14;18) chromosomal translocation, such as B-lineage acute lymphoblastic leukemia (ALL) cells. It has been reported that overexpression of BCL-2 is related to resistance to glucocorticoid-induced apoptosis. In this study, we aimed to evaluate whether in vitro culture with prednisolone (PDN) could modify the expression of BCL-2 protein. ALL cells from 21 patients were incubated for 72 hr with or without a minimally lethal (IC12) dose of PDN. In vitro culture with PDN did not affect the percentage of positive cells, even though the mean fluorescence index was significantly increased (P = 0.0001), thus indicating a higher level of protein production. These data could suggest a possible mechanism of drug resistance after treatment with PDN. © 1996 Wiley-Liss, Inc.     

Initial leukemic gene expression profiles of patients with poor in vivo prednisone response are similar to those of blasts persisting under prednisone treatment in childhood acute lymphoblastic leukemia

Response to initial glucocorticoid (GC) treatment is a strong prognostic factor in childhood acute lymphoblastic leukemia (ALL). Patients with a poor prednisone response (PPR) have a poor event-free survival as compared to those with a good prednisone response (PGR). Causes of prednisone resistance are still not well understood. We hypothesized that GC resistance is an intrinsic feature of ALL cells which is reflected in the gene expression pattern and analyzed genome-wide gene expression using microarrays. A case-control study was performed comparing gene expression profiles from initial ALL samples of 20 patients with PPR and those of 20 patients with PGR. Differential gene expression of a subset of genes was confirmed by real-time quantitative polymerase chain reaction analysis and validation was performed in a second independent patient sample (n=20). We identified 121 genes that clearly distinguished prednisone-resistant from sensitive ALL samples (FDR<5%, fold change>or=1.5). Differential gene expression of 21 of these genes could be validated in a second independent set. Of importance, there was a remarkable concordance of genes identified by comparing expression signatures of PPR and PGR cells at diagnosis and those previously described to be up- or downregulated in leukemic cells persisting under GC treatment. Thus, GC resistance seems at least in part to be an intrinsic feature of leukemic cells. Leukemic cells of patients with PPR are characterized by gene expression pattern which are similar to those of resistant cells persisting under glucocorticoid treatment.     

Classification of pediatric acute lymphoblastic leukemia by gene expression profiling

Contemporary treatment of pediatric acute lymphoblastic leukemia (ALL) requires the assignment of patients to specific risk groups. We have recently demonstrated that expression profiling of leukemic blasts can accurately identify the known prognostic subtypes of ALL, including T-cell lineage ALL (T-ALL), E2A-PBX1, TEL-AML1, MLL rearrangements, BCR-ABL, and hyperdiploid karyotypes with more than 50 chromosomes. As the next step toward developing this methodology into a frontline diagnostic tool, we have now analyzed leukemic blasts from 132 diagnostic samples using higher density oligonucleotide arrays that allow the interrogation of most of the identified genes in the human genome. Nearly 60% of the newly identified subtype discriminating genes are novel markers not identified in our previous study, and thus should provide new insights into the altered biology underlying these leukemias. Moreover, a proportion of the newly selected genes are highly ranked as class discriminators, and when incorporated into class-predicting algorithms resulted in an overall diagnostic accuracy of 97%. The performance of an array containing the identified discriminating genes should now be assessed in frontline clinical trials in order to determine the accuracy, practicality, and cost effectiveness of this methodology in the clinical setting.     

Inhibition of glycolysis modulates prednisolone resistance in acute lymphoblastic leukemia cells

Treatment failure in pediatric acute lymphoblastic leukemia (ALL) is related to cellular resistance to glucocorticoids (eg, prednisolone). Recently, we demonstrated that genes associated with glucose metabolism are differentially expressed between prednisolone-sensitive and prednisolone-resistant precursor B-lineage leukemic patients. Here, we show that prednisolone resistance is associated with increased glucose consumption and that inhibition of glycolysis sensitizes prednisolone-resistant ALL cell lines to glucocorticoids. Treatment of prednisolone-resistant Jurkat and Molt4 cells with 2-deoxy-D-glucose (2-DG), lonidamine (LND), or 3-bromopyruvate (3-BrPA) increased the in vitro sensitivity to glucocorticoids, while treatment of the prednisolone-sensitive cell lines Tom-1 and RS4; 11 did not influence drug cytotoxicity. This sensitizing effect of the glycolysis inhibitors in glucocorticoid-resistant ALL cells was not found for other classes of antileukemic drugs (ie, vincristine and daunorubicin). Moreover, down-regulation of the expression of GAPDH by RNA interference also sensitized to prednisolone, comparable with treatment with glycolytic inhibitors. Importantly, the ability of 2-DG to reverse glucocorticoid resistance was not limited to cell lines, but was also observed in isolated primary ALL cells from patients. Together, these findings indicate the importance of the glycolytic pathway in glucocorticoid resistance in ALL and suggest that targeting glycolysis is a viable strategy for modulating prednisolone resistance in ALL.     

Hypodiploidy is associated with a poor prognosis in childhood acute lymphoblastic leukemia

Leukemic cells from 31 (7.6%) of 409 children with newly diagnosed acute lymphoblastic leukemia (ALL) had a hypodiploid karyotype. The patients' ages ranged from 0.8 to 17 years (median, 5 years) and their initial leukocyte counts from 1.0 to 132 X 10(9)/L (median, 12.7 X 10(9)/L). Modal chromosome numbers for the leukemic stem lines were 45 in 26 cases, 28 in two cases, and 26, 36 and 43 in one case each. Seven cases had one to three additional abnormal lines due to clonal evolution. Chromosome 20 was lost most frequently (nine cases). Structural abnormalities--including chromosomal translocations (21 cases), deletions (ten cases), duplications (two cases), or inversions (one case)--were common findings; the nonrandom translocations consisted of the t(1;19)(q23;p13.3) in two pre-B cases and tdic(9;12)(p1?1;p1?2) in three cases of common ALL. When compared with hyperdiploid cases (greater than 50 chromosomes), ALL with hypodiploidy was found to have a poorer outcome and was more likely to be associated with chromosomal translocations, higher serum lactic dehydrogenase levels, and age less than 2 or greater than or equal to 10 years. Moreover, patients with hypodiploid ALL fared as poorly as those with pseudodiploid karyotypes, even though their leukocyte counts and serum lactic dehydrogenase levels were lower and they had a comparable frequency of leukemic cell translocations. Hypodiploidy is therefore an unfavorable karyotypic feature in childhood ALL.     

Changes in the MDR1 gene expression after short-term ex vivo therapy with prednisolone have prognostic impact in childhood acute lymphoblastic leukemia

Multidrug resistance 1 (MDR1) gene expression determined by real-time polymerase chain reaction and results of rhodamine assay were analyzed at diagnosis and after 3 days of ex vivo therapy with prednisolone in 36 pediatric patients with acute lymphoblastic leukemia (ALL). Only 62% patients with de novo ALL had significant decrease of MDR1 expression. These patients had over twofold lower rhodamine retention in the presence of cyclosporine A on day 3 than others and had better probability of disease-free survival. In this study, we have shown that changes in the expression of MDR1 gene after short-term incubation of lymphoblasts with prednisolone may have prognostic value in pediatric de novo ALL patients.     

Transient hypofibrinogenemia induced by prednisolone in a case of acute lymphoblastic leukemia

We report here a patient with acute lymphoblastic leukemia (ALL) in whom hypofibrinogenemia developed during chemotherapy. The patient was a 65-year-old female who was diagnosed as having common ALL, and she was treated with BHAC-DMPV (enocitabine: 160 mg, daunorubicin : 40 mg, 6-MP: 35 mg, prednisolone (PSL): 60 mg, and vincristine: 2 mg). Hypofibrinogenemia appeared promptly each chemotherapy, including PSL was given. To ascertain a correlation between hypofibrinogenemia and the drugs given in this patient, a trial administration of PSL was attempted during a complete remission state. The level of fibrinogen, in terms of the amount of antigen or coagulability, decreased during PSL treatment, although the levels of AT III, plasminogen, alpha 2PI.Plm complex, and FDP did not change. Thus, it is difficult to speculate that PSL induced destruction of leukemia cells and release of protease from the cells resulting in fibrinolysis and hypofibrinogenemia in this case. These findings also suggest that the administration of only PSL could induce hypofibrinogenemia.     

Expression of the mdr-1/P-170 gene in patients with acute lymphoblastic leukemia

Increased expression of the multidrug resistance gene (mdr-1/P-170) and the dihydrofolate reductase (DHFR) gene have been implicated in the development of in vitro drug resistance. Overexpression, with or without gene amplification, is seen in the development of drug resistance in culture and it has been postulated that genetic modulation of mdr-1/P-170 and DHFR may also be involved in the development of clinical drug resistance. We screened lymphoblasts from 28 patients with acute lymphoblastic leukemia (ALL) for evidence of overexpression of mdr-1/P-170 using RNAse protection, RNA in situ hybridization and immunohistochemistry. Overexpression of mdr-1/P-170 without gene amplification was detected in samples from four patients (three after multiple relapses, one at presentation). Overexpression of mdr-1/P-170 was heterogeneous within the population of malignant lymphoblasts as demonstrated by RNA in situ hybridization, immunohistochemistry, and drug uptake using daunomycin autofluorescence analysis. There was no evidence of overexpression of DHFR in any of the eight patient samples tested by RNAse protection nor was there any evidence of gene amplification in 11 patient samples on Southern blot analysis. From these observations it appears that overexpression without gene amplification of mdr-1/P-170 may be one mechanism of clinical drug resistance in ALL.     

Mutations in the p53 gene in acute myeloid leukemia patients correlate with poor prognosis

Inactivation of tumor suppressor genes, whose products exert an inhibitory influence on cell cycle progression, can lead to neoplastic transformation. In acute myeloid leukemia (AML), the frequency of p53 gene mutations ranges from 4 to 15% in populations from USA and Europe. In an attempt to investigate the frequency of point mutations in the p53 gene in AML Brazilian patients, DNA samples of 35 patients were studied using PCR-SSCP techniques, screening exons 4-10. Mutations were identified in bone marrow DNA in 5 of the 35 AML patients (14.3%), a frequency similar to those reported for Northern American and European populations. The overall survival of patients with mutations in the p53 gene was significantly shorter than for patients without mutations.     

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.     

Endocrine complications in pediatric patients with acute lymphoblastic leukemia

Endocrine complications of therapy for acute lymphoblastic leukemia (ALL) are common and are potentially debilitating both during and after therapy. Growth velocity slows during therapy for ALL, especially during the first year; however, children who do not receive cranial irradiation usually reach normal adult height. While growth hormone deficiency generally occurs in patients who have received 24Gy of cranial irradiation, it may also develop in those treated with lower doses (18Gy) of cranial radiation or with only high-dose methotrexate. Obesity commonly occurs during therapy and persists after completion of therapy. Osteopenia can occur early during therapy for ALL and can persist for many years. Adrenal insufficiency should be suspected in any child who has recently received glucocorticoid therapy, and stress doses of steroid should be administered in the event of metabolic stress. Screening of urine is useful for early detection of hyperglycemia during therapy with glucocorticoids and L-asparaginase. The syndrome of inappropriate secretion of anti-diuretic hormone is usually associated with vincristine therapy and may be aggravated by concurrent use of azole antifungals. Finally, patients who have received 18 or 24Gy of cranial irradiation may have clinical or subclinical deficiencies of thyroid hormones.