Prognostic significance of surface marker expression on blasts of patients with de novo acute myeloblastic leukemia

The prognostic significance of the expression of surface membrane antigens on the blasts of 123 consecutive patients with de novo acute myeloblastic leukemia (AML) was evaluated. For this purpose, reactivity of monoclonal antibodies (mAbs) CLB-ERY3 (antiblood-group H antigen), VIM-D5 (CD15), WT1 (CD7), MY7 (CD13), MY9 (CD33), VID-1 (antihuman leukocyte antigen locus DR [anti-HLA DR]), VIM-2 (CDw65L), VIM-13 (CD14), 63D3 (CD14) and anti-TdT with leukemic blast cell populations was prospectively analyzed with respect to the rates of complete remission (CR), continuous complete remission (CCR), and survival. The overall rate of CR was 65%, the 6-year rates of overall CCR and survival were 23% and 13%, respectively (median period of patient observation, 30 months). Of all Abs tested, four (CLB-ERY3, MY7, anti-TdT, and VIM-D5) were found to be of prognostic value. Reactivity of CLB-ERY3, MY7, and anti-TdT was predictive for CR (CLB-ERY3+, 43% v CLB-ERY3-, 73%, P less than .02; MY7+, 59% v MY7-, 91%, P less than .003; TdT+, 28% v TdT-, 71%, P less than .001, respectively) and probability of survival (significantly lower survival rates: CLB-ERY3+, P less than .02; MY7+, P less than .03; and TdT+ cases, P less than .001, respectively). Reactivity of VIM-D5 was significantly associated with a higher probability of CCR (P less than .01). Our results confirm earlier reports on the prognostic significance of expression of CD13 and TdT in AML and indicate CLB-ERY3 (antiblood-group H antibody) and VIM-D5 (CD15) as further markers predictive for the clinical outcome in patients with de novo AML.     

The use of enzyme marker analysis for subclassification of acute lymphocytic leukemia in childhood

Subclassification of acute lymphocytic leukemias in childhood by multiple marker analysis has proven the heterogeneity of this disease and this methodology has led to a better understanding of the cell-biological basis of ALL. Enzyme markers have become important tools in multiple marker analysis. This is especially true for TdT, purine metabolic enzymes, hexosaminidase I, acid phosphatase and carboxylic esterases. In spite of sophisticated methods and encouraging results multiple marker analysis has not been totally satisfactory in defining patients at risk. The same is true for a risk score established by clinical data. More efforts in the future are necessary for combining multiple marker analysis, cytogenetics, proliferation characteristics, basic clinical findings and the final outcome of the disease in these patients. Beyond that this kind of leukemia research will help to clarify the pathobiological basis of human leukemia and to develop new specific therapeutic modalities.     

The biology of acute myeloblastic leukemia

Many questions about the biology of AML remain to be answered. The initial genetic lesions that inhibit differentiation and increase the likelihood of self renewal have yet to be identified. Given the heterogeneity of this neoplasm, it is possible that many different mutational events may be capable of triggering leukemia. Alternatively, there may be only a small number of possible initial leukemic mutations, and the heterogeneous phenotype of the disease is determined by the evolution of different subclones that have acquired different secondary mutations. Studies with retroviral oncogenes have suggested that a common secondary event in an evolving myeloid tumor is the development of growth factor independence by either leukemic cell production of CSF or possibly constitutive activation of a CSF receptor. These mechanisms have not yet been established as important in human AML, although there is intriguing evidence to suggest that CSF genes are inappropriately activated in many cases.     

FLT3 and NPM1 gene mutations in childhood acute myeloblastic leukemia

Mutations of receptor tyrosine kinases are implicated in the constitutive activation and development of human hematologic malignancies. Mutations in fms-like tyrosine kinase 3 (FLT3) gene including internal tandem duplication (ITD) and point mutation in the tyrosine kinase domain (TKD) as well as in nucleoplasmin (NPM1) gene are associated with pathogenesis of acute myeloblastic leukemia (AML). Several reports have demonstrated high incidences of the FLT3 and NPM1 mutations in adult AML patients. Since the pathogenesis of pediatric AML is different from that of adult and the FLT3 and NPM1 mutations have not been well characterized in childhood AML. Therefore, the objective of this study was to determine the frequencies of FLT3 and NPM1 mutations in 64 newly diagnosed childhood AML patients. All blood and bone marrow samples were previously diagnosed with AML by using flow cytometry and/or cytochemistry. FLT3-ITD and FLT3-TKD were detected by PCR and PCR-RFLP methods, respectively. The NPM1 mutation was analyzed by PCR and direct DNA sequencing. The FLT3 mutations were detected in 7 of 64 (11.1%), including FLT3-ITD in 4 of 64 (6.3%) and FLT-TKD in 3 of 62 (4.8%). The NPM1 mutation was not detected in this cohort. By multivariate analysis, white blood cell counts, peripheral blood and bone marrow blast cell counts at diagnosis were significantly higher in children with FLT3-ITD (P<0.05). In addition, the median percentage of CD117 was significantly higher in leukemic blast cells with FLT3-ITD than those with wild type (P=0.01). We did not find any FLT3 mutations in children aged less than 5 years. The AML M3 cell type was most frequently associated with FLT3 gene mutations (50%). In conclusion, the FLT3 mutations was found in 11.1% but none of NPM1 mutation was detected in Thai children with AML. These data support the hypothesis of different biology and pathogenesis between adult and childhood AML.     

Chemoimmunotherapy for maintenance in acute myeloblastic leukemia.

Of 30 adult patients with acute myeloblastic leukemia, 14 achieved complete remission. Eight of these were given chemoimmunotherapy for maintenance. The immunotherapy consisted of intradermal pooled allogeneic leukemic cells (snap-frozen irradiated) and BCG vaccine given by Heaf gun, given twice in 4 weeks. The chemotherapy was given for 1 week in 4 weeks. The median duration of remission in these eight patients was 115+ weeks and the median duration of survival was 147+ weeks. The other six patients who were given chemotherapy only for maintenance had a median duration of remission of 15 weeks and a median survival of 52 weeks. The two groups cannot be compared properly, however, as allocation of patients was not random, and the chemotherapy differed significantly.     

Clonal expansion and progression in acute myeloblastic leukemia.

Diseaes originating in pluripotent stem cells, then developing through clonal expansion and clonal progression may properly be grouped together because of their common features. Among these, AML appears to be unique by reason of the presence within the clone of a blast cell population. Studies of cellular composition and regulation in AML clones require assays that measure not only myelopoiesis but also blast cell proliferation. The relation of the blast population to other components of AML clones remains uncertain. Resolution of the uncertainty is important in considering therapeutic strategies.     

Granulocyte colony-stimulating factor-dependent growth of an acute myeloblastic leukemia cell line.

We report herein the establishment and characterization of a granulocyte colony-stimulating factor (G-CSF)-dependent acute myeloblastic leukemia (AML) cell line. The cell line, designated as OCI/AML 1a, has been cultured in the presence of G-CSF and has shown exponential growth for over two years. The cells growing in suspension culture resembled myeloblasts on the basis of morphologic, cytochemical and surface phenotypic analyses. Other CSFs, interleukin-3 and granulocyte-macrophage colony-stimulating factor did not support the growth of OCI/AML 1a cells so well as G-CSF. The effect on the growth of OCI/AML 1a cells of G-CSF was almost completely abolished by neutralizing monoclonal anti-G-CSF antibody. These findings showed that OCI/AML 1a cells required G-CSF for growth. OCI/AML 1a cell line will be valuable for studies of the biological nature, proliferation and differentiation of leukemic cells. Furthermore, OCI/AML 1a cells should be useful for determining the mechanism by which G-CSF induces the growth of hemopoietic cells.     

p53 and redox state in etoposide-induced acute myeloblastic leukemia cell death

We investigated whether p53, being a redox-sensitive protein, has a role in the responsiveness of AML cells to etoposide. Two subclones of the OCI/AML-2 cell line, the etoposide-sensitive (ES) and the etoposide-resistant (ER), were used as models. Sensitivity to etoposide was measured by trypan blue and annexin V assays. Etoposide-induced peroxide formation was associated with the induction of cell death. Evident expression of mutated p53 was observed in both subclones in basal growth conditions as analysed by Western blotting and flow cytometry. After etoposide exposure for up to 24 hours, some nuclear accumulation of p53 was observed in the ER subclone, as analysed by Western blotting. The conformation of p53, however, was not changed from mutated toward wild-type during exposure in either of the subclones as analysed by flow cytometry. In conclusion, etoposide-induced change in cellular redox state was associated with apoptosis, but was not a sufficient stimulus for p53 to make its conformation active. Thus, mutated p53 seems to have no role in etoposide-induced apoptosis.     

Glycophorin a as a cell surface marker of early erythroid differentiation in acute leukemia

We show here that the leukemic blast cells from three patients of a total of 15 subsequently diagnosed as having acute leukemia express on their surface the major red cell sialoglycoprotein, glycophorin A (GP-A). This was demonstrated (1) by indirect immunofluorescence using rabbit anti GP-A antiserum and (2) by immune precipitation of GP-A from surface radiolabelled leukemic cells. Since GP-A is exclusively present on erythroid cells and their precursors, these findings indicate that a higher proportion of the blast leukemias than previously recognized show features of early erythroid differentiation.     

DNA repair mechanisms and acute myeloblastic leukemia

DNA repair mechanisms play a vital role in maintaining genomic integrity. With the wealth of knowledge gained recently on these processes it is becoming clear that defects in repair proteins and proteins associated with the regulation of repair are connected to many different human diseases including cancer. This paper has aimed to review the four major DNA repair processes and in particular concentrate on their association with acute myeloblastic leukemia (AML).     

Prognostic importance of systemic clearance of methotrexate in childhood acute lymphoblastic leukemia

Summary Pharmacokinetic studies of methotrexate have been carried out in 21 children with acute lymphoblastic leukemia diagnosed in 1981. Children were treated with intermediate dose (500 mg/m²) methotrexate in keeping with the 1981 ALL treatment Protocol of the Hungarian Childhood Leukemia Working Group. Of the 21 children, 8 relapsed, and 13 are in continuous complete remission. In the relapsed patients significantly increased systemic clearance of methotrexate was observed at the time of the second methotrexate treatment cycle compared with the calculated value after the first administration of the drug. No such change in the clearance was found in patients who are still in remission. There was no difference between children who relapsed or who are in remission in the elimination half-time of the drug. Age, sex, WBC at diagnosis, and systemic clearance of methotrexate were found to be connected with the probability of relapse in the patients studied. The possible reasons for the prognostic role of systemic methotrexate clearance are discussed.     

Allelotype analysis in relapsed childhood acute lymphoblastic leukemia

We performed for the first time the allelotype of relapsed childhood acute lymphoblastic leukemia (ALL). A total of 38 cases were screened for loss of heterozygosity (LOH) using 71 markers. In all, 26 (68%) patients showed LOH on at least one chromosomal arm, indicating that LOH is a frequent event at relapse. The most frequent loss was found on chromosomal arm 9p at the p16/INK4a locus (39%). LOH at the TEL gene locus on chromosomal arm 12p also occurred often (25%). Frequent loss was observed on chromosome arms 4q (20%), 6q (21%), and 17q (20%). Sequential analysis (i.e. samples obtained from both initial diagnosis and relapse) shows that some patients (63%) have the identical LOH status at both phases, suggesting the presence of the same clone. Other samples (37%) showed distinct LOH alterations, indicating clonal evolution at relapse. Despite the heterogeneous and complex changes, some shared LOH loci occurred in these matched samples, suggesting that many of the same tumor-suppressor genes are aberrant at both phases. In summary, novel tumor-suppressor genes on chromosome arms 4q, 6q, and 17q, as well as the p16 and TEL genes, have an important role in the relapse of childhood ALL.     

The successive development of acute myeloblastic leukemia, secondary non-Hodgkin's lymphoma and secondary myeloid/natural killer cell acute leukemia in a single patient

We report a very rare case of a female patient who was initially diagnosed with acute myeloid leukemia (AML) M2 who achieved complete remission (CR) after chemotherapy. Six years later she was still in continuous complete remission from leukemia, but developed a right nasal obstruction and based on the nasal and nasopharynx biopsies, a secondary B cell non-Hodgkin's lymphoma was diagnosed and treated with chemotherapy and involved field radiotherapy. One year and seven months after the completion of therapy she presented with fever, dyspnea and leukocytosis. The blasts were now negative for myeloperoxidase and immunophenotyping showed that they were positive for CD13 and CD56. Now the diagnosis of a secondary myeloid/NK cell acute leukemia was made. The patient died of multiorgan failure 1 month after the onset of leukemia. As far as we know, no other such patient has been described in the English literature until now.     

Phase II study of aclarubicin in acute myeloblastic leukemia

Aclarubicin is a new anthracycline antibiotic that produces substantially less cardiotoxicity in animals that does doxorubicin. Based upon prior Phase I and II trials in leukemia, a Phase II study in acute myeloblastic leukemia was developed to assess the response rate and toxicity in previously treated patients. Forty patients received aclarubicin 100 mg/m2 per day X 3 with repeated course on days 14-16 if marrow hypoplasia was not produced. Complete responses were achieved in 27.5% (11/40) with durations of 1.5, 2, 2, 2, 3, 3+, 4, 5+, 32+, 33+, and 34+ months. Toxic effects of this therapy included severe neutropenia and thrombocytopenia, nausea/vomiting, mucositis, and diarrhea. No patient developed significant changes in the left ventricular ejection fraction, as measured by radionuclide angiography, or any clinical cardiac symptoms. Alopecia was minimal. Aclarubicin can produce a significant response rate in previously treated patients with acute myeloblastic leukemia and should be considered for study in initial therapy.     

Carboplatin plus cytarabine in the treatment of high-risk acute myeloblastic leukemia.

Thirty-one patients (20 male and 11 female; median age 51 years (16-79)) with high-risk acute myeloblastic leukemia (AML) (20 refractory AML and 11 secondary AML (s-AML) (four to myelodysplastic syndrome, five to chemo/radiotherapy, one to aplastic anemia and one blastic chronic myelogenous leukemia (B-CML)) were treated with CBDCA (300 mg/m2/day x 5 days in continuous i.v. infusion) plus intermediate-dose Ara-C (500 mg/m2/day x 3 days in rapid i.v. infusion). Nine patients (29%) achieved CR (five s-AML (three myelodysplastic syndromes, one CML and one ALL) and four refractory AML) and 11 patients had resistant disease. There were 11 early deaths (35%). Median disease-free survival of the nine responders was 4 months. The main toxicity was hematological, febrile episodes took place in nearly all the patients (96%). The CBDCA plus Ara-C regimen showed an evident antileukemic activity in high-risk leukemia. However, the lack of long-term disease-free survivors shows the need for innovative postremission strategies. The high initial response rate seen in AML secondary to myelodysplastic syndromes (MDS) warrants further investigation of CBDCA in combination regimens for MDS patients.     

Cytogenetics for detection of minimal residual disease in acute myeloblastic leukemia.

Bone marrow samples collected from acute myeloblastic leukemia (AML) patients in complete clinical and hematological remission were studied for the persistence of cytogenetic abnormalities. AML patients from the three favorable cytogenetic categories [inv 16, t(8;21) and t(15;17)] and patients from the unfavorable cytogenetic categories (+8, -5, -7 and Philadelphia-positive) were studied. Seventy-one patients had evaluable metaphase spreads in remission marrows and 20 (28%) had one or more abnormal metaphases identical to that present in the pretreatment marrow. All 20 of these patients relapsed within 78 weeks, thus there were no false positive studies. Fifty-one patients had only diploid metaphases in their complete remission marrow, 25 relapsed, and 21 remained in continuous complete remission. Thus there was a 49% false negative rate of this study. These data indicate that the failure to detect residual chromosomally abnormal cells in the bone marrow does not guarantee continuous complete remission. Cytogenetic study was most useful in the favorable cytogenetic groups and least useful in the unfavorable groups. The persistence of normal metaphases in pretreatment marrows did not affect outcome or risk of recurrence. Twenty-five of 34 evaluable patients who relapsed after remission had either the identical cytogenetic abnormality present in the pretreatment marrow or showed the identical abnormality with additional chromosomal changes. Thus study indicates that cytogenetic examinations of complete remission bone marrow samples in patients with AML provides an objective method for detecting residual leukemia, and identifies patients with a potential for prolonged disease-free survival.     

The role of cell contact and autostimulatory soluble factors in the proliferation of blast cells in acute myeloblastic leukemia

The role of cellular interactions in stimulating leukemic cell proliferation was studied in AML. When peripheral blood blasts were cultured in suspension in flat-bottomed vessels at low cell numbers (15.6-500 x 10(3)/ml), the rate of DNA synthesis [( 3H]thymidine incorporation) was proportional to cell density. When cells were cultured under otherwise identical conditions in round-bottomed vessels, to induce cell crowding, the rate of DNA synthesis was independent of cell numbers and was significantly augmented (p less than or equal to 0.01). Physical separation of cells in round-bottomed (crowded) cultures by latex beads reduced DNA synthesis to the same rate as that in flat-bottomed (non-crowded) cultures. In contrast, addition of mitomycin-treated autologous blasts had little effect in crowded cultures but increased DNA synthesis in non-crowded cultures. DNA synthesis was also enhanced: by reducing culture volume to concentrate any diffusible factors produced by the cells or by blast cell conditioned medium from autologous blasts grown under crowded conditions. Cells cultured in suspension for 72 hr under crowded/non-crowded conditions both formed blast cell colonies in methyl cellulose; however, the number of colonies derived from crowded cultures was greater. These results indicate that cell proximity promotes proliferation of blasts and blast cell progenitors; both close cell contact and autostimulatory soluble factors are important in regulating growth of AML blasts.     

Breast-feeding and risk of childhood acute leukemia.

BACKGROUND: Breast-feeding is well known to have a protective effect against infection in infants. Although the long-term effects of breast-feeding on childhood cancer have not been studied extensively, a protective effect against childhood Hodgkin's disease and lymphoma has been suggested previously from small investigations. In this study, we tested the hypothesis that breast-feeding decreases the risk of childhood acute leukemia. METHODS: A total of 1744 children with acute lymphoblastic leukemia (ALL) and 1879 matched control subjects, aged 1-14 years, and 456 children with acute myeloid leukemia (AML) and 539 matched control subjects, aged 1-17 years, were included in the analysis. Information regarding breast-feeding was obtained through telephone interviews with mothers. All leukemias combined, histologic type of leukemia (ALL versus AML), immunophenotype of ALL (early pre-B cell, pre-B cell, or T cell), and morphology of AML were assessed separately in the data analysis. RESULTS: Ever having breast-fed was found to be associated with a 21% reduction in risk of childhood acute leukemias (odds ratio [OR] for all types combined = 0.79; 95% confidence interval [CI] = 0.70-0.91). A reduction in risk was seen separately for AML (OR = 0.77; 95% CI = 0.57-1.03) and ALL (OR = 0.80; 95% CI = 0.69-0.93). The inverse associations were stronger with longer duration of breast-feeding for total ALL and AML; for M0, M1, and M2 morphologic subtypes of AML; and for early pre-B-cell ALL. CONCLUSION: In this study, breast-feeding was associated with a reduced risk of childhood acute leukemia. If confirmed in additional epidemiologic studies, our findings suggest that future epidemiologic and experimental efforts should be directed at investigating the anti-infective and/or immune-stimulatory or immune-modulating effects of breast-feeding on leukemogenesis in children.     

Cytosine arabinoside phosphorylation and deamination in acute myeloblastic leukemia cells

Extracts of peripheral leukocytes from patients with AML and from controls were examined for enzymes involved in the metabolism of ara-C in order to investigate a possible relationship between enzymes activities and improvement of treatment with ara-C. The enzymes are deoxycytidine kinase and cytidine deaminase, which activates and inactivates ara-C respectively. Cytidine deaminase activity was found to be lower in AML cells than in normal cells, while deoxycytidine kinase was higher in AML cells. Furthermore, the ratio between kinase activity with ara-C as substrate and activity with deoxycytidine as substrate was higher in AML cells than in normal leukocytes. However, there was no significant difference between those patients with AML, who respond to ara-C treatment, and the patients who did not respond.The enzymatic differences between normal and AML leukocytes with regard to activity and substrate specificity might suggest that ara-C could be combined with advantage with deoxycytidine in the clinic.