Biomedical subjects
Publications and source records attributed to M Andreeff.
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Bone marrow and/or peripheral blood samples from 133 (75%) of a total of 177 consecutive previously untreated protocol patients with acute nonlymphoblastic leukemia (ANLL) were analyzed for terminal deoxynucleotidyl transferase (TdT) activity at the time of presentation. Twenty-nine (22%) were found to exhibit TdT activity (greater than or equal to 0.10 U/10(8) cells, TdT+) as measured in a biochemical microassay. There were no differences between TdT+ as compared with TdT-negative (TdT-) patients with respect to age, sex, French-American-British (FAB) classification, or the presence of Auer's rods. Remission induction rates were higher for the TdT- patients, with 68% v 48% for the TdT+ patients (P = .05). TdT- patients also experienced longer remissions (P = .003) than TdT+ patients, especially in the Auer's rod-positive subgroup (P = .002). None of five patients with TdT+ ANLL treated with vincristine and prednisone as initial therapy achieved complete remission; all required induction regimens containing daunorubicin or amsacrine in combination with cytosine arabinoside and 6-thioguanine. It is concluded that TdT activity in ANLL indicates biphenotypia or lineage infidelity and is associated with a poor prognosis on chemotherapy protocols currently used for the treatment of ANLL.
Human hematopoietic malignancies provide an excellent model for the study of the activity of cellular oncogenes in a context of known defects in cell proliferation and differentiation. A flow cytometric immunofluorescence assay was developed to quantitate the expression of the cellular ras oncogenes in relation to the cell cycle in individual leukemic cells. Specific binding of a monoclonal antibody to the 21-kd protein (p21ras) encoded by the Ha-ras, Ki-ras, and N-ras genes was measured by flow cytometry and confirmed by fluorescence microscopy. P21ras was detected in 416B, a murine hematopoietic precursor cell characterized by a high level of Ki-ras expression, and in the human leukemic cell lines P-12 and KG-1. The presence of p21ras in the cell lines was also shown by immunoprecipitation. Cellular DNA content was determined simultaneously to define cell cycle phases. There was an equal distribution of p21ras in G1, S, and G2M, with considerable heterogeneity of ras gene expression in the G1 compartment. The assay allows oncogene expression to be studied in populations of intact single cells in which cell heterogeneity is maintained, requires very few cells per sample, and directly correlates oncogene expression to cell kinetic data.
We examined the effects of various concentrations of etoposide (20-125 microM, 2-h incubation) on normal bone marrow and seven malignant cell lines: HL-60, K562, Namalva, MOLT-3, CEM-7, and the Hodgkin's cell lines L428KS and L428KSA. Tumor cell log-kill was dose dependent and greater than 4 for all cell lines but L428KSA (log-kill, 3.73). Median recovery of CFU-GM after purging with 75, 100, and 125 microM etoposide was 3.6%, 1.3%, and 1% of the controls, respectively. After one week of long-term marrow culture (LTMC), recovery increased 10-20 times/10(5) cells plated, reaching median values of 33.6%, 23.5%, and 20.7% of the controls in samples purged with 75, 100, and 125 microM etoposide, respectively. Flow cytometry for cell-cycle analysis and RNA content, and chromosomal studies of one-week-old LTMC from healthy donors detected no significant abnormalities in purged as compared to control cultures. After 3-4 weeks of LTMC, both control and etoposide-treated cells formed confluent monolayers. Using a panel of seven monoclonal antibodies (S3.13, S16.144, S4.7, S8.6, RIB-19, anti-HLA-DR, and VIL-A1), we investigated the expression of early and late differentiation antigens on LTMC after various culture times (0, 1, 2, 3, and 4 weeks). None of the early antigens S3.13, S16.144, and S8.6 was significantly reduced following etoposide treatment, either in nonadherent cells collected from the supernatant or in the adherent cell population obtained from trypsinized monolayers. Etoposide treatment dose dependently delayed the expression of RIB-19 (a late myeloid antigen) and, to a lesser extent, of S4.7 (an "intermediate" myelomonocytic marker). VIL-A1 expression was not affected by etoposide treatment. We conclude that etoposide purging of bone marrow in the 75-125 microM range spares a sufficient number of functionally intact stem cells to allow adequate in vivo reconstitution following autologous transplantation.
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The development of the leukemia-lymphoma complex was studied in AKR/J (H-2k) mice using flow cytometry and staining with acridine orange. Investigation of cytokinetics and of cellular RNA content showed that during the neonatal period all mice had a significant increase of S phase cells in the thymus, bone marrow, lymph nodes and spleen reflecting extramedullary hematopoiesis. Concomitantly, G0/G1 cells were significantly reduced in the thymus, lymph nodes and spleen when compared to 6-week old mice of the same strain. No changes in the cell cycle or in RNA content were observed until 10 months of age in congeneic AKR (H-2b) mice, which do not develop leukemia during the first year of life. In leukemia-prone AKR/J (H-2k) mice, however, it was shown that the first appearance of a leukemic process may be recognized in the thymus by a significant increase of cells in G1 phase of the cell cycle which have a high RNA content. These changes were first seen at 5 months of age before the increased expression of MuLV antigen signals preleukemic alterations at 6 months of age and long before morphological changes appear (8-10 months). Furthermore, at 6 months these mice showed a significant elevation of cells in S phase which always appeared initially in the thymus. By 10 months of age, when the mice were overtly leukemic, these changes had progressed in all lymphoid organs and in the peripheral blood. At the same time a unique population of cells was observed that was characterized by cells in S and G2M with very low RNA content. The method used is applicable to further analysis of the precise locus of development of leukemia in the thymus of AKR/J (H-2k) mice, analysis of the nature of the earliest malignant cells, and investigation of the influence of viruses in the pathogenesis of AKR leukemia.
Flow cytometry of cellular DNA and RNA content was employed to determine DNA ploidy, proliferation, and RNA content of hybridoma cultures shortly after cell fusion and sequentially thereafter. The parental mouse myeloma cell line P3UI was characterized by tetraploid DNA content, S-phase 50%, and high RNA, the parental mouse spleen cells by diploid DNA content, low proliferation, and low RNA content. Hybridoma cultures studied as early as 21 days after fusion were found to contain the sum of the parental cells' DNA content (hexaploid), or if less, more than that of the myeloma parental cells. Only one clone of 35 tested was shown to be hexaploid and the rest hypertetraploid or hyperpentaploid. Hybrid cell cultures were frequently found to contain a variable mixture of unfused parental cells. The high proliferation of hybridoma cells determined by flow cytometry indicates that these cells would eventually overgrow the parental cells. Flow cytometry also enabled an accurate estimation of the effect of various doses of dexamethasone added to HAT medium immediately after cell fusion on hybridoma formation. Cultures treated with 10(-5) mM of the hormone had a higher DNA ploidy than cultures grown in the presence of 10(-3) mM dexamethasone. No parental cells were observed in the hybridoma cultures studied with this hormone. Sequential DNA/RNA measurements of hybridoma clones showed a decrease in DNA ploidy over time with high dexamethasone doses and a minimal increase or no change with low hormone dose. Flow cytometry is suggested to be a useful technique for evaluating the effects of various agents on DNA ploidy and proliferation and on stability of fused cells.
Flow cytometry with acridine orange was used in 111 cases of childhood acute lymphoblastic leukaemia (ALL) to determine simultaneously DNA index, cell cycle distribution and cellular RNA content in bone marrow samples. The overall incidence of DNA aneuploidy in the population was 40%. Significantly higher incidence of DNA aneuploidy was present in the L2 (70.6%) as compared to the L1 (34.1%) group (P = 0.007), using the FAB classification. No difference in the frequency of normal and abnormal DNA stemlines was found between newly diagnosed and relapsed patients. The L2 group had significantly higher proliferation (S phase = 12.4%) than L1 (S phase = 5.8%) (P = 0.01), but RNA content was not significantly different. The aneuploid group had significantly more (P = 0.01) cases with L2 morphology (28.6%) compared to the diploid group (8%) and proliferation was higher in DNA aneuploid (S-phase = 9.5%) compared to DNA diploid (S-phase = 4.7%) leukaemias. Likewise, RNA content was significantly higher in aneuploid than in diploid ALL (P = 0.006). These correlations between morphology, cell kinetics and DNA index elucidate biological properties of leukaemic cells with potentially prognostic value.
Studies were carried out on the effect of dianhydrogalactitol on a human neuroblastoma cell line in culture. The drug was cytotoxic at concentrations of 5 micrograms/ml and 10 micrograms/ml for 1 hour, but not at 1 microgram/ml. Flow cytometry of DNA showed that cell kill was preceded by a transient accumulation of cells in early S at 10 hours, and in late S at 36 hours. Smaller increases in the percent of cells in S phase were seen after treatment with 1 microgram/ml, without cell kill. It may be concluded that dianhydrogalactitol causes an S-phase block, followed by death of cells in late S at concentrations above 5 micrograms/ml.
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We investigated the effect of OKT3 antibody and interleukin 2 (IL-2) on Tac antigen expression and the proliferation of human peripheral blood mononuclear leukocytes. OKT3 monoclonal antibody at low, nonmitogenic concentrations (25 pg/ml) or IL-2 alone at optimal concentrations (20 U/ml) did not induce IL-2 receptor expression, as measured by Tac antibody or by T cell proliferation. However, costimulation with these concentrations of OKT3 antibody and IL-2 led to Tac antigen expression and T cell proliferation. These data suggest that the T cells are activated in two steps: OKT3 antibody at 25 pg/ml does not induce Tac antigen expression, but preactivates T cells to become responsive to IL-2. The addition of exogenous IL-2 then leads to expression of the IL-2 receptor, as recognized by Tac antibody, and to subsequent proliferation.
Cerebrospinal fluid of 24 patients with acute leukemia was studied by DNA/RNA flow cytometry. In six of 15 patients with central nervous system (CNS) relapse, the spinal fluid cells had abnormal DNA stemlines, ranging from near haploid to hyperdiploid. In two additional cases, leukemic cells were identified by a abnormally high RNA content only. One patient had two different aneuploid cell populations in spinal fluid not distinguished by cytologic morphology. Another patient with initial diploid leukemia had CNS relapse characterized by the same DNA stemline long after a triploid DNA stemline emerged in the marrow. DNA/RNA flow cytometry detected leukemic cells that were not identified ("uniform" or "reactive") by cytological criteria in 5/6 patients studied and in addition differentiated lymphoblastic from nonlymphoblastic cell types according to low and high RNA content.
Serum lactate dehydrogenase activity (LDH) was examined in 66 children with acute lymphoblastic leukemia (ALL), 26 with acute non-lymphocytic leukemia (ANLL), and 116 with non-Hodgkin's lymphoma (NHL). The mean serum LDH value for the ALL and ANLL groups was not significantly different: 970 +/- 105 units/L (mean +/- standard error of the mean) and 817 +/- 161 units/L, respectively. The difference between the LDH values in patients with ALL (970 +/- 105 units/L) and NHL (551 +/- 51 units/L) was significant (P = .001). In 32% of the patients with ALL and 23% of the patients with ANLL, serum LDH values were above 1000 units/L, whereas only 13% of the cases with NHL had values above 1000 units/L. In patients with ALL the LDH levels were correlated with white blood cell counts at the time of diagnosis. In NHL, there was no difference in serum LDH levels among the various histologic subtypes. Values of LDH in stage IV NHL and in ALL were similar.
Histologic conversion of nodular lymphomas to more aggressive patterns has been well described and occurs in 15% to 40% of cases. Most conversions have involved changes from nodular to diffuse patterns and often from small to larger cell types. Conversions to undifferentiated lymphomas, but not specifically to the Burkitt's type, have infrequently been described. This report describes three cases of nodular poorly differentiated lymphocytic lymphoma that converted to a Burkitt's-like lymphoma. Conversion was associated with short survivals, increasing lactate dehydrogenase (LDH) levels, and changes in DNA stemline, RNA content, and S-phase values, as determined by flow cytometry. Karyotypes in two cases revealed a t(14;18). Surface immunoglobulin could be demonstrated on transformed Burkitt's-like cells from lymph node in one case, but in none of the cases on cells from bone marrow.
We have carried out a clinical trial in which patients with relapsed leukemia were treated with thymidine (dThd) prior to and concomitantly with the administration of 1-beta-D-arabinofuranosylcytosine (ara-C) in an effort to kinetically and biochemically modulate the leukemic cells with two objectives: (a) to increase the S-phase fraction before giving ara-C; and (b) to increase the uptake and phosphorylation of ara-C. Six patients with acute nonlymphocytic leukemia who had relapsed after conventional or experimental therapy were given continuous i.v. infusions of dThd in conjunction with ara-C. dThd was started at 75 g/sq m/day (producing 1 mM plasma levels) and was given for 5 to 8 days until the proportion of bone marrow cells in S-phase (measured by autoradiography and flow cytometry) had increased and/or stabilized; then ara-C at 200 mg/sq m/day was begun. Twenty-four hr after initiation of ara-C therapy, the dThd dose was reduced to 30 g/sq m/day (plasma dThd, 0.1 to 0.6 mM). Both drugs were continued for 6 to 12 days until marrow aplasia or unacceptable toxicity occurred. Four patients with a base-line labeling index lower than 30% experienced a sustained increase in the S-phase fraction; two patients with a base-line labeling index higher than 30% experienced a reduction in the S-phase compartment during dThd infusion, in one case followed by an increase. The degree of S-phase arrest did not correlate with remission induction. In 5 patients, the flash incorporation of labeled ara-C into nucleoside triphosphate and deoxycytidine into DNA of bone marrow cells was measured. Three patients had a significant increase in the incorporation of deoxycytidine into DNA. Two of them achieved a complete remission. Increases in ara-C uptake were less impressive. A new technique was used to measure the absolute number of blasts present in the bone marrow. A decrease in leukemic cells between 0.9 and 2.6 logs10/mm was found at the end of the infusions. Three patients experienced greater than 2-log reduction. Two of them entered complete remission that lasted 6 weeks and 3 months, respectively. These correlations should be interpreted with caution because of the small number of patients treated. This study suggests that dThd, although of very limited therapeutic value by itself, may have potentiated the antitumor activity of ara-C to some extent. Whether this effect is of significance can only be determined by further studies.
The physiological pyrimidine nucleoside thymidine (dThd) is cytotoxic to normal and neoplastic cells in culture that are exposed to concentrations in excess of 1 mM for prolonged periods. In order to explore the antileukemic potential of the compound, we have treated six patients with relapsed leukemia or lymphoma with marrow and blood involvement, by prolonged infusions of dThd, at dosages of 90 to 240 g/sq m/day for 14 to 29 days. Mean plasma dThd concentration ranged from 3.8 to 5.5 mM. Cerebrospinal fluid levels were measured on three occasions and ranged from 2 to 23.5% of simultaneous plasma levels. Diarrhea was dose limiting in one patient. The other side effects included nausea and vomiting in all patients, hepatotoxicity in two patients, electrolyte imbalance in one, progression of a pericardial effusion to tamponade in one, and mild central nervous system toxicity in five. In all cases, this therapy produced bone marrow aplasia. One patient with acute lymphoblastic leukemia, refractory to prior treatment, achieved a complete remission which lasted for 16 weeks. Another patient with lymphoblastic lymphoma had a greater than 50% reduction in his mediastinal mass which lasted for less than 1 month. At multiple points during therapy, the bone marrow S-phase fraction was measured by flow cytometry and autoradiography. In five patients, the proportion of cells in S phase increased during the first few days of the infusion but then returned to base line, concomitant with an overall reduction in the number of bone marrow blasts. Cytoreduction was evaluated by the technique of W. Hiddemann, B. D. Clarkson, T. Buchener, M. R. Melamed, and M. Andreeff (Blood, 59: 216-225, 1982). The magnitude of tumor cell kill ranged from 0.7 to 3.6 logs of blasts/cu mm of bone marrow. The data demonstrate that dThd is able to induce a complete remission in a patient with acute leukemia previously refractory to treatment. However, because of the very large drug quantities, fluid volumes, and the prolonged course required to produce the necessary tumor cell kill, this treatment approach is too impractical to be used extensively.
A patient with L3 leukemia is reported whose leukemic cells were characterized cytogenetically by a duplication of the long arm segment 1q25 leads to q42, trisomy for chromosome 7, t(8;22)(q24;q11), and two 14q+ chromosomes. Immunologic marker analysis revealed the leukemic cells to be of the non-T, non-B ("null") type. This observation, when considered in conjunction with a previous report of heterogeneity in cell lineages of L3 leukemias, raises the possibility of some lymphoid neoplasms arising in stem cells.