Differences in constitutive and post-methotrexate folylpolyglutamate synthetase activity in B-lineage and T-lineage leukemia

Folylpolyglutamate synthetase (FPGS) is responsible for the metabolism of natural folates and a broad range of folate antagonists to polyglutamate derivatives. Recent studies indicated increased accumulation of methotrexate (MTX) polyglutamates (MTX-PG) in blast cells as a predictor of favorable treatment outcome in childhood acute lymphoblastic leukemia (ALL). We determined the expression of FPGS activity in blasts from children with ALL at diagnosis and after treatment with MTX as a single agent, before conventional remission induction therapy. The levels of enzyme activity in ALL blasts at diagnosis (median of 689 pmol/h/mg protein) were significantly higher (P = .003) than those found in acute nonlymphoblastic leukemia (ANLL) blasts (median of 181 pmol/h/mg protein). Comparable lineage differences in normal lymphoid versus nonlymphoid cells suggest a lineage-specific control of FPGS expression, FPGS activity increased in ALL blasts after in vivo exposure to MTX. The median increase in FPGS activity was significantly higher (P = .003) in B-lineage ALL (188%) than in T-lineage ALL (37%). Likewise, the percentage of intracellular long chain MTX-PG (Glu3-6) was significantly higher (P = .02) in B- lineage ALL (92%) than in T-lineage ALL (65%), consistent with higher FPGS activity in B-lineage blasts. This finding could explain, at least in part, the superior outcome in children with B-lineage ALL treated with antimetabolite therapy.     

Infant leukemia: an analysis of nine Chinese patients

A study was made of the cellular and molecular characteristics of nine Chinese infants, consecutively presenting with acute leukemia. Five cases were acute lymphoblastic leukemia (ALL); four were acute nonlymphoblastic leukemia (ANLL). Hyperleukocytosis, hepatosplenomegaly, and poor response to conventional therapy were common features, and CNS involvement was detected at diagnosis in three cases. The blast cells from all five cases with ALL expressed early B-cell markers, i.e., HLA-DR+, CD19+, but CD10-. Terminal deoxynucleotidyl transferase (TdT) was present in blasts from four of the five cases and periodic acid-Schiff staining in blasts from two patients only. The leukemic cells of one patient also showed positive nonspecific esterase activity and expressed myeloid-associated antigens CD33 (My9), CD11 (OkM1), and CD14 (My4 and Mo2). Molecular analysis of leukemic cell DNA from this and two other patients showed rearrangement of the immunoglobulin (Ig) heavy-chain genes, but without any evidence of kappa light-chain gene rearrangement. T-cell receptor (TCR) genes remained in the germline configuration in these cases. Cytogenetic analysis showed translocation t(4;11) (q21;q23) in all four cases studied. In the group of ANLL, three cases belonged to the M4 and one to the M2 subtype. Chromosomal abnormality involving 11q23 was also detected in two patients: t(11;17)(q23;q11) and del(11)(q14q23) in each case respectively. Neither Ig nor TCR gene rearrangement was present in blast cells from patients with ANLL. The data indicate that chromosomal rearrangement of band 11q23 was quite common in Chinese infants with either form of leukemia, a finding that may have pathogenetic implications.     

Acute mixed lineage leukemia: clinicopathologic correlations and prognostic significance

The frequency and clinical significance of acute leukemia displaying both lymphoid and myeloid characteristics was determined in 123 consecutive children using a panel of lineage-associated markers. The leukemic blasts from 18 of 95 children (19%) with the diagnosis of acute lymphoblastic leukemia (ALL) by standard diagnostic criteria expressed myeloid-associated cell surface antigens. Despite immunological evidence of lymphoid differentiation (17 CALLA + and one T cell-associated antigen +) and findings of immunoglobulin gene rearrangement, blasts from these patients reacted with one to five monoclonal antibodies identifying myeloid-associated cell surface antigens (My-1, MCS.2, Mo1, SJ-D1, or 5F1). Dual staining with microsphere-conjugated antibodies and analysis by flow cytometry confirmed that some blasts were simultaneously expressing lymphoid- and myeloid-associated antigens. Conversely, blasts from seven of 28 patients (25%) with acute nonlymphocytic leukemia (ANLL), diagnosed by otherwise standard morphological and cytochemical criteria, expressed lymphoid-associated surface antigens. Dual staining of individual blasts demonstrated simultaneous expression of myeloperoxidase (MPO) (including Auer rods) in association with either T-11, CALLA, or terminal deoxynucleotidyl transferase. Blasts from one patient with ANLL demonstrated T cell receptor gene rearrangement, while blasts from another patient demonstrated characteristics associated with T (T-11), B (CALLA and heavy-chain immunoglobulin gene rearrangement), and myeloid (MPO) lineage. There were no consistent cytogenetic abnormalities, and no patient demonstrated independent leukemic clones. Each patient with typical ALL, except for myeloid-associated antigens, achieved complete remission with conventional induction therapy for ALL. By contrast, three of the seven children with ANLL whose blasts expressed the T-11 surface antigen failed ANLL induction therapy. These three patients subsequently achieved remission with ALL therapy.     

Aneuploidy and percentage of S-phase cells determined by flow cytometry correlate with cell phenotype in childhood acute leukemia

Cellular DNA content distributions of propidium-iodide-stained bone marrow blasts were determined by flow cytometry (FCM) for 225 untreated children with acute leukemia and were correlated with leukemia cell phenotype and karyotype. Aneuploidy of the primary malignant stem line was detected in 54 cases (24%): 51 hyperdiploid and 3 hypodiploid. A second stem line with approximately twice the DNA content of the primary stem line was recognized by FCM in 28 cases (23 ALL, 5 ANLL) and may be an important source of leukemia cell heterogeneity. The degree of DNA content abnormality detected by FCM was highly correlated (r = 0.98) with the number of whole chromosome gains or losses in the leukemia karyotype. Aneuploidy detectable by FCM was more frequent in acute lymphoblastic leukemia (ALL) (52 of 173, 30.1%) than in acute nonlymphoblastic leukemia (2 of 52, 3.8%) (p less than 0.001). In the ALL group, aneuploidy was significantly correlated with the cell surface expression of common ALL antigen: 46 of 127 antigen-positive cases were aneuploid compared to 6 of 46 antigen-negative cases (p less than 0.003). Only 2 of 21 cases of T-cell ALL without common ALL antigen had detectable aneuploidy, which was significantly less than in the common ALL group (p = 0.02). The median percentage of cells in S- phase was significantly greater for B-cell and erythrocyte rosette- positive T-cell ALL, than for the other phenotypic subgroups. We conclude that aneuploidy and S-phase cell percentage are correlated with the state of leukemia cell differentiation. The biologic basis for the correlation is not established, but may be linked to the process of malignant transformation.     

T-cell acute lymphoblastic leukemia with natural killer cell phenotype

To determine the type and proportion of cases within that type of acute lymphoblastic leukemia (ALL) that has a natural killer (NK) cell phenotype, we examined leukemic blasts from 31 children with ALL (14 with T-ALL, 17 with non-T-ALL) for expression of antigens detected by NK-specific monoclonal antibodies Leu 11b, Leu 7, and 1G2 (an antibody we have developed that cross-reacts with Leu 7). None of the patients had leukemic blasts that reacted with Leu 11b. However, leukemic blasts from four T-ALL patients were 1G2+ and/or Leu 7+. Blasts from two of these had spontaneous lytic activity against standard NK target cell line K562; blasts from one killed K562 only when incubated with interferon; blasts from the other had no lytic activity against K562 but did manifest antibody-dependent cell-mediated cytotoxicity against antibody-coated cells from NK-resistant cell line SB. Blasts from all four Leu 7+ patients had L2 morphology. In one, the leukemic blasts had azurophilic cytoplasmic granules similar to those found in NK-enriched normal populations of large granular lymphocytes. These findings suggest that a significant proportion of T-cell acute lymphoblastic leukemias may be malignancies of NK cell origin.     

The single cell gel electrophoresis: a potential tool for DNA analysis of the patients with hematological malignancies

Single cell gel electrophoresis (SCGE) was used to evaluate the level of DNA damage in peripheral blood (PB), bone marrow (BM), and lymphatic node (LN) cells of patients with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML) and non-Hodgkin's lymphoma (NHL). The level of DNA damage was compared with the level of basal DNA damage in control group, represented by healthy donors. Statistically significant increase of basal DNA damage was found in leukemia/lymphoma cells of patients suffered from AML, CML, ALL of T-cell subtype (T-ALL), and NHL, however, no difference in basal DNA damage was found in patients with ALL of early B-cell subtype (B-ALL) and CLL in comparison to control group. The mean basal DNA damage increased in the order CLL相似文献   

Effect of retinoic acid on the clonal growth of childhood myeloid and lymphoid leukemias: a pediatric oncology group study

We have studied the effects of retinoic acid (RA) on bone marrow leukemic cells from children with acute nonlymphocytic leukemia (ANLL) at the time of diagnosis, and on cells from four ALL/lymphoma cell lines (common-ALL, pre-B-ALL, T-ALL, and Burkitt's lymphoma) derived from children with these diseases. Cells were cultured in methylcellulose medium with clinically attainable concentrations (0.25-2.0 microM) of RA for two weeks prior to colony and cluster quantitation. Myeloid progenitor cells (CFU-GM) obtained from children with hematologically normal bone marrows were also cultured with RA. Of 19 patients with ANLL whose cells formed colonies, 16 (84%) were inhibited by RA; three patients showed either increased or unchanged colony numbers with RA. RA had a similar effect on both ANLL cluster and colony growth. RA (1-2 microM) also inhibited colony growth of the pre-B-ALL, common-ALL, and Burkitt's lymphoma lines; the T-ALL line and normal bone marrow CFU-GM were not inhibited. The inhibitory effects of RA on pediatric ANLL bone marrow cells and on some ALL/lymphoma cell lines compared with CFU-GM indicate that RA may be of value in the treatment of these malignancies in children.     

急性白血病细胞体外对Jurkat细胞凋亡的影响

为探讨白血病细胞凋亡相关基因 (Fas)的功能性表达情况 ,将 2 7例白血病患者的白血病细胞与 Jurkat细胞体外孵育后 ,用原位末端标记法 (TUNEL )测定单纯 Jurkat细胞 (对照组 )、不加和加入不同浓度抗凋亡相关基因配体 (Fas L)的 Jurkat细胞凋亡率。结果显示 ,不加抗 Fas L 急性非淋巴细胞白血病组 (ANL L 组 )与对照组比较 ,凋亡率明显增高 (P<0 .0 1) ,急性淋巴细胞白血病组 (AL L 组 )与对照组比较仅轻度增高 (P<0 .0 5 ) ,两者均随细胞浓度的增加而凋亡率增高 ;ANL L组加与不加抗 Fas L 相比凋亡率明显减少 (P<0 .0 1) ,随抗 Fas L浓度的增加而凋亡率减少 ,AL L 组加与不加抗 Fas L 相比凋亡率轻度减少 (P<0 .0 5 ) ,也随抗 Fas L 浓度的增高而减少 ,但不如 ANL L组凋亡率变化明显。提示急性白血病细胞存在 Fas L的功能性表达 ,ANL L较 AL L的 Fas L表达高     

Expression of CD56/NCAM on hematopoietic malignant cells. A useful marker for acute monocytic and megakaryocytic leukemias.

We investigated the expression of CD56 (a neural cell adhesion molecule, NCAM) and CD57 in various hematopoietic and non-hematopoietic malignant cells, using Leu-19 and Leu-7 monoclonal antibodies. Although both molecules are commonly defined as a natural killer cell marker, we found that CD56 was highly expressed on blasts from patients with acute monocytic (4/6) and megakaryocytic (3/3) leukemias. In the latter, FACS two-color analysis revealed that leukemic megakaryoblasts simultaneously expressed CD56 and platelet-related antigens. Among leukemic cell lines, one myelocytic, three monocytic, and two megakaryocytic lines were positive for CD56. On the other hand, except for one large granular lymphocytic leukemia and one multiple myeloma cell line, none of the lymphoid leukemia cell lines or lymphoblasts from patients with acute lymphocytic leukemia (ALL) (0/15), non-Hodgkin's lymphoma (NHL) (0/2), and central nervous system (CNS) leukemia (0/2) reacted with Leu-19 antibody for CD56. The expression of CD56 in leukemia cells was not significantly affected by 12-O-tetradecanoylphorbol-13-acetate (TPA). By contrast, all hematopoietic materials were negative for CD57, while non-hematopoietic neuroblastoma cell lines expressed this molecule (4/5) as well as CD56 (5/5). Cytogenetically, the NCAM gene is located at chromosome 11q23, and chromosome breaks were often observed at this location in various leukemias. Blasts from all five acute non-lymphocytic leukemia (ANLL) patients and cell lines with 11q23-proximal chromosomal breaks were positive, while those from one ALL patient with an 11q23 abnormality were negative for CD56, necessitating further studies to clarify the link between the 11q23 abnormality and CD56 expression.     

Flow cytometric determination of the S-phase compartment in adult acute leukemia

Flow-cytometric analysis of bone marrow aspirates and blood samples was performed in 106 adult patients with acute leukemia in order to assess the size and the prognostic significance of the percentage of S-phase cells in the bone marrow (%Sbm). A correction procedure was applied for the fraction of contaminating peripheral nucleated cells in bone marrow aspirates (%Fpb). In 82 out of 106 patients studied, the %Sbm could be reliably determined, and was compared to the %Sbm in 25 healthy controls. The %Sbm in these healthy controls ranged from 8.4 to 14.6%. The median %Sbm in 31 patients with acute nonlymphocytic leukemia (ANLL) at diagnosis (11.3%) and in 14 patients with ANLL at relapse (11.8%) did not differ significantly from the median %Sbm in normal bone marrow (11.7%). On the other hand, in 12 out of 23 patients with acute lymphocytic leukemia (ALL) at diagnosis and in 6 out of 11 patients with ALL at relapse the %Sbm was much higher and ranged from 17.8 to 44.0%. The prognosis of patients with ALL with a high %Sbm (greater than 15%) was significantly worse. Blast cells with an abnormal DNA content (aneuploid cells) were noticed in 7.7% of the patients with acute leukemia at diagnosis. This incidence, however, was significantly higher in ALL patients at relapse (i.e. 42.1%).     

Acute leukaemia with mixed lymphoid and myeloid phenotype

S ummary Three children with acute leukaemia had blasts that expressed both lymphoid and myeloid markers. The blasts met immunological criteria for acute lymphoblastic leukaemia (ALL)—common ALL antigen⁺, HLA-DR⁺, terminal deoxynucleotidyl transferase⁺–but their cytochemical features, including positive myeloperoxidase and Sudan black B, were those of acute nonlymphoblastic leukaemia (ANLL) as defined by the French-American-British Group. 30% of the blasts from one of two patients tested reacted with a monoclonal antibody specific for nonlymphoid cells (MCS-2). The wide overlap in the percentages of blasts expressing lymphoid or myeloid markers indicates that some leukaemic cells in each child had a mixed phenotype. There were no consistent cytogenetic findings, and the Philadelphia chromosome was not present. Complete remission was induced by treatment effective for either ALL (two patients) or ANLL. These three cases appear to represent a rare leukaemia subtype that we have designated acute leukaemia with mixed lymphoid and myeloid phenotype. Its recognition may be important in treatment, since two patients achieved remission with standard therapy for ALL. These cases demonstrate further the phenotypic heterogeneity that may be seen in leukaemic cell differentiation.     

Serum lactic dehydrogenase (LDH) levels in acute leukemia: marked elevations in lymphoblastic leukemia

Serum total lactic dehydrogenase (LDH) levels were examined in 42 patients with acute leukemia, 9 patients with chronic myeloid leukemia, 6 of them in blastic crisis, and 53 patients with lymphoma and other lymphoproliferative disorders. The mean range of serum LDH leveles in Hodgkin's and non-Hodgkin's lymphoma was 402 +/- 210 IU/liter and 313 +/- 113 IU/liter, while that of patients with nonmalignant disorders was 308 +/- 74 IU/liter. In acute nonlymphoblastic leukemia (ANLL), the range was 126-684 IU/liter (mean value 413 +/- 146 IU/liter). In 6 of the patients (11.3%) with lymphoma and in 6 cases (26.8%) with ANLL, the LDH levels were above 500 IU/liter. None of these patients had levels over 900 IU/liter. Patients with acute lymphoblastic leukemia (ALL) had a range of 402-3582 IU/liter (mean value of 1669 + 1038 IU/liter). In 15 of the 19 patients (78.9%) with ALL, serum LDH values were above 900 IU/liter. In addition, 3 patients with chronic myeloid leukemia (CLM) in blastic crisis had levels of 970-1940 IU/liter. One of these 3 patients had lymphoblastic crisis, while the second case responded clinically to vincristine and prednisone, but was not regarded as ALL. The differences in serum LDH levels between ALL and ANLL are statisticaly significant (p < 0.001). It appears that markedly elevated serum LDH levels in acute leukemia are suggestive of ALL, and that in individual patients, the LDH levels were correlated with the number of blasts during remission and relapse.     

白血病细胞形态(M)、细胞化学(C)及免疫表型(Ⅰ)对急性白血病的断诊意义

报告了按细胞形态学(M)分类的急性非淋巴细胞白血病(ANLL)44例,急性淋巴细胞白血病(ALL)28例。经细胞化学(C)及免疫表型(I)检测,最后诊断为ANLL38例,ALL19例,过氧化物酶阴性急非淋白血病(POX-ANLL)4例,杂合细胞急性白血病(HAL)5例,急性未分化细胞白血病(AUL)6例。发现形态(M)分类的ANLL及ALL的准确性只分别为MCI联合检测分类法的86.4％及67.9％。对急性白血病MCI检测法的诊断意义,对HAL、POX-ANLL及AUL略做讨论,强调了MCI检测法对诊断分类急性白血病的重要性.     

Analysis of the cellular DNA and RNA content in acute leukemias by flow cytometry

Summary In the present study bone marrow samples from 573 patients with newly diagnosed acute myeloid (AML) and lymphoblastic or undifferentiated leukemias (ALL/AUL), were analysed for their cellular DNA und DNA/RNA content, respectively, by means of flow cytometry. From 237 patients with AML 35.4% revealed aneuploid DNA stemlines. While no relation of DNA aneuploidy with other pretherapeutic parameters, including FAB subtype, white blood cell count, lactate dehydrogenase, S-phase index and percentage of blasts in the bone marrow, was observed, cases with aneuploid DNA stemlines revealed a tendency towards longer remission duration. In ALL/AUL 21.8% of 280 patients expressed DNA aneuploidies, which were less frequently found in T-cell ALL (11.1%) as compared to common(C)-ALL (21.4%) or null-cell(null)-ALL (23.5%). DNA aneuploidy was not related with other clinically defined risk factors such as age, white blood cell count, and rapid achievement of remission. Patients with DNA indices <1.0, however, tended to have shorter remissions. A significant difference in RNA indices was observed between AML and ALL/AUL with median values of 14.4 and 10.1, respectively (P<0.05). These data indicate the usefulness of flow cytometric analyses of cellular DNA and RNA content for the characterization and classification of acute leukemias, complementing the identification of clinical risk factors, immuno-phenotyping and cytogenetics.Abbreviations AML acute myeloid leukemia - ALL acute lymphoblastic leukemia - AUL acute undifferentiated leukemia - T-ALL, C-ALL and Null-ALL T-cell, common and Null-cell ALL     

Differences in capping behavior between immunological phenotypes in childhood acute lymphoblastic leukemia. A study with ConA and an anti-HLA-ABC backbone

Capping with concanavalin A (ConA) and monoclonal anti-HLA-ABC backbone was studied in childhood acute lymphoblastic leukemia (ALL). Capping with ConA and HLA gave quite different results, both in common ALL and T-ALL. With ConA most cases capped poorly, comparable to results described in chronic lymphatic leukemia and lymphoma, but in several cases capping was comparable to that of normal lymphocytes. In HLA capping T-ALL cells capped better than common ALL cells. HLA capping of T-ALL cells is comparable to that of normal lymphocytes. HLA capping results in handmirror cell formation giving support to the hypothesis that capping and motility are associated events.     

Sudan black B positive acute lymphoblastic leukaemia

S ummary. The presence of greater than 3% Sudan black B (SBB) positivity in leukaemic blasts has been considered diagnostic of acute non-lymphocytic leukaemia (ANLL). A rare report has indicated that this finding may not be specific for ANLL. In order to determine whether SBB could be found in acute lymphoblastic leukaemia (ALL) the data on 350 patients with newly diagnosed ALL were reviewed. Six patients (1.6%) were found to have 5% or greater SBB positive blasts. The diagnosis of ALL was supported by morphology, cytochemistries, immunologic markers, therapeutic response, and in one case immunoglobulin gene rearrangement. It is important to recognize the fact that SBB is not specific for AML and may be found in the blasts of patients with ALL.     

Characterization of acute undifferentiated leukemia by combined analysis of plasma membrane-associated gamma-glutamyltranspeptidase and soluble terminal transferase

gamma-Glutamyltranspeptidase (gamma-GT) is a plasma membrane-associated enzyme present in blasts of certain acute leukemias. We analyzed 90 cases of undifferentiated and differentiated acute leukemias for gamma- GT, using a colorimetric assay. Blasts of all patients with common acute lymphoblastic leukemia (ALL) and T-ALL were negative for gamma-GT (less than 5 units). In contrast, gamma-GT was significantly elevated in acute myeloblastic or monoblastic leukemia blasts (P less than .001). In 16 cases of acute undifferentiated leukemia (AUL) studied, the levels of gamma-GT ranged from 0 to 93 units; in eight cases, gamma- GT was positive (greater than 5 units), and six of these had 2% to 5% Sudan black-positive leukemic cells in the blast-enriched suspension. Combined gamma-GT/TdT analysis revealed that both enzyme markers were mutually exclusive in 75% of AUL cases, suggesting that gamma-GT+/TdT- blasts are of nonlymphoid origin, and gamma-GT-/TdT+ blasts are of lymphoid origin. Two cases were devoid of both enzyme activities and could represent truly undifferentiated leukemia. Thus, combined gamma- GT/TdT analysis underlines the heterogeneity of AUL and appears to be useful in defining the lineage commitment of undifferentiated leukemic blasts.     

Cyclin E overexpression in relapsed adult acute lymphoblastic leukemias of B-cell lineage

Using Western blot analysis, we examined cyclin E and cyclin A protein levels in 19 patients with acute lymphoblastic leukemia ([ALL] 15 B-ALL and four T-ALL). Whereas normal, nonproliferating peripheral blood mononuclear cells (PBMCs) expressed low levels of the 50-kD cyclin E, ALL blasts in the peripheral blood, although showing low-level or no proliferation as judged by FACS/cell-cycle analysis and cyclin A protein levels, expressed high levels of cyclin E, with a mean value similar to that of the proliferating Burkitt's lymphoma cell line, Akata. The accumulation of a protein shown to shorten the G1 phase of the cell cycle, cyclin E, in growth-delayed leukemic blasts may reflect the malignant status of these cells. Before treatment, B-ALL cells expressed predominantly the 50-kD cyclin E. T-ALL samples displayed the 50-kD cyclin E protein and a smaller, approximately 43-kD cyclin E species. In paired B-ALL samples taken before treatment and at relapse, we found a significant overexpression of the 50-kD protein in relapsed samples (P < .006), plus the presence of up to four additional smaller- molecular-weight species of cyclin E, illustrating clear diagnosis versus relapse differences. Cyclin E expression in ALL blasts may correlate to the relative malignant status of the cells, with higher protein levels reflecting a more advanced stage of the disease and a greater potential to proliferate under permissive conditions.