Key treatment questions in childhood acute lymphoblastic leukemia: results in 5 consecutive trials performed by the ALL-BFM study group from 1981 to 2000.

Between 1981 and 2000, 6 609 children (<18 years of age) were treated in 5 consecutive trials of the Berlin-Frankfurt-Münster (BFM) study group for childhood acute lymphoblastic leukemia (ALL). Patients were treated in up to 82 centers in Germany, Austria, and Switzerland. Probability of 10-year event-free survival (survival) improved from 65% (77%) in study ALL-BFM 81-78% (85%) in ALL-BFM 95. In parallel to relapse reduction, major efforts focused on reducing acute and late toxicity through advanced risk adaptation of treatment. The major findings derived from these ALL-BFM trials were as follows: 1) preventive cranial radiotherapy could be safely reduced to 12 Gy in T-ALL and high-risk ALL patients and eliminated in non-high-risk non-T-ALL patients, if it was replaced by high-dose and intrathecal methotrexate; 2) omission of delayed reintensification severely impaired outcome of low-risk patients; 3) 6 months less maintenance therapy caused an increase in systemic relapses; 4) slow response to an initial 7-day prednisone window was identified as adverse prognostic factor; 5) condensed induction therapy resulted in a significant improvement of outcome; 6) the daunorubicin dose in induction could be safely reduced in low-risk patients; 7) intensification of consolidation/reintensification treatment led to considerable improvement of outcome in high-risk patients.

Lineage classification of childhood acute lymphoblastic leukemia according to the EGIL recommendations: results of the ALL-BFM 2000 trial.

BACKGROUND: Flow cytometry immunophenotyping (FCM) is an undispensable tool for the diagnosis and for the treatment stratification of childhood acute lymphoblastic leukemia. The correlation of the EGIL-classification with prognostically relevant parameters like age, prednisone response and risk group is analyzed. PATIENTS: Between March 2000 and June 2009 12 patients less than 1 year of age, 1 836 patients with 1 to less than 6 years, 620 patients with 6 to less than 10 years, 615 patients with 10 to less than 15 years and 275 patients with 15 to less than 19 years were analyzed with a comprehensive 4-color antibody panel and classified according to the EGIL recommendations. METHODS: Bone marrow or peripheral blood mononuclear cells were isolated by ficoll gradient centrifugation, washed and stained with fluorochrome-conjugated antigen-specific monoclonal antibodies. Cell preparations were acquired and analyzed on a flow cytometer. RESULTS: Centralized FCM was performed for 2 775 patients (82.6%) with B-cell precursor acute lymphoblastic leukemia, 493 patients (14.7%) with T-cell acute lymphoblastic leukemia and 90 patients (2,7%) with biphenotypic acute leukemia. There was a slight overall predominance of male (56.1%) over female (43.9%) patients. Patients with B-cell precursor ALL had a slightly more favourable outcome with a 10 y pEFS of 78 ± 1.0%, compared to patients with a T-ALL or BAL (biphenotypic acute leukemia) phenotype with a 10 y pEFS of 74 ± 1.8% (n.s.) or 69 ± 9.0% (p<0.009), respectively. CONCLUSIONS: FCM according to the EGIL recommendations not only provides diagnostic lineage determination and subclassification but also enables an initial prognostic orientation before MRD (minimal residual disease)-based risk stratification becomes available.

[Autologous bone-marrow stem-cell transplantation for induction of arteriogenesis for limb salvage in critical limb ischaemia]. / Extremitätenerhalt durch autologe Knochenmarksstammzelltransplantation zur Induktion der Arteriogenese bei kritischer, nicht-revaskularisierbarer Extremitätenischämie.

BACKGROUND: Bone marrow cell transplantation has been shown to induce angiogenesis and thus improve ischaemic artery disease. This study evaluates the effects of intramuscular bone marrow cell transplantation in patients with limb-threatening critical limb ischaemia with a very high risk for major amputation. METHODS AND RESULTS: After failed or impossible operative and / or interventional revascularisation and after unsuccessful maximum conservative therapy, 51 patients with impending major amputation due to severe critical limb ischaemia had autologous bone marrow cells (BMC) transplant-ed into the ischaemic leg. Patients 1-12 received Ficoll-isolated bone marrow mononuclear cells (total cell number 1.1 +/- 1.1 x 10(9)), patients 13-51 received point of care isolated bone marrow total nucleated cells (3.0 +/- 1.7 x 10(9)). Limb salvage was 59 % at 6 months and 53 % at last follow-up (mean: 411 +/- 261 days, range: 175-1186 days). Perfusion measured with the ankle-brachial-index (ABI) and transcutaneous oxygen tension (tcpO2) at baseline and after 6 months increased in -patients with consecutive limb salvage (ABI 0.33 +/- 0.18 to 0.46 +/- 0.15, tcpO2 12 +/- 12 to 25 +/- 15 mmHg) and did not change in patients eventually undergoing major amputation. No differences in clinical outcome between the isolation methods were seen. Clinically most important, patients with limb salvage improved from a mean Rutherford category of 4.9 at baseline to 3.3 at 6 months (p = 0.0001). Analgesics consumption was reduced by 62 %. -Total walking distance improved in non-amputees from zero to 40 metres. Three severe peri-procedural adverse events resolved without sequelae, and no unexpected long-term adverse events occurred. CONCLUSIONS: In no-option patients with end-stage critical limb ischaemia due to peripheral -artery disease, bone marrow cell transplantation is a safe procedure which can improve leg perfusion sufficiently to reduce major amputations and permit durable limb salvage.

Monitoring treatment response of childhood precursor B-cell acute lymphoblastic leukemia in the AIEOP-BFM-ALL 2000 protocol with multiparameter flow cytometry: predictive impact of early blast reduction on the remission status after induction.

Treatment response is a strong outcome predictor for childhood acute lymphoblastic leukemia (ALL). Here, we evaluated the predictive impact of flow cytometric blast quantification assays (absolute blast count, BC, and blast reduction rate, BRR) in peripheral blood (pB) and/or bone marrow (BM) at early time points of induction therapy (days 0, 8 and 15) on the remission status in the AIEOP-BFM-ALL 2000 protocol. At the single parameter level (905 patients), the strongest predictive parameter for the remission status as a dichotomous minimal residual disease (MRD) parameter (positive/negative) has been provided by the BC at day 15 in BM (cutoff: 17 blasts/microl; 50 vs 15%; odds ratio: 5.6; 95% confidence interval: 4.1-7.6, P<0.001), followed by the BRR at day 15 in BM and by the BC at day 8 in pB (odds ratios: 3.8 and 2.6, respectively). In the multiple regression analysis (440 patients), BC in pB (d0 and d8) and in BM (d15) as well as BRR at day 8 in pB provided significantly contributing variables with an overall correct prediction rate of 74.8%. These data show that the quantitative assessment of early response parameters, especially absolute BCs at day 15 in BM, has a predictive impact on the remission status after induction therapy.

Discriminant function analysis as decision support system for the diagnosis of acute leukemia with a minimal four color screening panel and multiparameter flow cytometry immunophenotyping.

Despite several recommendations for standardization of multiparameter flow cytometry (MFC) the number, specificity and combinations of reagents used by diagnostic laboratories for the diagnosis and classification of acute leukemias (AL) are still very diverse. Furthermore, the current diagnostic interpretation of flow cytometry readouts is influenced arbitrarily by individual experience and knowledge. We determined the potential value of a minimal four-color combination panel of 13 monoclonal antibodies (mAbs) with a CD45/sideward light scatter-gating strategy for a standardized MFC immunophenotyping of the clinically most relevant subgroups of AL. Bone marrow samples from 155 patients with acute myeloid leukemia (AML, n=79), B-cell precursor acute lymphoblastic leukemia (BCP-ALL, n=29), T-cell precursor acute lymphoblastic leukemia (T-ALL, n=12) and normal bone marrow donors (NBMD, n=35) were analyzed. A knowledge-based learning algorithm was generated by comparing the results of the minimal panel with the actual diagnosis, using discriminative function analysis. Correct classification of the test sample according to lineage, that is, BCP-ALL, T-ALL, AML and differentiation of NBMD was achieved in 97.2% of all cases with only six of the originally applied 13 mAbs of the panel. This provides evidence that discriminant function analysis can be utilized as a decision support system for interpretation of flow cytometry readouts.

Gene expression shift towards normal B cells, decreased proliferative capacity and distinct surface receptors characterize leukemic blasts persisting during induction therapy in childhood acute lymphoblastic leukemia.

In childhood acute lymphoblastic leukemia (ALL), persistence of leukemic blasts during therapy is of crucial prognostic significance. In the present study, we address molecular and cell biologic features of blasts persisting after 1 week of induction glucocorticoid therapy. Genome-wide gene expression analysis of leukemic samples from precursor B-cell ALL patients (n=18) identified a set of genes differentially expressed in blasts at diagnosis day 0 (d0) and persisting on day 8 (d8). Expression changes indicate a shift towards mature B cells, inhibition of cell cycling and increased expression of adhesion (CD11b/ITGAM) and cytokine (CD119/IFNGR1) receptors. A direct comparison with normal B cells, which are largely therapy resistant, confirmed the differentiation shift at the mRNA (n=10) and protein (n=109) levels. Flow cytometric analysis in independent cohorts of patients confirmed both a decreased proliferative activity (n=13) and the upregulation of CD11b and CD119 (n=29) in d8 blasts. The differentiation shift and low proliferative activity in d8 blasts may account for the persistence of blasts during therapy and affect their sensitivity to further therapeutic treatment. CD11b and CD119 are potential specific markers for d8 blast persistence and detection of minimal residual disease, which warrant further investigation.

Drug-induced immunophenotypic modulation in childhood ALL: implications for minimal residual disease detection.

Assessment of minimal residual disease (MRD) by flow cytometry is considered to be based on the reproducibility of the leukemic immunophenotype detected at diagnosis. However, we previously noticed modulation of surface antigen expression in acute lymphoblastic leukemia (ALL) during the early treatment. Hence, we investigated this in 30 children with B-cell precursor ALL consecutively enrolled in the AIEOP-BFM ALL 2000 protocol. Quantitative expression of seven antigens useful in MRD monitoring was studied at diagnosis and compared to that measured at different time points of remission induction therapy. Downmodulation in the expression of CD10 and CD34 occurred at follow-up. By contrast, upmodulation of CD19, CD20, CD45RA, and CD11a was observed, while the expression of CD58 remained stable. Despite this, we could unambiguously discriminate leukemic cells from normal residual B cells. This holds true when bone marrow (BM) samples from similarly treated T-ALL patients, but not from healthy donors, were used as reference. Our results indicate that immunophenotypic modulation occurs in ALL during the early phases of BFM-type protocols. However, the accuracy of MRD detection by flow cytometry seems not negatively affected if adequate analysis protocols are employed. Investigators should take this phenomenon into account in order to avoid pitfalls in flow cytometric MRD studies.

CD99 expression in T-lineage ALL: implications for flow cytometric detection of minimal residual disease.

Expression of CD99 is higher on immature than on mature T cells. We postulated that this marker could be used to assess minimal residual disease (MRD) in T-lineage acute lymphoblastic leukemia (T-ALL). In diagnostic bone marrow (BM) samples from 27 children with T-ALL, expression of CD99 on leukemic lymphoblasts by flow cytometry was in median 7.7 times higher than on normal T lymphocytes from within the same sample. In 85% of cases, leukemic MFI values were higher than the mean MFI+2 s.d. of normal populations. We applied CD99 to study MRD in 39 follow-up samples from 15 consecutive T-ALL patients, and compared the results with those obtained with the well-established MRD-marker terminal deoxynucleotidyl transferase (TdT). Either antibody was combined in four-color flow cytometry with CD7, surfaceCD3, and cytoplasmicCD3. We found that CD99 was a valid complement to TdT in quantifying T-ALL MRD. Given a considerable interpatient variability, CD99 could be favorably used in nine patients, and TdT in other five patients. Both approaches showed a similar very low nonspecific background throughout 12 weeks from diagnosis (in median 0.002% of nucleated BM cells in patients with non-T ALL). We conclude that CD99 is a highly informative tool for MRD detection in T-ALL, bearing the advantage of surface expression in contrast to TdT.

Impact of CD133 (AC133) and CD90 expression analysis for acute leukemia immunophenotyping.

BACKGROUND AND OBJECTIVES: AC133 is a novel monoclonal antibody (Moab) reacting with a population of immature/primitive or granulo-monocytic committed CD34+ve cells. Up to now, only few studies with small numbers of cases have examined AC133 (recently designated CD133) expression in acute leukemia. To determine the value of this Moab for acute leukemia immunophenotyping, we investigated a large series of leukemic cell samples for their reactivity with Moab AC133. DESIGN AND METHODS. A total of 298 cell samples from patients with de novo acute myeloid leukemia (AML) (n=142), acute lymphoblastic leukemia (ALL) (n=119), CD34+ve biphenotypic acute leukemia (n=13), and CD34+ve CML blast crisis (=BC; 21 myeloid BC/3 lymphoid BC) were investigated by flow cytometry for Moab AC133 reactivity.CD133 expression was compared with CD90(Thy-1) expression, another CD34-associated antigen. RESULTS: Fifteen (5%) samples expressed CD90, whereas 114 (38%) samples were positive for Moab AC133 (20% cut-off level). No significant differences in CD133 and CD90 expression levels between AML and ALL were observed. In AML, but not ALL, CD133 was more often expressed in CD34+ve cases than in CD34-ve ones (p<0.00001). However, CD133 expression was not restricted to CD34+ve leukemic cells in individual cell samples. All 8 pro-B-ALL cell samples with 11q23-anomalies and MLL (mixed lineage leukemia) gene translocations were positive for CD133, whereas only 2 of 9 pro-B-ALL without MLL gene translocations expressed CD133 (p<0.002). In contrast, none of the 5 AML cell samples with a t(9;11) and MLL gene translocation reacted with Moab AC133. CD34+ve CML cells in myeloid BC were less often positive for CD133 than CD34+ve de novo AML cells (p<0.0001). INTERPRETATION AND CONCLUSIONS: CD133 and CD90 expression analysis is not helpful for lineage determination in acute leukemia immunophenotyping. However, MoabAC133 may be an informative marker for the detection and further characterization of immature AML cells, as well as pro-B-ALL cells with MLL gene translocations, by flow cytometry.

Constitutive expression levels of CD95 and Bcl-2 as well as CD95 function and spontaneous apoptosis in vitro do not predict the response to induction chemotherapy and relapse rate in childhood acute lymphoblastic leukaemia.

CD95 (Fas/APO-1) expression and function and Bcl-2 expression, as well as spontaneous apoptosis in vitro, have been shown to be predictive markers for the in vivo response to chemotherapy in acute myeloid leukaemia (AML). To determine the clinical significance of apoptosis-regulating factors in acute lymphoblastic leukaemia (ALL), we investigated cell samples of children with ALL who had been included in the German ALL Berlin-Frankfurt-Münster (BFM) study using flow cytometry for constitutive expression levels of CD95 (n = 110) and Bcl-2 (n = 110). Furthermore, we determined the extent of spontaneous apoptosis in vitro (n = 102) and susceptibility to anti-CD95-induced apoptosis (CD95-sensitivity) (n = 97). We correlated these findings with the functional activity of the multidrug resistance (MDR)-associated P-glycoprotein (P-gp), as detected by the rhodamine123 efflux test, immunophenotype, cytogenetics and clinical data of the patients examined. Good responders to initial prednisone therapy ('prednisone response') revealed significantly higher Bcl-2 expression levels [5.4 +/- 3.4 relative fluorescence intensity (RFI), n = 68] than poor responders (3.7 +/- 2.6 RFI, n = 42; P = 0.002). There was no significant correlation between the other investigated parameters and prednisone response. Moreover, neither the CD95 and Bcl-2 expression levels nor the extent of spontaneous apoptosis in vitro, CD95 sensitivity or P-gp function were correlated with the response to induction chemotherapy or relapse rate, either for B-cell precursor ALL or T-cell ALL. No consistent pattern of change in CD95 (n = 10) and Bcl-2 expression (n = 9) was noted in cases studied at both initial diagnosis and relapse. In conclusion, our findings underline the different cell biological features of primary AML and ALL cells.

Clinical significance of P-glycoprotein expression and function for response to induction chemotherapy, relapse rate and overall survival in acute leukemia.

BACKGROUND AND OBJECTIVES: A multidrug-resistance (MDR) phenotype mediated by P-glycoprotein (P-gp) contributes to chemotherapy failure in acute leukemia. However, the exact prognostic significance of this resistance mechanism is still unclear, mostly due to methodologic problems in P-gp detection. We therefore investigated, whether P-gp expression levels or functional P-gp activity better predict response to induction chemotherapy, relapse rate and overall survival in acute leukemia. DESIGN AND METHODS: We examined cell samples of 121 adults with de novo acute myeloid leukemia (AML) and 102 children with newly diagnosed acute lymphoblastic leukemia (ALL) for P-gp expression and functional P-gp activity by flow cytometry. P-gp function was determined by the rhodamine 123 (rh123)-efflux test (AML n=121, ALL n=102) and P-gp expression levels using the P-gp specific monoclonal antibodies (moabs) MRK-16 (AML n=51, ALL n=31), 4.E3 (AML n=35, ALL n=32), or UIC-2 (AML n=68, ALL n=50). We correlated our findings with the immunophenotype, FAB morphology, cytogenetics and clinical data of the examined patients. RESULTS: P-gp expression levels as detected by MRK-16 and 4.E3 were very low and did not differ between AML and ALL as estimated using relative fluorescence intensity (RFI) values and D-values by Kolmogorow-Smirnov (KS) statistics. For moab UIC-2, P-gp expression levels were higher in AML than in ALL. Within AML, moab UIC-2 mainly reacted with myelomonocytic-differentiated leukemic cells of the FAB M4/5 subtypes. No correlation between P-gp expression levels as detected by MRK-16, 4.E3 or UIC-2 and the response to induction chemotherapy or relapse rate, both in AML and ALL, was observed. However, a prognostic impact of P-gp expression levels on overall survival in AML was seen for moab MRK-16. Moreover, within AML, P-gp function was higher in immature blast cells as defined by immunophenotype and FAB morphology and correlated with response to induction chemotherapy, relapse rate, overall survival as well as cytogenetic risk groups. In ALL, the overall functional P-gp activity was lower than in AML and did not correlate with immunophenotypical subgroups, response to induction chemotherapy, relapse rate or overall survival. INTERPRETATION AND CONCLUSIONS: Our data demonstrate a prognostic impact of P-gp in AML but not ALL and indicate that the functional rh123-efflux assay should be preferred for flow-cytometric P-gp evaluation in acute leukemia compared with P-gp expression analysis by monoclonal antibodies.

Detection of acute leukemia cells with mixed lineage leukemia (MLL) gene rearrangements by flow cytometry using monoclonal antibody 7.1.

Translocations involving 11q23 are among the most common genetic abnormalities in hematologic malignancies, occurring in approximately 5-10% of acute lymphoblastic leukemia (ALL) and 5% of acute myeloblastic leukemia (AML). In 11q23 translocations, the mixed lineage leukemia (MLL) gene on chromosome 11, band q23, is usually disrupted. The human homologue of the rat NG2 chondroitin sulfate proteoglycan molecule, as detected by the monoclonal antibody (moab) 7.1, was shown to be expressed on leukemic cells with MLL rearrangements of children with acute leukemia. We further investigated the reactivity of the moab 7.1 on 533 cell samples of adults (n = 215) and children (n = 318) with acute leukemias (271 AML, 217 B-lineage ALL, 37 T-lineage ALL, eight CD7+ CD56+ myeloid/natural killer cell precursor acute leukemias) by flow cytometry. In AML, 38 samples were positive for moab 7.1 ('20%-cut-off-level'). These moab 7.1-positive AML cases revealed a myelomonocytic-differentiated immunophenotype with coexpression of the NK cell marker CD56 in 33 of 38 cases. Two of eight cell samples of the recently described CD7+ CD56+ myeloid/natural killer cell precursor acute leukemia entity reacted with moab 7.1. In ALL, 35 samples mostly of the pro-B-ALL subtype (33 pro-B-ALL, one common-ALL, one pre-B-ALL) were positive for moab 7.1. 58 (81%) of 72 samples with MLL rearrangements were positive for moab 7.1 including 28/31 with a t(4;11), 16/17 with a t(9;11), 3/5 with a t(11;19), and 2/6 with a del(11)(q23). All moab 7.1-positive ALL (n = 34) and childhood AML (n = 17) cases revealed MLL rearrangements as detected by Southern blot analysis and RT-PCR. However, 11 adults with AML, and one adult with moab 7.1-positive CD7+ CD56+ myeloid/natural killer cell precursor acute leukemia were negative for MLL rearrangements as proved by Southern blot analysis. We conclude that moab 7.1 is a sensitive but not entirely specific marker for the identification of 11q23-associated AML and ALL by flow cytometry in children and adults.

Salvage therapy for relapsed mediastinal B-cell lymphoma with allogeneic HLA-identical related donor bone marrow transplantation, donor lymphocyte infusion and IDEC-C2B8.

Primary B-cell lymphoma of the mediastinum is an aggressive non-Hodgkin's lymphoma with distinct clinicopathologic features. Response rates are between 60-80% following intensive chemotherapy regimens. Poor responders or patients with an early relapse usually do not achieve a prolonged second remission with conventional salvage therapy protocols and therefore qualify for intensive or experimental approaches. Here we describe two patients of same age, gender and stage with primary mediastinal B-cell lymphoma and an early relapse after the first courses of combination chemotherapy and irradiation of the mediastinum. One patient relapsed after a salvage therapy with allogeneic donor-related bone marrow transplantation and donor lymphocyte infusion but responded again with a continuing good partial remission after infusion of the chimeric anti-CD20 antibody IDEC-C2B8. For the other patient an allogeneic bone marrow transplantation was not possible. He finally failed to respond to salvage therapy with IDEC-C2B8 and died of progressive disease. The anti-CD20 antibody IDEC-C2B8 induced a partial remission in a patient with primary mediastinal B-cell lymphoma refractory to other therapeutic approaches, including allogeneic bone marrow transplanatation (alloBMT), donor lymphocyte infusion (DLI) and irradiation. The role of IDEC-C2B8 as a component of salvage regimens appears to be worthy for further evaluation in high-risk patients with primary mediastinal B-cell lymphoma

Clinical significance of CD95, Bcl-2 and Bax expression and CD95 function in adult de novo acute myeloid leukemia in context of P-glycoprotein function, maturation stage, and cytogenetics.

Resistance to chemotherapy-induced apoptosis and a multidrug-resistance (MDR) phenotype, mainly mediated by P-glycoprotein (P-gp), contribute to chemotherapy failure in hematologic malignancies. To study apoptosis-regulating factors in acute myeloid leukemia (AML), we investigated cell samples of adults with de novo AML by flow cytometry for constitutive expression levels of the apoptosis-related molecules CD95 (n = 135), Bcl-2 (n = 131), and Bax (n = 66), as well as spontaneous apoptosis in vitro (n = 104) and susceptibility to anti-CD95-induced apoptosis (CD95 sensitivity) (n = 93). We correlated these findings with P-gp function as detected by the rhodamine123-efflux test (n = 121), immunophenotype, FAB morphology, cytogenetics, and clinical data of the examined patients. Immature FAB M0/1 AML cells expressed significantly more Bcl-2 (P < 0.0002) and less CD95 (P < 0.0003) compared with AML cells of the more mature FAB M2-5 subtypes. No maturation-dependent difference in Bax expression was observed. FAB M2-5 AML cells were more susceptible to anti-CD95-induced apoptosis (P < 0.008) and showed a lower P-gp function (P < 0.002) than FAB M0/1 AML cells. Leukemic cells of AML patients who achieved a complete remission (CR) after induction chemotherapy expressed less Bcl-2 than non-responder (NR) (69 CR, 23 NR; P = 0.05). CR was associated with a higher extent of spontaneous apoptosis in vitro (58 CR, 17 NR; P=0.05) and a tendency towards a higher CD95 expression (73 CR, 23 NR; P = 0.08) compared to NR. CR also correlated with a low P-gp function (70 CR, 21 NR; P = 0.008) and a tendency towards CD34 negativity (73 CR, 23 NR; P = 0.08). No correlation between Bax expression and response to induction chemotherapy (49 CR, 12 NR) was observed. In stepwise logistic regression analyses, P-gp function and the extent of spontaneous apoptosis in vitro as well as CD95 sensitivity but not Bcl-2, CD95, Bax, and CD34 expression levels emerged as significant markers for response to induction chemotherapy. We conclude that the constitutive expression of CD95 and Bcl-2, as well as CD95 sensitivity and P-gp function but not constitutive Bax expression depend on the maturation stage of leukemic cells in adult de novo AML. P-gp function, the extent of spontaneous apoptosis in vitro and CD95 sensitivity are more predictive for response to induction chemotherapy in adult de novoAML than the constitutive expression levels of the apoptosis-related molecules CD95, Bcl-2 and Bax.

Immunophenotype and clinical characteristics of CD45-negative and CD45-positive childhood acute lymphoblastic leukemia.

To evaluate the expression pattern of the leukocyte common antigen CD45 in acute leukemias and to investigate whether the lack of CD45 expression in childhood acute lymphoblastic leukemia (ALL) is associated with other immunophenotypic features and a distinct clinical behavior, we have carried out extensive immunophenotypic analyses of bone marrow and peripheral blood samples from 638 patients with childhood B-cell precursor (n=529) or T-lineage ALL (n=109). All 638 patients were enrolled in the German ALL-BFM 90 and ALL-BFM 95 trials. CD45 was detected on the surface of childhood ALL cells (cut-off > or = 20% positive cells) in only 88.7% (n=566) of all cases. Among 529 patients with childhood B-cell precursor ALL, 12.9% (n=68) did not express CD45, compared with only 3.7% (n=4) of patients with childhood T-lineage ALL (p < 0.001). In the B-cell precursor ALL subtypes, the highest frequency of CD45- cases (15.1%) was observed in common ALL (56/372) compared with only 7.2% in pro-B ALL (3/41) and 7.8% in pre-B ALL (9/116). Assessment of clinical parameters (age, organ enlargement, WBC, etc.) and event-free survival did not reveal significant differences between CD45- and CD45+ patients. Myeloid antigen coexpression was not correlated with CD45 expression. The mean percentage of antigen expression for CD34, CD10, TdT, CD22, and CD24 was significantly higher in children with CD45- B-cell precursor ALL than in those with CD45+ B-cell precursor ALL. In 28 patients with B-cell precursor ALL, cell cycle analyses of freshly isolated leukemic cells were performed with propidium iodide (PI) staining and flow-cytometric analysis. The percentage of cells in S-phase was inversely correlated to the percentage of CD45+ cells (r=-0.48, p < 0.05). With two-parameter analysis of CD45-fluorescein isothiocyanate (FITC)- and PI-stained cells in nine patients with a percentage of CD45+ cells between 40 and 60%, two populations were distinguishable in a single patient. It was shown that the CD45- subpopulation had a higher percentage of cells in S-phase than the CD45+ subpopulation (10.7 +/- 4.0 vs. 2.7 +/- 1.8, p < 0.007). We conclude that the lack of CD45 expression contributes to the identification of a distinct functional and immunological subgroup of B-cell precursor ALL, but that it has no significant impact on clinical behavior or on therapy outcome in childhood ALL.

Immunophenotypic and clinical features of T-cell receptor gammadelta+ T-lineage acute lymphoblastic leukaemia.

The immunophenotypic and clinical features of TCR-gammadelta+ T-lineage acute lymphoblastic leukaemia (T-ALL) were prospectively analysed in 52 children with membrane CD3+ T-ALL. We observed a relatively high incidence of TCR-gammadelta+ T-ALL (26/52 patients). Leukaemic blasts from 22 children demonstrated TCR-alphabeta positivity, and simultaneous expression of the TCR-beta and -delta chain was found in four children. Clinical and haematological features of TCR-alphabeta and gammadelta+ T-ALL did not differ significantly, except that haemoglobin levels were significantly lower in TCR-gammadelta+ cases. Event-free survival at 4 years was significantly better in TCR-gammadelta+ compared with TCR-alphabeta+ T-ALL, but expression of TCR molecules did not emerge as an independent prognostic factor in multivariate analysis.

Incidence and clinical outcome of children with BCR/ABL-positive acute lymphoblastic leukemia (ALL). A prospective RT-PCR study based on 673 patients enrolled in the German pediatric multicenter therapy trials ALL-BFM-90 and CoALL-05-92.

A variety of oncogenes are activated by specific chromosomal translocations, which are associated with distinct subtypes of leukemia. The identification of these rearrangements provides critical diagnostic and prognostic information, which may contribute to the selection of specific anti-leukemic therapy. The translocation t(9;22), the equivalent of the BCR/ABL rearrangement, is associated with a poor prognosis. We therefore used RT-PCR to detect this molecular event in a prospective study including 890 children. 673 of them suffered from acute lymphoblastic leukemia (ALL) at primary diagnosis and a transcription of the chimeric gene was detected in 21 of 648 with a successful analysis (3.2%). All children were treated by one of the two German multicenter childhood ALL therapy studies ALL-BFM-90 or COALL-05-92, respectively. Comparison of clinical features between BCR/ABL-positive and -negative children showed no significant differences regarding WBC, percentage of blasts, splenomegaly, hepatomegaly and age. Immunophenotypic studies at diagnosis in 21 BCR/ABL-positive children identified common ALL in 16 patients (76.2%), pre-B-ALL in four (19.0%), and an early T-lineage ALL in one (4.8%). Coexpression of myeloid antigens (CD13 and/or CD33) was observed in six of 16 common ALL patients as well as in the one child with early T-lineage ALL phenotype. The type of breakpoint (m-BCR/ABL: n = 14; M-BCR/ABL: n = 7) showed no correlation with clinical parameters. A comparison of cytogenetic and molecular data was performed in 16 positive patients and was concordant in all of them. We analyzed the response to the prednisone pretreatment and found a higher incidence of poor responders among the BCR/ABL-positive children. Regarding the event-free survival (EFS) of BCR/ABL-positive (0.53) and -negative patients (0.79) after a follow-up of 2 years, significant differences (P < 0.05) between both groups could be demonstrated.

[Morphological, immunological and cytogenetic diagnosis in acute leukemias]. / Morphologische, immunologische und zytogenetische Diagnostik akuter Leukämien.

Current diagnosis of acute leukemia includes traditional morphology and cytochemistry supplemented with immunophenotypic, cytogenetic and molecular biologic analyses. This multiparameter approach has revealed the biological heterogeneity of acute leukemias and has enabled the identification of leukemic syndromes with distinct clinical and biological features. Morphology and cytochemistry are of particular importance for the classification of acute myeloid leukemia, except for certain subtypes such as minimally differentiated acute myeloid leukemia [AML-M0] or acute megakaryoblastic leukemia [AML-M7], requiring additional immunophenotypic or ultrastructural analyses. In acute lymphoblastic leukemia [ALL], immunophenotyping is essential for the diagnosis and lineage assignment [B- and T-lineage ALL] of leukemic blasts. Furthermore, it allows the characterization of the maturation stage and certain subtypes, i.e. ALL with coexpression of myeloid antigens [My+ ALL]. Cytogenetic and molecular analyses of leukemic cells have contributed important informations to the understanding of pathogenetic mechanisms in leukemogenesis and have led to the definition of prognostic risk groups and the development of subtype-specific or risk-adapted therapy strategies.