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The article presents a study which analyzes the presence of ERGdel gene coding in a large cohort of children with acute lymphoblastic leukemia (ALL) treated according to the Austrian-German-Swiss ALL-BFM-2000 protocol. It mentions the clonal stability of ERGdel from diagnosis to relapse in paired samples that occurred at a frequency of 4.5% in childhood ALL. The authors note that their observations support a positive impact of ERGdel on treatment susceptibility in ALL-BFM 2000.

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/μl; 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.Leukemia (2009) 23, 528–534; doi:10.1038/leu.2008.324; published online 20 November 2008 [ABSTRACT FROM AUTHOR]

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.Leukemia (2007) 21, 1204–1211. doi:10.1038/sj.leu.2404675; published online 5 April 2007 [ABSTRACT FROM AUTHOR]

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.Leukemia (2007) 21, 897–905. doi:10.1038/sj.leu.2404613; published online 1 March 2007 [ABSTRACT FROM AUTHOR]

Recently, we have shown that a novel recombinant bispecific single-chain antibody construct (bscCD19 ×CD3), induces highly efficacious lymphoma-directed cytotoxicity mediated by unstimulated peripheral T lymphocytes. Functional analysis of bscCD19×CD3 has so far been exclusively performed with human B lymphoma cell lines and T cells from healthy donors. Here we analysed the properties of bscCD19×CD3 using primary B cells and autologous T cells from healthy volunteers or patients with B-cell chronic lymphocytic leukaemia (B-CLL). We show that bscCD19×CD3 induces T-cell-mediated depletion of nonmalignant B cells in all four cases and depletion of primary lymphoma cells in 22 out of 25 cases. This effect could be observed at low effector-to-target (E:T) ratios and in the majority of cases without additional activation of autologous T cells by IL-2. Even in samples derived from patients heavily pretreated with different chemotherapy regimens, strong cyto-toxic effects of bscCD19×CD3 could be observed. The addition of bscCD19×CD3 to patients' cells resulted in an upregulation of activation-specific cell surface antigens on autologous T cells and elevated levels of CD95 on lymphoma B cells. Although anti-CD95 antibody CH-11 failed to induce apoptosis in lymphoma cells, we provide evidence that B-CLL cell depletion by bscCD×CD3 is mediated at least in part by apoptosis via the caspase pathway. [ABSTRACT FROM AUTHOR]

ORGANIZING COMMITTEE::G.J.L. Kaspers, VU University Medical Center, Amsterdam, The NetherlandsJ.J.M. van Dongen, Erasmus MC, Rotterdam, The NetherlandsM. Schrappe, Medizinische Hochschule Hannover, GermanyA.J.P. Veerman, VU University Medical Center, Amsterdam, The NetherlandsLeukemia (2003) 17, 657-674. doi:10.1038/sj.leu.2402898 [ABSTRACT FROM AUTHOR]

The TNF-related apoptosis-inducing ligand (TRAIL) is currently under evaluation as a possible (co-)therapeutic in cancer treatment. We therefore examined 129 cell samples from patients with de novo acute leukemia as to their constitutive susceptibility to TRAIL-induced apoptosis In vitro. Only 21 (16%) cell samples revealed at least 10% TRAIL-susceptible cells/sample as detected by flow cytometric annexinV staining after 24 h culture compared with medium control. Precursor B cell ALL samples (11 (27%) of 41) were more TRAIL-susceptible compared with AML (5 (9%) of 54; P < 0.05) but not compared with precursor T cell ALL (5 (15%) of 34; P = 0.20). Furthermore, we examined constitutive mRNA expression levels of TRAIL receptors R1-R4 by semi-quantitative RT-PCR (n = 58). Expression levels were heterogeneous, however, there was no significant correlation between the expression of the signal-transducing receptors (R1, R2) as well as of the decoy receptors (R3, R4) and TRAIL sensitivity in this series. Constitutive NF-kappa B activity has been shown to influence TRAIL susceptibility of leukemic cells. In 39 leukemic cell samples examined, we found a generally high NF-kappa B activity as detected by electrophoretic mobility shift assay which did not differ between TRAIL-susceptible and TRAIL-resistant cases. Finally, 49 acute leukemic cell samples were coincubated with doxorubicin in vitro. Doxorubicin sensitized four of 35 initially TRAIL-resistant samples and augmented TRAIL-induced apoptosis in two of 14 TRAIL-susceptible samples. In summary, constitutive TRAIL susceptibility differs between leukemia subtypes and does not correlate with mRNA expression levels of the TRAIL receptors R1-R4 as well as constitutive NF-kappa B activation status. The observed sensitization of leukemic cells to TRAIL by doxorubicin in vitro indicates that TRAIL should be further evaluated as to its possible role as an in vivo cotherapeutic in acute leukemia. [ABSTRACT FROM AUTHOR]

In the present study, we investigated the in vitro apoptotic response of leukemic cells to the cellular stress induced by homoharringtonine (HHT), a plant alkaloid with antileukemic activity which is currently being tested for treatment of acute and chronic leukemias. A comparison of leukemic cell lines with different p53 gene status revealed a considerably higher sensitivity to HHT-induced apoptosis in the cells with a wt p53, and apoptotic events in wt p53 leukemia cells (MOLT-3 cell line) were studied in more detail. To this end, we examined components of apoptotic cascades including Bax expression and its intracellular localization, changes of mitochondrial membrane potential (MMP), reactive oxygen species (ROS) levels, cytochrome c release from mitochondria and activation of caspases. Bax protein levels did not increase despite an up-regulation of bax at mRNA level. However, Bax translocation from cytosol towards mitochondria was observed. In addition, we observed a release of cytochrome c from the mitochondria, and the localization changes of both Bax and cytochrome c were found already at the early, annexin V-negative stage of HHT-induced apoptosis. HHT-treated MOLT-3 cells revealed loss of MMP as well as activation of caspases demonstrated by DEVD-, IETD- and LEHD-tetrapeptide cleavage activity in the cell lysates. ROS levels only slightly increased in HHT-treated cells and antioxidants did not prevent apoptosis and MMP changes. Therefore, wt p53 leukemic cells respond to HHT-specific cellular stress by induction of ROS-independent apoptotic pathway characterized by translocation of Bax, mitochondrial cytochrome c release and activation of caspases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

CD95 (Fas/APO-1) is a cell surface receptor able to trigger apoptosis in a variety of cell types. The expression and function of the CD95 antigen on leukemic blasts from 42 patients with B lineage and 53 patients with T lineage acute lymphoblastic leukemia (ALL) were investigated using immunofluorescence staining and apoptosis assays. The CD95 surface antigen was expressed in most ALL cases, with the T lineage ALL usually showing a higher intensity of surface CD95 expression as compared with the B lineage ALL cells (relative fluorescence intensity, RFI: 4.8 +/- 0.47 vs 2.2 +/- 0.23, respectively, P < 0.01). Functional studies disclosed that upon oligomerization by anti-CD95 monoclonal antibodies the CD95 protein was either not able to initiate apoptosis of leukemic cells (75% of cases) or induced low rates of apoptosis (20% of cases). Only in 5% of cases did the apoptosis rate exceed the 20% level of the CD95-specific apoptosis. Most of the CD95-sensitive cases were found among T lineage ALLs (38% of T lineage vs 10% of B lineage ALLs). Overall, the extent of CD95-induced apoptosis did not correlate with the expression level of CD95. Similarly, no significant correlation between expression level and functionality of CD95 in human leukemia cell lines of B and T cell origin could be observed. Bcl-2 protein has been associated with prolonged cell survival and has been shown to block partially CD95-mediated apoptosis, but for ALL cells no correlation between bcl-2 expression and spontaneous or CD95-mediated apoptosis could be found. The results obtained in this study indicate that, despite constitutive expression of CD95, the ALL cells are mainly resistant to CD95-triggering. More detailed investigations of the molecular mechanisms involved in the intracellular apoptotic signal transduction, such as interactions of the bcl-2 and the other members of the bcl-2 family, and functionality of the interleukin-1beta converting enzyme (ICE) like-proteases, may give new insights into key events responsible for the resistance or sensitivity to the induction of apoptosis in acute leukemia. [ABSTRACT FROM AUTHOR]

A correction to the article "ERG deletion is associated with CD2 and attenuates the negative impact of IKZF1 deletion in childhood acute lymphoblastic leukemia" that was published in the January 2014 issue is presented.