ALBERT

Description: FC is still not employed in MRD based treatment protocols. One problem is lack of standardization suitable for prospective trials involving multiple clinical centers and FC laboratories. Therefore, we established a MiniMini Project, an international collateral study within the ALL IC BFM 2002 treatment protocol for childhood acute lymphoblastic leukemia (ALL). The MiniMini Project provides a mainframe of minimal panel of monoclonal antibody combinations to evaluate MRD by FC. Patients (pts) are stratified according non-MRD criteria (prednisone response at day 8 in peripheral blood (PB), percentage of blasts at day 15 and day 33 in bone marrow (BM), leukocytosis, and age at diagnosis and presence of BCR/ABL or MLL/AF4 fusions). Identical immunophenotypic populations are reported in all pts regardless presenting phenotype. Each laboratory investigates at least 2 pts with B lineage ALL by the T ALL combinations and vice versa. These “cross-lineage controls” together with data on subpopulations that are negative at diagnosis were used to set the specificity cutoff values at each time point (diagnosis, day 8 BM and PB, day 15, day 33 day 52 BM). MRD levels obtained by Ig/TCR rearrangements RQ-PCR in 32 pts (24 pts BCP ALL, 8 pts T ALL) were used to define specificity thresholds. 185 pts were investigated in the participating laboratories. We used data from first Czech cohort of pts (92 pts in total, 16 pts T lineage, 74 pts B lineage, in standard risk group (SRG), n=36, IRG, n=40 and HRG, n=16) in whom clinical data as well as standard FC analysis results were available. We compared morphological percentage of blasts (used for stratification) to a level of residual disease by FC. There was high concordance in SRG of both methods, except 1 patient redirected into IRG group (M3 BM vs. only 14% of blasts by FC). In IRG, concordance was in 92.5% of pts, 3 pts should be placed in HRG group according FC. 98.9% of pts morphologically in complete remission at day 33 were confirmed by FC. Although FC data confirm a significant difference between PGR and PPR in PB specimens at day 8 (p=0.0014), there is an overlap in percentage of leukemic cells between these categories. In total, MRD level above 0.1% was observed in BM of 100, 99, 84, 32 and 3.5 % pts in days 0, 8, 15, 33 and 52, respectively and in PB of 95% pts at day 8. Our first results show feasibility of FC standardization. The choice of subpopulations and the cutoff points will be validated in an independent cohort within the same Project.

Description: The cryptic translocation t(12;21), resulting in TEL/AML1 fusion, is the single most frequent non-random genetic aberration in childhood ALLs (up to 25%). Both partner genes are important for the development and maintenance of normal haematopoiesis. TEL part of the fusion protein contains domains interacting with the mSin3, N-CoR and HDAC-3 corepressors. Part of AML1 gene involved in the fusion carries a DNA binding domain that controls the interaction with the cis-regulatory elements of the target genes. TEL/AML1 is supposed to negatively regulate the expression of the genes that, under normal conditions, are transactivated by AML1. TEL/AML1 chimaeric protein seems to affect genes responsible for the differentiation during haematopoiesis by chromatin remodelling via association with histone deacetylases (HDAC). We used HDAC inhibitors (HDACi) to overcome the presumptive differentiation block mediated by HDAC recruited by TEL/AML1. Trichostatin A (TSA) and valproic acid (VPA), common antiepileptic drug with attributed HDACi activity, were used as HDACi. We tested different concentrations of TSA (120nM, 240nM and 600nM) and VPA (0.5mM, 1.0mM and 2.0mM; concentrations close to VPA plasmatic level used in the treatment of epilepsy). We determined the cell cycle and the state of differentiation using flow cytometry 24 and 48 hours after HDACi administration. We compared TEL/AML1[+] (REH) and TEL/AML1[-] (Nalm-6) leukaemic cell lines to the non-leukaemic lymphoid cell line (NC-NC). Under HDACi treatment, leukaemic cells were arrested in proliferation and entered apoptosis. In REH cell line, the apoptosis rate was 11% using 1mM of VPA, and 30% using 240nM of TSA in contrast to 3.7% in control. Similarly, Nalm-6 apoptosis rate reached 16% with VPA, and 39% with TSA compared to 0.6% in control. In contrast, NC-NC cell line showed only negligible shift in cell cycle. Nalm-6 and REH cell lines reduced their proliferation activity by 36% and 19%, respectively. Furthermore, after both TSA and VPA treatment, surviving TEL/AML1[+] cells showed drift in expression of CD10 and CD20 surface antigens and in intracellular level of RAG-1 and TdT proteins. The CD 10 decreased to 88% and 64%, TdT decreased to 46% and 29%, RAG-1 decreased to 57% and 38% compared to the levels in untreated controls, after 48-hour treatment with 1.0mM VPA and 240nM TSA, respectively (all p

Description: Expression profiling (EP) studies in acute lymphoblastic leukemia (ALL) investigate thousands of genes per sample to predict principal ALL subtypes and/or prognosis. So far, insufficient amount of information is available on transferring the knowledge from EP to other methods. We have screened publicly available data of two EP studies (Yeoh et al., Cancer Cell.1(2), 2002; Ross et al., Blood.102(8), 2003) for genes that can be taken into flow cytometry (FC) diagnostics. The genes, which were identified as the best correlating with the childhood ALL subgroups (E2A/PBX1, MLL, TEL/AML1, BCR/ABL and hyperdiploid genotypes; relapse and therapy-induced AML) were individually screened. After recalculation of the data just for the B-cell precursor (BCP) ALL cases, we selected the genes in which the difference in expression was likely to be observed on protein level. Next, we selected molecules with available monoclonal antibody (mAb). Reactivity and specificity of mAbs was tested in healthy peripheral blood cells and/or in cell lines. Next, selected molecules (CD44, CD27, CD247, CD49f, CD103) were investigated by 4-color FC in diagnostic BM samples. A total of 62 children with BCP ALL and 13 children with T lineage ALL were investigated. CD44 expression (identified as predictor of poor prognosis T-ALL by EP) correlated with the risk group categories of T-ALL (p=0.0321). Strong positive correlation of CD27 (p

Description: INTRODUCTION. There has been a controversy about the prognostic impact of myeloid antigens (MyAgs) in ALL. The issue has now regained significance since a monoclonal antibody (mAb) is available for clinical treatment, which is considered for AML as well as for ALL with CD33 expression. Previous studies on prognostic impact of MyAgs often combined the information on several MyAgs using logical OR or AND. Since regulation and mutual relationships of MyAg expression are unknown, a study on prognostic impact of each MyAg alone is needed. In addition, recent in vitro data showed a higher resistance for ALL blasts with MyAgs, contrasting with clinical studies showing no prognostic value of MyAgs. DESIGN. We evaluated the prognostic impact of CD33, CD13, CD15 and CD65 in uniformly treated Czech patients. PATIENTS AND METHODS. From 9/1996 to 10/2002, 343 children with ALL were enrolled in a nationwide study ALL BFM 95. In 327 patients (96%) immunophenotyping and molecular genetics was performed centrally in our reference labs. DNA index 1.16–1.6 determined hyperdiploidy. Prednisone good responders (PGR) with WBC at diagnosis