Ondřej Hrušák

EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes

Most consensus leukemia & lymphoma antibody panels consist of lists of markers based on expert opinions, but they have not been validated. Here we present the validated EuroFlow 8-color antibody panels for immunophenotyping of hematological malignancies. The single-tube screening panels and multi-tube classification panels fit into the EuroFlow diagnostic algorithm with entries defined by clinical and laboratory parameters. The panels were constructed in 2–7 sequential design–evaluation–redesign rounds, using novel Infinicyt software tools for multivariate data analysis. Two groups of markers are combined in each 8-color tube: (i) backbone markers to identify distinct cell populations in a sample, and (ii) markers for characterization of specific cell populations. In multi-tube panels, the backbone markers were optimally placed at the same fluorochrome position in every tube, to provide identical multidimensional localization of the target cell population(s). The characterization markers were positioned according to the diagnostic utility of the combined markers. Each proposed antibody combination was tested against reference databases of normal and malignant cells from healthy subjects and WHO-based disease entities, respectively. The EuroFlow studies resulted in validated and flexible 8-color antibody panels for multidimensional identification and characterization of normal and aberrant cells, optimally suited for immunophenotypic screening and classification of hematological malignancies.

EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols

The EU-supported EuroFlow Consortium aimed at innovation and standardization of immunophenotyping for diagnosis and classification of hematological malignancies by introducing 8-color flow cytometry with fully standardized laboratory procedures and antibody panels in order to achieve maximally comparable results among different laboratories. This required the selection of optimal combinations of compatible fluorochromes and the design and evaluation of adequate standard operating procedures (SOPs) for instrument setup, fluorescence compensation and sample preparation. Additionally, we developed software tools for the evaluation of individual antibody reagents and antibody panels. Each section describes what has been evaluated experimentally versus adopted based on existing data and experience. Multicentric evaluation demonstrated high levels of reproducibility based on strict implementation of the EuroFlow SOPs and antibody panels. Overall, the 6 years of extensive collaborative experiments and the analysis of hundreds of cell samples of patients and healthy controls in the EuroFlow centers have provided for the first time laboratory protocols and software tools for fully standardized 8-color flow cytometric immunophenotyping of normal and malignant leukocytes in bone marrow and blood; this has yielded highly comparable data sets, which can be integrated in a single database.

Real-time quantitative PCR detection of WT1 gene expression in children with AML: prognostic significance, correlation with disease status and residual disease detection by flow cytometry

The clinical significance of WT1 gene expression at diagnosis and during therapy of AML has not yet been resolved. We analysed WT1 expression at presentation in an unselected group of 47 childhood AML patients using real-time quantitative reverse-transcription PCR. We also showed that within the first 30 h following aspiration RQ-RT-PCR results were not influenced by transportation time. We observed lower levels of WT1 transcript in AML M5 (P = 0.0015); no association was found between expression levels and sex, initial leukocyte count and karyotype-based prognostic groups. There was significant correlation between very low WT1 expression at presentation and excellent outcome (EFS P = 0.0014). Combined analysis of WT1 levels, three-colour flow cytometry residual disease detection and the course of the disease in 222 samples from 28 children with AML showed remarkable correlation. Fourteen patients expressed high WT1 levels at presentation. In eight of them, who suffered relapse or did not reach complete remission, dynamics of WT1 levels clearly correlated with the disease status and residual disease by flow cytometry. We conclude that very low WT1 levels at presentation represent a good prognostic factor and that RQ-RT-PCR-based analysis of WT1 expression is a promising and rapid approach for monitoring of MRD in approximately half of paediatric AML patients.

TEL/AML1 positivity in childhood ALL: average or better prognosis?

The presence of TEL/AML1 fusion gene in childhood acute lymphoblastic leukaemia (ALL) defines a subgroup of patients with better than average outcome. However, the prognostic significance of this aberration has recently been disputed by the Berlin–Frankfurt–Münster (BFM) study group due to its relatively high incidence found in relapsed patients (19.6% and 21.9%, in two cohorts). In contrast, only four out of 45 (8.9%) unselected relapsed patients (all of whom had been treated according to BFM protocols) in the Czech Republic carry this fusion. From March 1995 to June 1998, 41 out of 190 (21.6%) newly diagnosed children with ALL were TEL/AML1-positive. There is a statistically significant difference between the incidence of TEL/AML1 fusion at diagnosis and at relapse within our group (P = 0.035). Interim analysis of the minimal residual disease (MRD) detection shows heterogeneity within the group of newly diagnosed TEL/AML1-positive leukaemias – 10 out of 24 patients tested at the end of induction therapy had detectable levels of MRD. However, only one of these patients reached relapse-predictive level (10−3) of MRD. In conclusion, we corroborate low frequency of TEL/AML1 positivity among relapsed patients with ALL among Czech children who are treated by the BFM protocols. Moreover, we demonstrate different patterns of bone marrow clean-up in TEL/AML1-positive patients.

Upregulation of asparagine synthetase fails to avert cell cycle arrest induced by L-asparaginase in TEL/AML1-positive leukaemic cells

L-Asparaginase is a standard component in chemotherapy of childhood acute lymphoblastic leukaemia (ALL). Leukaemic cells carrying TEL/AML1 fusion gene are more sensitive to treatment with L-asparaginase compared to other subtypes of ALL. We demonstrate in vitro the prolonged growth suppression of TEL/AML1[+] cells compared to TEL/AML1[−] leukaemic cells after L-asparaginase treatment simulating treatment protocol. Cell cycle analysis revealed TEL/AML1[+] cells to accumulate in G1/G0 phase (81–98%) compared to TEL/AML1[−] cells (47–60%). Quantitative analysis of asparagine synthetase (AsnS) expression showed the ability of TEL/AML1[+] cells to increase AsnS mRNA levels after L-asparaginase treatment to the same extent as TEL/AML1[−] leukaemic and nonleukaemic lymphoid cells. We hypothesise that TEL/AML1[+] cells are unable to progress into the S phase of cell cycle under nutrition stress caused by L-asparaginase, despite the ability of AsnS upregulation. Significantly higher expression of AsnS was found in untreated leukaemic cells from children with TEL/AML1[+] ALL (n=20) in comparison with the group of age-matched children with ALL bearing no known fusion gene (n=25; P=0.0043). Interestingly, none of the TEL/AML1[+] patients with high AsnS level relapsed, whereas 10/15 patients with AsnS below median relapsed (P=0.00028). Therefore, high AsnS levels in TEL/AML1[+] patients correlate with better prognosis, possibly reflecting the stretched metabolic demand of the lymphoblast.

Transfer of genomics information to flow cytometry: expression of CD27 and CD44 discriminates subtypes of acute lymphoblastic leukemia.

Transfer of genomics information to flow cytometry: expression of CD27 and CD44 discriminates subtypes of acute lymphoblastic leukemia

Slower molecular response to treatment predicts poor outcome in patients with TEL/AML1 positive acute lymphoblastic leukemia: Prospective real-time quantitative reverse transcriptase-polymerase chain reaction study

The translocation t(12;21)(p13;q22), which produces the TEL/AML1 fusion gene, is the most frequent chromosomal abnormality in patients with childhood acute lymphoblastic leukemia (ALL) and generally is associated with a favorable prognosis. Furthermore, real‐time quantitative‐polymerase chain reaction (RQ‐PCR)‐based detection of TEL/AML1 represents an accurate technique for the reproducible assessment of minimal residual disease (MRD).

Detection of residual B precursor lymphoblastic leukemia by uniform gating flow cytometry

Residual disease (RD) is an important prognostic factor in acute lymphoblastic leukemia (ALL). Flow cytometry (FC)‐based RD detection is easy to perform, but interpretation requires expert analysis due to individual differences among patients.

Acute lymphoblastic leukemia incidence during socioeconomic transition: selective increase in children from 1 to 4 years

Pre-school acute lymphoblastic leukemia (ALL) peak is consistent in developed but not in developing countries and its magnitude apparently correlates with the socioeconomic status. A population-based study describing ALL incidence during socioeconomic transition has been lacking. Central European post-communist countries (with very low foreign migration and centralized statistics) offer reliable data for the period before and during major socioeconomic changes. Population-based data on Czech ALL patients younger than 18 years were taken from two independent Czech national registries partially overlapping in time (1980–1998, n = 1236 and 1991–1999, n = 570). During the 1980s and 1990s, ALL incidence among children 1–4 years increased 1.5 times (P = 0.01). This increase was more prominent in females than in males (slopes 0.13 and 0.09, P values 0.03 and >0.05, respectively). No significant change was observed in other age groups (0, 5–9, 10–14, 15–17 years or all others combined). We discuss possible underlying socioeconomic factors including infant care and breast-feeding, hygiene, birth order, industry and pollution. Moreover, we try to pinpoint the immunophenotypic/molecular-genetic subsets of ALL that might be socioeconomically affected. Selective increase of ALL in children 1–4 years old provides epidemiological evidence that etiology and/or trigger mechanisms are different for a considerable proportion of these children and that these mechanisms are exogenous.

Generation of functional dendritic cells for potential use in the treatment of acute lymphoblastic leukemia

Abstract. Immunotherapy of malignant diseases mediated by dendritic cells (DC) pulsed with tumor antigens ex vivo is a promising new tool in the individual treatment of malignant diseases. The present study focuses on the problem of how to optimize in vitro culture conditions and induce the maturation of DC with the capacity to induce antitumor immunity toward leukemic cells. DC were generated from peripheral mononuclear cells by co-cultivation with granulocyte/macrophage–colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). Tumor antigens were added for 2 h after 7 days in culture. Irradiated leukemic blasts, blast lysate, apoptotic cells from the Jurkat cell line (T ALL) and their lysate were used in various concentrations for antigen pulsing. Harvested DC were phenotyped by flow cytometry, and viability was assessed using trypan blue exclusion (Annexin test). After the cells had been pulsed with tumor antigens and co-cultured with autologous lymphocytes, the production of interferon-gamma (IFN-γ) and IL-12 was analyzed, and lymphocyte proliferative response and cytotoxicity against the target tumor cell line were assessed. The cultivation of monocytes under the described conditions led to the expression of surface markers typical of DC (i.e. CD83, CD86, HLA-DR, CD11c and CD40). Pulsation by antigens from leukemic cells further increased the cell populations expressing these markers. Antigen pulsation decreased the viability of generated DC depending on the increase in concentration of tumor antigens. Pulsed DC–lymphocyte interaction increased the proliferative response of lymphocytes and IFN-γ production depending on the type of tumor antigens used for pulsation. The highest proliferative response was detected with DC pulsed with Jurkat cell-line lysate. Similarly to the proliferation assay, cytotoxic testing showed the highest efficiency of DC pulsed with Jurkat cell-line lysate in killing the target malignant cells. Our results show that an appropriate antigen concentration used for DC pulsing is one of the crucial factors in an effective treatment strategy, as high concentrations of tumor antigens induce apoptosis of DC, thereby rendering them non-functional. Under optimal conditions, pulsation by lysate from leukemic blasts induced the maturation of DC and led to an increase in the proliferation of autologous lymphocytes, to the production of Th1-cytokines and to the induction of cytotoxicity toward the leukemic cell line. These results are encouraging for the possible application of pulsed DC in the therapy of acute lymphoblastic leukemia.