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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. [ABSTRACT FROM AUTHOR]

The article focuses on the treatment of children with acute lymphoblastic leukemia (ALL) in the Czech Republic through the gradual introduction of protocol ALL-BFM 83 at the individual departments of pediatrics of regional and/or teaching hospitals. Imunophenotyping and cytogenetics were performed on all patients enrolled into the study at selected centers. In children with medium- and high-risk disease, prophylactic cranial radiotherapy was applied.

The ALL IC-BFM 2002 protocol was created as an alternative to the MRD-based AIEOP-BFM ALL 2000 study, to integrate early response criteria into risk-group stratification in countries not performing routine PCR-based MRD testing. ALL IC stratification comprises the response to prednisone, bone marrow (BM) morphology at days 15 and 33, age, WBC and BCR/ABL or MLL/AF4 presence. Here, we compared this stratification to the MRD-based criteria using MRD evaluation in 163 patients from four ALL IC member countries at days 8, 15 and 33 and week 12. MRD negativity at day 33 was associated with an age of 1–5 years, WBC<20 000 μl−1, non-T immunophenotype, good prednisone response and non-M3 morphology at day 15. There were no significant associations with gender or hyperdiploidy in the study group, or with TEL/AML1 fusion within BCP-ALL. Patients with M1/2 BM at day 8 tended to be MRD negative at week 12. Patients stratified into the standard-risk group had a better response than intermediate-risk group patients. However, 34% of them were MRD positive at day 33 and/or week 12. Our findings revealed that morphology-based ALL IC risk-group stratification allows the identification of most MRD high-risk patients, but fails to discriminate the MRD low-risk group assigned to therapy reduction.Leukemia (2008) 22, 989–997; doi:10.1038/leu.2008.22; published online 28 February 2008 [ABSTRACT FROM AUTHOR]

Data on secondary acute lymphoblastic leukaemia (sALL) following ALL treatment are very rare. However, the incidence might be underestimated as sALLs without a significant lineage shift might automatically be diagnosed as relapses. Examination of immunoglobulin and T-cell receptor gene rearrangements brought a new tool that can help in discrimination between relapse and sALL. We focused on the recurrences of childhood ALL to discover the real frequency of the sALL after ALL treatment. We compared clonal markers in matched presentation and recurrence samples of 366 patients treated according to the Berlin–Frankfurt–Munster (BFM)-based protocols. We found two cases of sALL and another three, where the recurrence is suspicious of being sALL rather than relapse. Our proposal for the ‘secondary ALL after ALL’ diagnostic criteria is as follows: (A) No clonal relationship between diagnosis and recurrence; (B) significant immunophenotypic shift – significant cytogenetic shift – gain/loss of a fusion gene. For the sALL (A) plus at least one (B) criterion should be fulfilled. With these criteria, the estimated frequency of the sALL after ALL is according to our data 0.5–1.5% of ALL recurrences on BFM-based protocols. Finally, we propose a treatment strategy for the patients with secondary disease.Leukemia (2007) 21, 1431–1435; doi:10.1038/sj.leu.2404718; published online 26 April 2007 [ABSTRACT FROM AUTHOR]

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.Leukemia (2004) 18, 434-441. doi:10.1038/sj.leu.2403259 Published online 15 January 2004 [ABSTRACT FROM AUTHOR]

Multi-parameter flow cytometry, molecular genetics, and cytogenetic studies have all contributed to new classification of leukemia. In this review we discuss immunophenotypic characteristics of major genotypic leukemia categories. We describe immunophenotype of: B-lineage ALL with MLL rearrangements, TEL/AML1, BCR/ABL, E2A/PBX1 translocations, hyperdiploidy, end myc fusion genes; T-ALL with SCL gene aberrations and t(5;14) translocation; and AML with AML1/ETO, PML/RARα, OTT/MAL and CBFβ/MYH11 translocations, trisomies 8 or 11 and aberrations of chromosomes 7 and 5. Whereas some genotypes associate with certain immunophenotypic features, others can present with variable immunophenotype. Single molecules (as NG2, CBFβ/SMMHC and PMIJRARα proteins) associated with or derived from specific translocations have been described. More often, complex immunophenotype patterns have been related to the genotype categories. Most known associations between immunophenotype and genotype have been defined empirically. Therefore, these associations should be validated in independent patient cohorts before they can be widely used for prescreening of leukemia. Progress in our knowledge on leukemia will show how the moleculargenetic changes modulate the immunophenotype as well as how the expressed protein molecules further modulate cell behavior. [ABSTRACT FROM AUTHOR]

The clinical significance of WT1 gene expression at diagnosis and during therapy of AML has not yet been resolved. We analyeed WT1 expression at presentation in an unselected group of 47 childhood AML patients using reel-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 karyotypa-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. [ABSTRACT FROM AUTHOR]

Studies acute lymphoblastic leukemia incidence during socioeconomic transition. Selective increase in children from one to four years old; Analysis of pertinent topics and relevant issues; Implications on the study of leukemia.

Presents a letter to the editor about acute lymphoblastic leukemia.

Examines the genetic changes in children with acute lymphocytic leukemia and TEL/acute myeloid leukemia 1 fusion gene. Immunophenotype; Final abnormal karyotype; Conventional cytogenetics.

Presents a letter to the editor about the role of TEL/AML1 real-time quantitative reverse transcriptase polymerase chain reaction on the residual disease assessment in childhood ALL.

Presents a letter to the editor about upregulation of asparagine synthetase and cell cycle arrest in t (12;21)-positive acute leukemia.

Presents a letter to the editor about statistical difference in asparagine synthetase expression levels in TEL/AML1 paediatric acute leukemia patients with or without subsequent relapses.