Automated database-guided expert-supervised orientation for immunophenotypic diagnosis and classification of acute leukemia.

Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide towards the relevant classification panel (T-cell acute lymphoblastic leukemia (T-ALL), B-cell precursor (BCP)-ALL and/or acute myeloid leukemia (AML)) and final diagnosis. Now we built a reference database with 656 typical AL samples (145 T-ALL, 377 BCP-ALL, 134 AML), processed and analyzed via standardized protocols. Using principal component analysis (PCA)-based plots and automated classification algorithms for direct comparison of single-cells from individual patients against the database, another 783 cases were subsequently evaluated. Depending on the database-guided results, patients were categorized as: (i) typical T, B or Myeloid without or; (ii) with a transitional component to another lineage; (iii) atypical; or (iv) mixed-lineage. Using this automated algorithm, in 781/783 cases (99.7%) the right panel was selected, and data comparable to the final WHO-diagnosis was already provided in >93% of cases (85% T-ALL, 97% BCP-ALL, 95% AML and 87% mixed-phenotype AL patients), even without data on the full-characterization panels. Our results show that database-guided analysis facilitates standardized interpretation of ALOT results and allows accurate selection of the relevant classification panels, hence providing a solid basis for designing future WHO AL classifications.

Quantitative expression of regulatory and differentiation-related genes in the key steps of human hematopoiesis: The LeukoStage Database.

Differentiation during hematopoiesis leads to the generation of many cell types with specific functions. At various stages of maturation, the cells may change pathologically, leading to diseases including acute leukemias (ALs). Expression levels of regulatory molecules (such as the IKZF, GATA, HOX, FOX, NOTCH and CEBP families, as well as SPI-1/PU1 and PAX5) and lineage-specific molecules (including CD2, CD14, CD79A, and BLNK) may be compared between pathological and physiological cells. Although the key steps of differentiation are known, the available databases focus mainly on fully differentiated cells as a reference. Precursor cells may be a more appropriate reference point for diseases that evolve at immature stages. Therefore, we developed a quantitative real-time polymerase chain reaction (qPCR) array to investigate 90 genes that are characteristic of the lymphoid or myeloid lineages and/or are thought to be involved in their regulation. Using this array, sorted cells of granulocytic, monocytic, T and B lineages were analyzed. For each of these lineages, 3-5 differentiation stages were selected (17 stages total), and cells were sorted from 3 different donors per stage. The qPCR results were compared to similarly processed AL cells of lymphoblastic (n=18) or myeloid (n=6) origins and biphenotypic AL cells of B cell origin with myeloid involvement (n=5). Molecules characteristic of each lineage were found. In addition, cells of a newly discovered switching lymphoblastic AL (swALL) were sorted at various phases during the supposed transdifferentiation from an immature B cell to a monocytic phenotype. As demonstrated previously, gene expression changed along with the immunophenotype. The qPCR data are publicly available in the LeukoStage Database in which gene expression in malignant and non-malignant cells of different lineages can be explored graphically and differentially expressed genes can be identified. In addition, the LeukoStage Database can aid the functional analyses of next-generation sequencing data.

CD2-positive B-cell precursor acute lymphoblastic leukemia with an early switch to the monocytic lineage.

Switches from the lymphoid to myeloid lineage during B-cell precursor acute lymphoblastic leukemia (BCP-ALL) treatment are considered rare and thus far have been detected in MLL-rearranged leukemia. Here, we describe a novel BCP-ALL subset, switching BCP-ALL or swALL, which demonstrated monocytosis early during treatment. Despite their monocytic phenotype, 'monocytoids' share immunoreceptor gene rearrangements with leukemic B lymphoblasts. All swALLs demonstrated BCP-ALL with CD2 positivity and no MLL alterations, and the proportion of swALLs cases among BCP-ALLs was unexpectedly high (4%). The upregulation of CEBPα and demethylation of the CEBPA gene were significant in blasts at diagnosis, prior to the time when most of the switching occurs. Intermediate stages between CD14(neg)CD19(pos)CD34(pos) B lymphoblasts and CD14(pos)CD19(neg)CD34(neg) 'monocytoids' were detected, and changes in the expression of PAX5, PU1, M-CSFR, GM-CSFR and other genes accompanied the switch. Alterations in the Ikaros and ERG genes were more frequent in swALL patients; however, both were altered in only a minority of swALLs. Moreover, switching could be recapitulated in vitro and in mouse xenografts. Although children with swALL respond slowly to initial therapy, risk-based ALL therapy appears the treatment of choice for swALL. SwALL shows that transdifferentiating into monocytic lineage is specifically associated with CEBPα changes and CD2 expression.

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.

Uncommon leukemic case of anaplastic large cell lymphoma diagnosed through a typical chromosomal abnormality t(2;5) with a null phenotype and aberrant expression of NG2 and CD13.

Anaplastic large cell lymphoma represents approximately 10-15 % of pediatric non-Hodgkin lymphomas. Leukemic presentation is very rare, and in particular, the null phenotype ALCL without typical anaplastic morphology together with aberrant expression of CD13 and/or CD11b represents a diagnostic challenge. We report a case of a 9 year-old boy with leukemic presentation of ALCL with the typical translocation t(2;5)(p23;q35); in this patient, the only positive antigens identified by immunophenotyping were CD13, NG2 HLA-DR, and CD38. To our knowledge, aberrant expression of NG2 has never been reported in ALCL cases (Tab. 1, Fig. 6, Ref. 20).

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.

Quantification of fusion transcript reveals a subgroup with distinct biological properties and predicts relapse in BCR/ABL-positive ALL: implications for residual disease monitoring.

Minimal residual disease (MRD) monitoring is an essential tool for risk group stratification in current treatment protocols for childhood acute lymphoblastic leukaemia (ALL). Although quantitative detection of clonal immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements is currently considered to be the standard method, leukaemia fusion genes provide other possible targets for MRD follow-up, as already demonstrated in TEL/AML1-positive ALLs. We analysed and compared MRD levels quantified by BCR/ABL transcript detection and by the standard Ig/TCR-based method in 218 bone marrow specimens from 17 children with BCR/ABL-positive ALL. We found only a limited overall correlation of MRD levels as assessed by the two methods (correlation coefficient R(2)=0.64). The correlation varied among patients from excellent (R(2)=0.99) to very poor (R(2)=0.17). Despite identical sensitivity of the approaches, 20% of the samples were negative by the Ig/TCR approach whereas positive by the BCR/ABL method. We show that multilineage involvement is at least partly responsible for the discrepancy. Moreover, our data demonstrate that BCR/ABL monitoring enables better and earlier prediction of relapse compared to the standard Ig/TCR methodology. We conclude that BCR/ABL-based MRD monitoring of childhood ALL is a clinically relevant tool and should be performed in parallel with the standard Ig/TCR follow-up.

Allo-SCT in children with high-risk leukemia using unmanipulated grafts from alternative donors.

Allogeneic HSCT is a curative treatment for high-risk leukemia. In Europe, approximately 15% of children have an HLA-matched sibling, but in 65-70% HLA allele-matched (9-10/10) unrelated donors (UD) can be identified. Transplantation using an HLA partially mismatched donor, unrelated cord blood or haploidentical family donor with graft manipulation is then considered with preference on the basis of local experience and/or availability. Here we evaluate the outcomes of 87 consecutive patients with leukemia transplanted with unmanipulated graft from matched or partially mismatched UD or cord blood (CB) at our institution between January 2001 and December 2007. Within the median follow-up of 30 months, the acute GVHD grade II was diagnosed in 70.9% patients; grades III-IV only in 4.6%. The overall incidence of chronic GVHD was 43.3% (extensive in 34.9%). The probability of 3-year EFS was 59.5% and that of 3-year overall survival was 66.9%. TRM at day +100 was 4.5%, and overall it was 13.8%. Fourteen patients (16.1%) died as a consequence of post-transplant leukemia relapse. We conclude that the prognosis of patients transplanted for leukemia using unmanipulated grafts from HLA-matched or partially mismatched UD or CB is comparable and satisfactory. TRM and relapse rate are lower than in the earlier period.

B-cell reconstitution after allogeneic SCT impairs minimal residual disease monitoring in children with ALL.

Minimal residual disease (MRD) detection using quantification of clone-specific Ig or TCR rearrangements before and after transplantation in children with high-risk ALL is an important predictor of outcome. The method and guidelines for its interpretation are very precise to avoid both false-negative and -positive results. In a group of 21 patients following transplantation, we observed detectable MRD positivities in Ig/TCR-based real-time quantitative PCR (RQ-PCR) leading to no further progression of the disease (11 of 100 (11%) total samples). We hypothesized that these positivities were mostly the result of nonspecific amplification despite the application of strict internationally agreed-upon measures. We applied two non-self-specific Ig heavy chain assays and received a similar number of positivities (20 and 15%). Nonspecific products amplified in these RQ-PCR systems differed from specific products in length and sequence. Statistical analysis proved that there was an excellent correlation of this phenomenon with B-cell regeneration in BM as measured by flow cytometry and Ig light chain-kappa excision circle quantification. We conclude that although Ig/TCR quantification is a reliable method for post transplant MRD detection, isolated positivities in Ig-based RQ-PCR systems at the time of intense B-cell regeneration must be viewed with caution to avoid the wrong indication of treatment.

Minimal residual disease (MRD) analysis in the non-MRD-based ALL IC-BFM 2002 protocol for childhood ALL: is it possible to avoid MRD testing?

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 microl(-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.

CD44 and CD27 delineate B-precursor stages with different recombination status and with an uneven distribution in nonmalignant and malignant hematopoiesis.

The expression of CD27 and CD44 correlate with the genotype of B-precursor acute lymphoblastic leukemia (ALL). Based on the expression of these antigens, we identified counterparts of TEL/AML1(pos) and TEL/AML1(neg) leukemic cells in nonmalignant bone marrow. Although CD27 is known as a marker of mature memory B cells, we recently showed that CD27 is also expressed by malignant and nonmalignant B precursors. Here, we show that CD27 and CD44 delineate stages of B-precursor development. Well-established differentiation markers showed that the developmental sequence starts from undetermined progenitors, expressing CD44. Upon B-lineage commitment, cells gain CD27 and lose CD44. The CD27(pos)CD44(neg) (CD27 single positive, 27SP) cells are the earliest stage within CD10(pos)CD19(pos) B precursors and express RAG-1 and TDT. These cells correspond to TEL/AML1(pos) ALL (1/4 pediatric B-precursor ALL). The development follows to CD27/CD44 double-positive (27/44DP) stage, 44SP stage and CD27/CD44 double-negative (27/44DN) stage. Before exit to periphery, CD44 is reexpressed. The 27/44DP cells are mostly large and profoundly suppress RAG-1. Despite their presumably high proliferation potential, 27/44DP cells rarely dominate in leukemia. At 44SP stage, which corresponds to TEL/AML1(neg) leukemias, RAG-1 is reexpressed and Ig light chain gene starts to be rearranged.

Childhood secondary ALL after ALL treatment.

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.

[Familial haemophagocytic lymphohistiocytosis caused by perforin deficit can be successfully treated by haematopoietic stem cell transplantation--the first diagnosed case in the Czech Republic]. / Familiární hemofagocytující lymfohistiocytóza na podklade deficitu perforinu uspesne lécená transplantací hematopoetických kmenových bunek--první diagnostikovaný prípad v Ceské republice.

Familial haemophagocytic lymphohistiocytosis (FHL) is an inherited disorder characterized by an impaired cytotoxicity of T lymphocytes and NK cells typically manifesting within first few months after birth. If not treated adequately, it is inevitably fatal within several months. The incidence in Caucasians has been estimated to 1: 50 000 births. Haematopoietic stem cell transplantation represents the only curative treatment for FHL. Recently, several genetic defects underlying molecular defects in FHL have been identified. In approximately 30% of patients FHL is caused by mutations in PRF1 gene coding for perforin. Further 30% of patients were found to have mutations in UNC13D coding for hMunc13-4 protein. Very recent report has identified another cause of FHL, mutations in STX11 gene on chromosome 6, coding for syntaxin 11. Absence of any of those proteins severely impairs the process of exocytosis of cytotoxic granules. We describe patient with clinical symptoms of FHL. Immunological and molecular biology methods led to the identification of perforin mutation as a cause of the disease. Patient received an allogeneic SCT from HLA-matched unrelated donor. SCT was followed by rapid normalization of clinical symptoms and laboratory findings. In patient described in this study, FHL manifested with typical clinical and laboratory symptoms. Adequate immunosuppressive treatment and subsequent SCT led to the sustained remission of FHL and correction of molecular defect. This is the first case of FHL in Czech Republic where perforin mutation was identified as a molecular cause both at cellular and molecular level.