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.

TCRα rearrangements identify a subgroup of NKL-deregulated adult T-ALLs associated with favorable outcome.

T-cell acute lymphoblastic leukemia (T-ALL) results from leukemic transformation of T-cell precursors arrested at specific differentiation stages, including an 'early-cortical' thymic maturation arrest characterized by expression of cytoplasmic TCRß but no surface T-cell receptor (TCR) and frequent ectopic expression of the TLX1/3 NK-like homeotic proteins (NKL). We designed a TCRα VJC PCR to identify clonal TCRα rearrangements in 32% of 127 T-ALLs, including 0/52 immature/TCRγδ lineage cases and 41/75 (55%) TCRαß lineage cases. Amongst the latter, TCRα rearrangements were not identified in 30/54 (56%) of IMß/pre-αß early-cortical T-ALLs, of which the majority (21/30) expressed TLX1/3. We reasoned that the remaining T-ALLs might express other NKL proteins, so compared transcript levels of 46 NKL in T-ALL and normal thymic subpopulations. Ectopic overexpression of 10 NKL genes, of which six are unreported in T-ALL (NKX2-3, BARHL1, BARX2, EMX2, LBX2 and MSX2), was detectable in 17/104 (16%) T-ALLs. Virtually all NKL overexpressing T-ALLs were TCRα unrearranged and ectopic NKL transcript expression strongly repressed Eα activity, suggesting that ectopic NKL expression is the major determinant in early-cortical thymic T-ALL maturation arrest. This immunogenetic T-ALL subtype, defined by TCRß VDJ but no TCRα VJ rearrangement, is associated with a favorable outcome in GRAALL-treated adult T-ALLs.

Age-related clinical and biological features of PTEN abnormalities in T-cell acute lymphoblastic leukaemia.

The tumour suppressor gene PTEN is commonly altered in T-cell acute lymphoblastic leukaemia but its prognostic impact is still debated. We screened a cohort of 573 fully characterised adult and paediatric T-cell acute lymphoblastic leukaemia (T-ALL) patients for genomic PTEN abnormalities. PTEN-inactivating mutations and/or deletions were identified in 91 cases (16%), including 18% of paediatric (49/277) and 14% of adult cases (42/296). Thirty-four patients harboured only mutations, 12 cases demonstrated only large deletions and 9 only microdeletions. About 36 patients had combined alterations. Different mechanisms of PTEN inactivation predicted differences in the clinical outcome for both adult and paediatric patients treated according to the GRAALL03/05 and FRALLE2000 protocols. Whereas large deletions predicted lower 5-year overall survival (P=0.0053 in adults, P=0.001 in children) and disease-free survival (P=0.0009 in adults, P=0.0002 in children), mutations were not associated with a worse prognosis. The prognostic impact of PTEN loss is therefore linked to the underlying type of genomic abnormality, both in adult and paediatric T-ALLs, demonstrating that detailed analysis of the type of abnormality type would be useful to refine risk stratification.

Acute lymphoblastic leukemia relapsing after first-line pediatric-inspired therapy: a retrospective GRAALL study.

The outcome of adult patients with Philadelphia chromosome-negative acute lymphoblastic leukemia (Ph- ALL) relapsing after pediatric-inspired front-line therapy is ill known. Here 229 relapsing Ph- ALL younger adults (18-63 years) treated within the Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL)-2003/-2005 trials were considered. Salvage regimens consisted of potentially curative therapies in 194 cases, low-intensity therapies in 21, allogeneic stem cell transplant (allo-SCT) in 6 and best supportive care in 8. Overall, 77 patients received allo-SCT after relapse. The median follow-up was 3.1 years. A second complete remission (CR2) was achieved in 121 patients (53%). In multivariate analysis, only younger age <45 years (P=0.008) and CR1 duration ⩾18 months (P=0.009) predicted CR2. Overall survival (OS) at 2 and 5 years was 19.3% (14-24%) and 13.3% (8-18%), respectively. In CR2 patients, disease-free survival (DFS) at 2 and 5 years was 29.0% (21-38%) and 25% (17-33%). In multivariate analysis, CR1 duration ⩾18 months and allo-SCT after relapse were associated with longer DFS (P<0.009 and P=0.004, respectively) and longer OS (P=0.004 and P<0.0001, respectively). In conclusion, although younger adults relapsing after pediatric-inspired ALL therapies retain a poor outcome, some of them may be cured if CR1 duration ⩾18 months and if allo-SCT can be performed in CR2. New therapies are definitely needed for these patients.

Oncogenic PTEN functions and models in T-cell malignancies.

PTEN is a protein phosphatase that is crucial to prevent the malignant transformation of T-cells. Although a numerous mechanisms regulate its expression and function, they are often altered in T-cell acute lymphoblastic leukaemias and T-cell lymphomas. As such, PTEN inactivation frequently occurs in these malignancies, where it can be associated with chemotherapy resistance and poor prognosis. Different Pten knockout models recapitulated the development of T-cell leukaemia/lymphoma, demonstrating that PTEN loss is at the center of a complex oncogenic network that sustains and drives tumorigenesis via the activation of multiple signalling pathways. These aspects and their therapeutic implications are discussed in this review.

Methodological aspects of minimal residual disease assessment by flow cytometry in acute lymphoblastic leukemia: A French multicenter study.

Background: Minimal residual disease (MRD) assessment provides a powerful prognostic factor for therapeutic stratification in acute lymphoblastic leukemia (ALL). Multiparameter flow cytometry (MFC) has the potential for a rapid and sensitive identification of high risk patients. Our group has previously published that MRD levels analyzed by clone specific Ig/TcR-QPCR and MFC were concordant at a sensitivity of 10-4 . Here we report the MFC methodological aspects from this multi-center experience. Methods: MRD was assessed by MFC in 1030 follow-up samples from 265 pediatric and adult patients with de novo ALL treated in the FRALLE, EORTC or GRALL clinical trials. MRD assessment as applied by the eight participating MFC laboratories is described in detail regarding cell preparation, leukemia-associated immunophenotype (LAIP) markers and data analysis. Samples were obtained from bone marrow (BM) and peripheral blood (PB). Immunostaining was performed after erythrocyte lysis or Ficoll enrichment. Results: This study confirms the applicability of MFC-based MRD assessment in 97% of patients with ALL at the 10-4 cut-off. MRD values after Ficoll enrichment and erythrocyte lysis were found comparable. Higher MRD values were obtained in BM than in PB, especially for B-lineage ALL. Conclusions: Measurement of MRD by MFC at the 10-4 cut-off is applicable within a few hours for almost all patients and using a comparable analytical strategy allows for multicenter collaborative studies. The method can be introduced in a strategy aimed at defining the risk of failure of patients with childhood or adult ALL. © 2014 Clinical Cytometry Society.

Flow cytometry and IG/TCR quantitative PCR for minimal residual disease quantitation in acute lymphoblastic leukemia: a French multicenter prospective study on behalf of the FRALLE, EORTC and GRAALL.

Minimal residual disease (MRD) quantification is widely used for therapeutic stratification in pediatric acute lymphoblastic leukemia (ALL). A robust, reproducible, sensitivity of at least 0.01% has been achieved for IG/TCR clonal rearrangements using allele-specific quantitative PCR (IG/TCR-QPCR) within the EuroMRD consortium. Whether multiparameter flow cytometry (MFC) can reach such inter-center performance in ALL MRD monitoring remains unclear. In a multicenter study, MRD was measured prospectively on 598 follow-up bone marrow samples from 102 high-risk children and 136 adult ALL patients, using IG/TCR-QPCR and 4/5 color MFC. At diagnosis, all 238 patients (100%) had at least one suitable MRD marker with 0.01% sensitivity, including 205/238 samples (86%) by using IG/TCR-QPCR and 223/238 samples (94%) by using MFC. QPCR and MFC were evaluable in 495/598 (83%) samples. Qualitative results (<0.01% or ≥0.01%) concurred in 96% of samples and overall positivity (including <0.01% and nonquantifiable positivity) was concurrent in 84%. MRD values ≥0.01% correlated highly (r(2)=0.87) and 69% clustered within half-a-log(10). QPCR and MFC can therefore be comparable if properly standardized, and are highly complementary. MFC strategies will benefit from a concerted approach, as does molecular MRD monitoring, and will contribute significantly to the achievement of 100% MRD informativity in adult and pediatric ALL.

Receptor kinase profiles identify a rationale for multitarget kinase inhibition in immature T-ALL.

Constitutively activated FLT3 signaling is common in acute myeloid leukemia, and is currently under evaluation for targeted therapy, whereas little data is available in T-cell acute lymphoblastic leukemia (T-ALL). We analyzed 357 T-ALL cases for FLT3 mutations and transcript expression. FLT3 mutations (3% overall) and overexpression (FLT3 high expresser (FLT3(High))) were restricted to immature/TCRγδ T-ALLs. In vitro FLT3 inhibition induced apoptosis in only 30% of FLT3(High) T-ALLs and did not correlate with mutational status. In order to investigate the mechanisms of primary resistance to FLT3 inhibition, a broad quantitative screen for receptor kinome transcript deregulation was performed by Taqman Low Density Array. FLT3 deregulation was associated with overexpression of a network of receptor kinases (RKs), potentially responsible for redundancies and sporadic response to specific FLT3 inhibition. In keeping with this resistance to FLT3 inhibition could be reversed by dual inhibition of FLT3 and KIT with a synergistic effect. We conclude that immature T-ALL may benefit from multitargeted RK inhibition and that exploration of the receptor kinome defines a rational strategy for testing multitarget kinase inhibition in malignant diseases.

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.

Fragmented red blood cells automated measurement is a useful parameter to exclude schistocytes on the blood film.

INTRODUCTION:   The diagnosis of thrombotic microangiopathies (TMA) or disorders that may mimic their features remains difficult. Mechanical hemolytic anemia with the detection of shistocytes on the blood smear is a cornerstone finding to assess the diagnosis, but microscopic evaluation of shistocytes is still problematic with wide interobserver variations. Some of the latest generation automated blood cell counters (ABCC) propose an original quantitative approach of fragmented red cells (FRC), aiming to be equivalent to the microscopic count. This parameter has been poorly evaluated. METHODS:   To assess the predictive value (PV) of this test, we conducted studies comparing automated and microscopic counts of FRC/schistocytes, based on the analysis of thousands samples in four university hospitals and using the 2 ABCC currently available (Siemens ADVIA series, Sysmex XE-2100). RESULTS: Reference range for FRC was <0.3% for the ADVIA and <0.5% for the XE-2100. The presence of FRC below a threshold determined at 1% (ADVIA and XE-2100) had a negative PV close to 100% to exclude the presence of schistocyte on the blood smear, but in relationship with a poor PV value. CONCLUSIONS: Our study validated the utility of the immediately available FRC parameter on ABCC to exclude schistocytes and the diagnosis of TMA.

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.

Caspase-activated ROCK-1 allows erythroblast terminal maturation independently of cytokine-induced Rho signaling.

Stem cell factor (SCF) and erythropoietin are strictly required for preventing apoptosis and stimulating proliferation, allowing the differentiation of erythroid precursors from colony-forming unit-E to the polychromatophilic stage. In contrast, terminal maturation to generate reticulocytes occurs independently of cytokine signaling by a mechanism not fully understood. Terminal differentiation is characterized by a sequence of morphological changes including a progressive decrease in cell size, chromatin condensation in the nucleus and disappearance of organelles, which requires transient caspase activation. These events are followed by nucleus extrusion as a consequence of plasma membrane and cytoskeleton reorganization. Here, we show that in early step, SCF stimulates the Rho/ROCK pathway until the basophilic stage. Thereafter, ROCK-1 is activated independently of Rho signaling by caspase-3-mediated cleavage, allowing terminal maturation at least in part through phosphorylation of the light chain of myosin II. Therefore, in this differentiation system, final maturation occurs independently of SCF signaling through caspase-induced ROCK-1 kinase activation.

Standardized MRD quantification in European ALL trials: proceedings of the Second International Symposium on MRD assessment in Kiel, Germany, 18-20 September 2008.

Assessment of minimal residual disease (MRD) has acquired a prominent position in European treatment protocols for patients with acute lymphoblastic leukemia (ALL), on the basis of its high prognostic value for predicting outcome and the possibilities for implementation of MRD diagnostics in treatment stratification. Therefore, there is an increasing need for standardization of methodologies and harmonization of terminology. For this purpose, a panel of representatives of all major European study groups on childhood and adult ALL and of international experts on PCR- and flow cytometry-based MRD assessment was built in the context of the Second International Symposium on MRD assessment in Kiel, Germany, 18-20 September 2008. The panel summarized the current state of MRD diagnostics in ALL and developed recommendations on the minimal technical requirements that should be fulfilled before implementation of MRD diagnostics into clinical trials. Finally, a common terminology for a standard description of MRD response and monitoring was established defining the terms 'complete MRD response', 'MRD persistence' and 'MRD reappearance'. The proposed MRD terminology may allow a refined and standardized assessment of response to treatment in adult and childhood ALL, and provides a sound basis for the comparison of MRD results between different treatment protocols.