Barbara Buldini

Risk of Relapse of Childhood Acute Lymphoblastic Leukemia Is Predicted By Flow Cytometric Measurement of Residual Disease on Day 15 Bone Marrow

PURPOSE Speed of blast clearance is an indicator of outcome in childhood acute lymphoblastic leukemia (ALL). Availability of measurement of minimal residual disease (MRD) at an early time point with a reduced-cost method is of clinical relevance. In the AIEOP-BFM-ALL (Associazione Italiana Ematologia Oncologia Pediatrica and Berlin-Frankfurt-Münster Study Group) 2000 trial, patients were stratified by levels of polymerase chain reaction (PCR) MRD at day +33 and +78. AIEOP studied the prognostic impact of MRD measured by flow cytometry (FCM) at day 15 of induction therapy. PATIENTS AND METHODS Bone marrow samples from 830 Italian patients were collected on day 15, after 14 days of steroids, and one dose of intrathecal methotrexate, vincristine, daunorubicine, and asparaginase. Cells were analyzed by four-color FCM for detection of leukemia-associated immunophenotypes. RESULTS Three patient risk groups were identified by FCM: standard (< 0.1% blast cells; 42% of the total), intermediate (0.1 to < 10%; 47%), and high (> or = 10%; 11%). Their 5-year cumulative incidences of relapse were 7.5% (SE, 1.5), 17.5% (SE, 2.1), and 47.2% (SE, 5.9), respectively. In multivariate analysis, FCM was the most important prognostic factor among those available by day 15, with two-fold and five-fold increase in the risk of relapse compared with patients with less than 0.1%. PCR MRD, when added to the model, had significant prognostic impact; yet high levels of FCM MRD retained an independent ability to detect a significantly higher risk of relapse. CONCLUSION Measurement of FCM MRD in day 15 bone marrow was the most powerful early predictor of relapse, applicable to virtually all patients; it may complement PCR MRD-based stratification including later time points, thus allowing additional treatment tailoring.

Safety and efficacy of a caspofungin-based combination therapy for treatment of proven or probable aspergillosis in pediatric hematological patients.

BackgroundFungal infections are diagnosed increasingly often in patients affected by hematological diseases and their mortality has remained high. The recent development of new antifungal drugs gives the clinician the possibility to assess the combination of antifungal drugs with in-vitro or in animal-model synergistic effect.MethodsWe analyzed retrospectively the safety and efficacy of caspofungin-based combination therapy in 40 children and adolescents, most of them were being treated for a malignant disease, who developed invasive aspergillosis (IA) between November 2002 and November 2005.ResultsThirteen (32.5%) patients developed IA after hematopoietic stem cell transplantation (HSCT), 13 after primary diagnosis, usually during remission-induction chemotherapy, and 14 after relapse of disease. Severe neutropenia was present in 31 (78%) out of the 40 patients. IA was classified as probable in 20 (50%) and documented in 20 (50%) patients, respectively. A favorable response to antifungal therapy was obtained in 21 patients (53%) and the probability of 100-day survival was 70%. Different, though not significant, 100-day survival was observed according to the timing of diagnosis of IA: 51.9% after HSCT; 71.4% after relapse; and 84.6% after diagnosis of underlying disease, p 0.2. After a median follow-up of 0.7 years, 20 patients are alive (50%). Overall, the combination therapy was well tolerated. In multivariate analysis, the factors that were significantly associated to a better overall survival were favorable response to antifungal therapy, p 0.003, and the timing of IA in the patient course of underlying disease, p 0.04.ConclusionThis study showed that caspofungin-based combination antifungal therapy is an effective therapeutic option also for pediatric patients with IA. These data need to be confirmed by prospective, controlled studies.

Time point-dependent concordance of flow cytometry and real-time quantitative polymerase chain reaction for minimal residual disease detection in childhood acute lymphoblastic leukemia.

Background Flow cytometric analysis of leukemia-associated immunophenotypes and polymerase chain reaction-based amplification of antigen-receptor genes rearrangements are reliable methods for monitoring minimal residual disease. The aim of this study was to compare the performances of these two methodologies in the detection of minimal residual disease in childhood acute lymphoblastic leukemia. Design and Methods Polymerase chain reaction and flow cytometry were simultaneously applied for prospective minimal residual disease measurements at days 15, 33 and 78 of induction therapy on 3565 samples from 1547 children with acute lymphoblastic leukemia enrolled into the AIEOP-BFM ALL 2000 trial. Results The overall concordance was 80%, but different results were observed according to the time point. Most discordances were found at day 33 (concordance rate 70%) in samples that had significantly lower minimal residual disease. However, the discordance was not due to different starting materials (total versus mononucleated cells), but rather to cell input number. At day 33, cases with minimal residual disease below or above the 0.01% cut-off by both methods showed a very good outcome (5-year event-free survival, 91.6%) or a poor one (5-year event-free survival, 50.9%), respectively, whereas discordant cases showed similar event-free survival rates (around 80%). Conclusions Within the current BFM-based protocols, flow cytometry and polymerase chain reaction cannot simply substitute each other at single time points, and the concordance rates between their results depend largely on the time at which they are used. Our findings suggest a potential complementary role of the two technologies in optimizing risk stratification in future clinical trials.

Early T-cell precursor acute lymphoblastic leukaemia in children treated in AIEOP centres with AIEOP-BFM protocols: a retrospective analysis

BACKGROUND Early T-cell precursor acute lymphoblastic leukaemia was recently recognised as a distinct leukaemia and reported as associated with poor outcomes. We aimed to assess the outcome of early T-cell precursor acute lymphoblastic leukaemia in patients from the Italian Association of Pediatric Hematology Oncology (AIEOP) centres treated with AIEOP-Berlin-Frankfurt-Münster (AIEOP-BFM) protocols. METHODS In this retrospective analysis, we included all children aged from 1 to less than 18 years with early T-cell precursor acute lymphoblastic leukaemia immunophenotype diagnosed between Jan 1, 2008, and Oct 31, 2014, from AIEOP centres. Early T-cell precursors were defined as being CD1a and CD8 negative, CD5 weak positive or negative, and positive for at least one of the following antigens: CD34, CD117, HLADR, CD13, CD33, CD11b, or CD65. Treatment was based on AIEOP-BFM acute lymphoblastic leukaemia 2000 (NCT00613457) or AIEOP-BFM acute lymphoblastic leukaemia 2009 protocols (European Clinical Trials Database 2007-004270-43). The main differences in treatment and stratification of T-cell acute lymphoblastic leukaemia between the two protocols were that in the 2009 protocol only, pegylated L-asparaginase was substituted for Escherichia coli L-asparaginase, patients with prednisone poor response received an additional dose of cyclophosphamide at day 10 of phase IA, and high minimal residual disease at day 15 assessed by flow cytometry was used as a high-risk criterion. Outcomes were assessed in terms of event-free survival, disease-free survival, and overall survival. FINDINGS Early T-cell precursor acute lymphoblastic leukaemia was diagnosed in 49 patients. Compared with overall T-cell acute lymphoblastic leukaemia, it was associated with absence of molecular markers for PCR detection of minimal residual disease in 25 (56%) of 45 patients; prednisone poor response in 27 (55%) of 49 patients; high minimal residual disease at day 15 after starting therapy in 25 (64%) of 39 patients (bone marrow blasts ≥ 10%, by flow cytometry); no complete remission after phase IA in 7 (15%) of 46 patients (bone marrow blasts ≥ 5%, morphologically); and high PCR minimal residual disease (≥ 5 × 10(-4)) at day 33 after starting therapy in 17 (85%) of 20 patients with markers available. Overall, 38 (78%) of 49 patients are in continuous complete remission, including 13 of 18 after haemopoietic stem cell transplantation, with three deaths in induction, five deaths after haemopoietic stem cell transplantation, and three relapses. Severe adverse events in the 2009 study were reported in 10 (30%) of 33 patients with early T-cell precursor acute lymphoblastic leukaemia versus 24 (15%) of 164 patients without early T-cell precursor acute lymphoblastic leukaemia and life-threatening events in induction phase IA occurred in 4 (12%) of 33 patients with early T-cell precursor acute lymphoblastic leukaemia versus 7 (4%) of 164 patients without early T-cell precursor acute lymphoblastic leukaemia. No difference was seen in the subsequent consolidation phase IB of protocol I. INTERPRETATION Early T-cell precursor acute lymphoblastic leukaemia is characterised by poor early response to conventional induction treatment. Consolidation phase IB, based on cyclophosphamide, 6-mercaptopurine, and ara-C at conventional (non-high) doses is effective in reducing minimal residual disease. Although the number of patients and observational time are limited, patients with early T-cell precursor acute lymphoblastic leukaemia treated with current BFM stratification and treatment strategy have a favourable outcome compared with earlier reports. The role of innovative therapies and haemopoietic stem cell therapy in early T-cell precursor acute lymphoblastic leukaemia needs to be assessed. FUNDING None.

Minimal residual disease analysis by eight-color flow cytometry in relapsed childhood acute lymphoblastic leukemia

Multiparametric flow cytometry is an alternative approach to the polymerase chain reaction method for evaluating minimal residual disease in treatment protocols for primary acute lymphoblastic leukemia. Given considerable differences between primary and relapsed acute lymphoblastic leukemia treatment regimens, flow cytometric assessment of minimal residual disease in relapsed leukemia requires an independent comprehensive investigation. In the present study we addressed evaluation of minimal residual disease by flow cytometry in the clinical trial for childhood relapsed acute lymphoblastic leukemia using eight-color flow cytometry. The major challenge of the study was to reliably identify low amounts of residual leukemic cells against the complex background of regeneration, characteristic of follow-up samples during relapse treatment. In a prospective study of 263 follow-up bone marrow samples from 122 patients with B-cell precursor acute lymphoblastic leukemia, we tested various B-cell markers, adapted the antibody panel to the treatment protocol, and evaluated its performance by a blinded parallel comparison with the polymerase chain reaction data. The resulting eight-color single-tube panel showed a consistently high overall concordance (P<0.001) and, under optimal conditions, sensitivity similar to that of the reference polymerase chain reaction method. Overall, evaluation of minimal residual disease by flow cytometry can be successfully integrated into the clinical management of relapsed childhood acute lymphoblastic leukemia either as complementary to the polymerase chain reaction or as an independent risk stratification tool. ALL-REZ BFM 2002 clinical trial information: NCT00114348

MLL partner genes drive distinct gene expression profiles and genomic alterations in pediatric acute myeloid leukemia: an AIEOP study

MLL partner genes drive distinct gene expression profiles and genomic alterations in pediatric acute myeloid leukemia: an AIEOP study

Advanced pediatric myelodysplastic syndromes: Can immunophenotypic characterization of blast cells be a diagnostic and prognostic tool?

The diagnosis of myelodysplastic syndromes (MDS) is mainly based on morphology and cytogenetic analysis. Several efforts to analyze MDS by flow cytometry have been reported in adults. These studies have focused on the identification of abnormalities in the maturation pathway of antigen expression of myelo‐monocytic cells, and characterization of blast populations. Therefore, phenotype has been proposed as a diagnostic and prognostic criterion tool for adult MDS. The current article provides data concerning the blast phenotype in pediatric MDS.

Minimal residual disease monitored after induction therapy by RQ-PCR can contribute to tailor treatment of patients with t(8;21) RUNX1-RUNX1T1 rearrangement.

Disease relapse still remains the most important cause of treatment failure in childhood acute myeloid leukemia (AML). Molecular monitoring of response to treatment by minimal residual disease (MRD) provides important information, widely used to tailor treatment in childhood acute lymphoblastic leukemia.1–3 On the contrary, prognostic relevance of MRD in pediatric AML has only been recently proposed and needs to be further investigated and confirmed.4–6 So far, the prognostic impact of the quality of response measured by flow cytometry after induction and consolidation therapy has been shown to provide independent prognostic information in pediatric AML,5 able to permit a refinement of risk stratification and to potentially improve AML patient outcome.

Detection of PICALM-MLLT10 (CALM-AF10) and outcome in children with T-lineage acute lymphoblastic leukemia.

Detection of PICALM-MLLT10 ( CALM-AF10 ) and outcome in children with T-lineage acute lymphoblastic leukemia

AIEOP-BFM consensus guidelines 2016 for flow cytometric immunophenotyping of Pediatric acute lymphoblastic leukemia

Immunophenotyping by flow cytometry (FCM) is a worldwide mainstay in leukemia diagnostics. For concordant multicentric application, however, a gap exists between available classification systems, technologic standardization, and clinical needs. The AIEOP‐BFM consortium induced an extensive standardization and validation effort between its nine national reference laboratories collaborating in immunophenotyping of pediatric acute lymphoblastic leukemia (ALL). We elaborated common guidelines which take advantage of the possibilities of multi‐color FCM: marker panel requirements, immunological blast gating, in‐sample controls, tri‐partite antigen expression rating (negative vs. weak or strong positive) with capturing of blast cell heterogeneities and subclone formation, refined ALL subclassification, and a dominant lineage assignment algorithm able to distinguish “simple” from bilineal/“complex” mixed phenotype acute leukemia (MPAL) cases, which is essential for choice of treatment. These guidelines are a first step toward necessary inter‐laboratory standardization of pediatric leukemia immunophenotyping for a concordant multicentric application.