CD371-positive pediatric B-cell acute lymphoblastic leukemia: propensity to lineage switch and slow early response to treatment.

ABSTRACT: In the effort to improve immunophenotyping and minimal residual disease (MRD) assessment in acute lymphoblastic leukemia (ALL), the international Berlin-Frankfurt-Münster (iBFM) Flow Network introduced the myelomonocytic marker CD371 for a large prospective characterization with a long follow-up. In the present study, we aimed to investigate the clinical and biological features of CD371-positive (CD371pos) pediatric B-cell precursor ALL (BCP-ALL). From June 2014 to February 2017, 1812 pediatric patients with newly diagnosed BCP-ALLs enrolled in trial AIEOP-BFM ALL 2009 were evaluated as part of either a screening (n = 843, Italian centers) or validation cohort (n = 969, other iBFM centers). Laboratory assessment at diagnosis consisted of morphological, immunophenotypic, and genetic analysis. Response assessment relied on morphology, multiparametric flow cytometry (MFC), and polymerase chain reaction (PCR)-MRD. At diagnosis, 160 of 1812 (8.8%) BCP-ALLs were CD371pos. This correlated with older age, lower ETV6::RUNX1 frequency, immunophenotypic immaturity (all P < .001), and strong expression of CD34 and of CD45 (P < .05). During induction therapy, CD371pos BCP-ALLs showed a transient myelomonocytic switch (mm-SW: up to 65.4% of samples at day 15) and an inferior response to chemotherapy (slow early response, P < .001). However, the 5-year event-free survival was 88.3%. Among 420 patients from the validation cohort, 27 of 28 (96.4%) cases positive for DUX4-fusions were CD371pos. In conclusion, in the largest pediatric cohort, CD371 is the most sensitive marker of transient mm-SW, whose recognition is essential for proper MFC MRD assessment. CD371pos is associated to poor early treatment response, although a good outcome can be reached after MRD-based ALL-related therapies.

FCM marker importance for MRD assessment in T-cell acute lymphoblastic leukemia: An AIEOP-BFM-ALL-FLOW study group report.

T-lineage acute lymphoblastic leukemia (T-ALL) accounts for about 15% of pediatric and about 25% of adult ALL cases. Minimal/measurable residual disease (MRD) assessed by flow cytometry (FCM) is an important prognostic indicator for risk stratification. In order to assess the MRD a limited number of antibodies directed against the most discriminative antigens must be selected. We propose a pipeline for evaluating the influence of different markers for cell population classification in FCM data. We use linear support vector machine, fitted to each sample individually to avoid issues with patient and laboratory variations. The best separating hyperplane direction as well as the influence of omitting specific markers is considered. Ninety-one bone marrow samples of 43 pediatric T-ALL patients from five reference laboratories were analyzed by FCM regarding marker importance for blast cell identification using combinations of eight different markers. For all laboratories, CD48 and CD99 were among the top three markers with strongest contribution to the optimal hyperplane, measured by median separating hyperplane coefficient size for all samples per center and time point (diagnosis, Day 15, Day 33). Based on the available limited set tested (CD3, CD4, CD5, CD7, CD8, CD45, CD48, CD99), our findings prove that CD48 and CD99 are useful markers for MRD monitoring in T-ALL. The proposed pipeline can be applied for evaluation of other marker combinations in the future.

Early-life liver cirrhosis and variable clinical presentation in telomere disease.

AIM: Telomeres are DNA sequences of tandem TTAGGG repeats that protect chromosome ends from degradation and instability. Constitutional loss-of-function telomerase mutations result in rapid telomere shortening, premature senescence and cell death. Liver cirrhosis is rare and has only been reported in adults. We present five family members of Bedouin-Muslim origin, all of which carry the same mutation, and yet demonstrate an extremely variable phenotypical presentation, including liver cirrhosis during early childhood. METHODS: A multidisciplinary long-term follow-up of two healthy and three affected patients was analysed. The mutation (r.95G>C) was identified in all patients using Sanger sequencing. Telomere length samples were obtained and analysed. RESULTS: Clinical phenotypes were extremely variable, including age at first symptoms, organ involvement, disease severity and patient prognosis. The most prominent clinical phenotype is liver involvement, including end-stage liver disease early in life, which affects three members of the family. Affected patients had markedly shorter telomeres. CONCLUSION: We describe an unusual presentation of early liver failure in telomere disease patients. Little, if any, is known about the association between the genotype and phenotype among children with telomere disease and whether the mutation we have described (r.95G>C) is predisposed to early severe hepatic involvement.

An Extensive Quality Control and Quality Assurance (QC/QA) Program Significantly Improves Inter-Laboratory Concordance Rates of Flow-Cytometric Minimal Residual Disease Assessment in Acute Lymphoblastic Leukemia: An I-BFM-FLOW-Network Report.

Monitoring of minimal residual disease (MRD) by flow cytometry (FCM) is a powerful prognostic tool for predicting outcomes in acute lymphoblastic leukemia (ALL). To apply FCM-MRD in large, collaborative trials, dedicated laboratory staff must be educated to concordantly high levels of expertise and their performance quality should be continuously monitored. We sought to install a unique and comprehensive training and quality control (QC) program involving a large number of reference laboratories within the international Berlin-Frankfurt-Münster (I-BFM) consortium, in order to complement the standardization of the methodology with an educational component and persistent quality control measures. Our QC and quality assurance (QA) program is based on four major cornerstones: (i) a twinning maturation program, (ii) obligatory participation in external QA programs (spiked sample send around, United Kingdom National External Quality Assessment Service (UK NEQAS)), (iii) regular participation in list-mode-data (LMD) file ring trials (FCM data file send arounds), and (iv) surveys of independent data derived from trial results. We demonstrate that the training of laboratories using experienced twinning partners, along with continuous educational feedback significantly improves the performance of laboratories in detecting and quantifying MRD in pediatric ALL patients. Overall, our extensive education and quality control program improved inter-laboratory concordance rates of FCM-MRD assessments and ultimately led to a very high conformity of risk estimates in independent patient cohorts.

Pediatric myelodysplastic syndrome with inflammatory manifestations: Diagnosis, genetics, treatment, and outcome.

BACKGROUND: Inflammatory manifestations (IM) are well described in adult patients with myelodysplastic syndrome (MDS), but the presentation is highly variable and no standardized treatment exists. This phenomenon is rarely reported in children. As more pediatric patients are hematopoietic stem cell transplantation (HSCT) candidates, the role of anti-inflammatory treatment in relation to HSCT should be defined. PROCEDURE: Here, we report a series of five children from a tertiary center. We describe the clinical presentation, molecular findings, and treatment options. RESULTS: All patients presented with advanced MDS with blast percentages ranging 10-30%, all had severe IM. One patient had MDS secondary to severe congenital neutropenia, the other four patients had presumably primary MDS. All four were found to harbor a PTPN11 gene driver mutation, which is found in 35% of cases of juvenile myelomonocytic leukemia (JMML). The mutation was present in the myeloid lineage but not in T lymphocytes. Three had symptoms of Behcet's-like disease with trisomy 8 in their bone marrow. All patients were treated with anti-inflammatory medications (mainly systemic steroids) in an attempt to bring them to allogeneic HSCT in a better clinical condition. All demonstrated clinical improvement as well as regression in their MDS status post anti-inflammatory treatment. All have recovered from both MDS and their inflammatory symptoms post HSCT. CONCLUSION: Primary pediatric MDS with IM is driven in some cases by PTPN11 mutations, and might be on the clinical spectrum of JMML. Anti-inflammatory treatment may reverse MDS progression and improve the outcome of subsequent HSCT.

Blinatumomab as a bridge to further therapy in cases of overwhelming toxicity in pediatric B-cell precursor acute lymphoblastic leukemia: Report from the Israeli Study Group of Childhood Leukemia.

Tremendous progress in the therapy of pediatric acute lymphoblastic leukemia (ALL) has been achieved through combination cytotoxic chemotherapy, leading to high cure rates, at the cost of significant life-threatening toxicity. The bispecific T-cell engager blinatumomab, recently approved for relapsed/refractory ALL, has a unique nonmyelotoxic toxicity profile. As blinatumomab causes B-cell depletion, the safety of its use during severe chemotherapy-induced toxicity is unclear. We report 11 pediatric patients with ALL, treated with blinatumomab following overwhelming chemotherapy-associated toxicity, with recovery of all patients and successful bridging to further antileukemia therapy. Blinatumomab can be considered for rare patients who cannot tolerate cytotoxic therapy.

International cooperative study identifies treatment strategy in childhood ambiguous lineage leukemia.

Despite attempts to improve the definitions of ambiguous lineage leukemia (ALAL) during the last 2 decades, general therapy recommendations are missing. Herein, we report a large cohort of children with ALAL and propose a treatment strategy. A retrospective multinational study (International Berlin-Frankfurt-Münster Study of Leukemias of Ambiguous Lineage [iBFM-AMBI2012]) of 233 cases of pediatric ALAL patients is presented. Survival statistics were used to compare the prognosis of subsets and types of treatment. Five-year event-free survival (EFS) of patients with acute lymphoblastic leukemia (ALL)-type primary therapy (80% ± 4%) was superior to that of children who received acute myeloid leukemia (AML)-type or combined-type treatment (36% ± 7.2% and 50% ± 12%, respectively). When ALL- or AML-specific gene fusions were excluded, 5-year EFS of CD19+ leukemia was 83% ± 5.3% on ALL-type primary treatment compared with 0% ± 0% and 28% ± 14% on AML-type and combined-type primary treatment, respectively. Superiority of ALL-type treatment was documented in single-population mixed phenotype ALAL (using World Health Organization and/or European Group for Immunophenotyping of Leukemia definitions) and bilineal ALAL. Treatment with ALL-type protocols is recommended for the majority of pediatric patients with ALAL, including cases with CD19+ ALAL. AML-type treatment is preferred in a minority of ALAL cases with CD19- and no other lymphoid features. No overall benefit of transplantation was documented, and it could be introduced in some patients with a poor response to treatment. As no clear indicator was found for a change in treatment type, this is to be considered only in cases with ≥5% blasts after remission induction. The results provide a basis for a prospective trial.

Modulating ALA-PDT efficacy of mutlidrug resistant MCF-7 breast cancer cells using ALA prodrug.

Multi-drug resistance of breast cancer is a major obstacle in chemotherapy of cancer treatments. Recently it was suggested that photodynamic therapy (PDT) can overcome drug resistance of tumors. ALA-PDT is based on the administration of 5-aminolevulinic acid (ALA), the natural precursor for the PpIX biosynthesis, which is a potent natural photosensitizer. In the present study we used the AlaAcBu, a multifunctional ALA-prodrug for photodynamic inactivation of drug resistant MCF-7/DOX breast cancer cells. Supplementation of low doses (0.2mM) of AlaAcBu to the cells significantly increased accumulation of PpIX in both MCF-7/WT and MCF-7/DOX cells in comparison to ALA, or ALA + butyric acid (BA). In addition, our results show that MCF-7/DOX cells are capable of producing higher levels of porphyrins than MCF-7/WT cells due to low expression of the enzyme ferrochelatase, which inserts iron into the tetra-pyrrol ring to form the end product heme. Light irradiation of the AlaAcBu treated cells activated efficient photodynamic killing of MCF-7/DOX cells similar to the parent MCF-7/WT cells, depicted by low mitochondrial enzymatic activity, LDH leakage and decreased cell survival following PDT. These results indicate that the pro-drug AlaAcBu is an effective ALA derivative for PDT treatments of multidrug resistant tumors.

The centrality of PBGD expression levels on ALA-PDT efficacy.

Successful 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) is dependent on efficient porphyrin synthesis in the inflicted cancer tissue, which is regulated by several enzymes. Irradiation of the tumor excites the light-sensitive porphyrins and results in ROS production and cell death. In this study we investigated the effect of the expression levels of two main enzymes in heme biosynthesis, ALA dehydratase (ALAD) and porphobilinogen deaminase (PBGD), on the capacity of K562 cells to undergo cell death following ALA-PDT. We manipulated PBGD and ALAD expression levels by shRNAs and PBGD overexpressing plasmid. PBGD down-regulation induced an elevation in ALAD activity, while overexpression of PBGD reduced ALAD activity, indicating a novel regulation feedback of PBGD on ALAD activity. This feedback mechanism enabled partial PpIX synthesis under PBGD silencing, whereas ALAD silencing reduced PpIX production to a minimum. ALA-PDT efficacy was directly correlated to PpIX levels. Thus, only ALAD-silenced cells were not affected by ALA+ irradiation, while following PBGD silencing, the accumulated PpIX, though decreased, was sufficient for successful ALA-PDT. The alterations in ALAD activity level initiated by changes in PBGD expression indicates PBGD's central role in heme synthesis. This enables efficient ALA-PDT, even when PBGD is not fully active. Conversely, ALAD loss resulted in reduced PpIX synthesis and consequently failure in ALA-PDT, due to the absence of compensation mechanism for ALAD.

Silencing of ALA dehydratase affects ALA-photodynamic therapy efficacy in K562 erythroleukemic cells.

Synthesis of protoporphyrin IX (PpIX) by malignant cells is essential for the success of ALA-based photodynamic therapy (PDT). Two key enzymes that were described as affecting PpIX accumulation during ALA treatment are porphobilinogen deaminase (PBGD) and ferrochelatase. Here, we show that down regulation of ALA dehydratase (ALAD) expression and activity by specific shRNA induced a marked decrease in PpIX synthesis in K562 erythroleukemic cells. Photo-inactivation efficacy following ALA-PDT was directly correlated with ALAD-silencing and cellular levels of PpIX. MTT metabolism following ALA-PDT was shown to be 60% higher in ALAD-silenced cells in comparison to control cells, indicating that mitochondria were protected in the silenced cells. Morphological analysis by scanning electron microscopy (SEM) of cells treated by ALA-PDT showed no morphological changes in ALAD-silenced cells, in contrast to controls exhibiting cell deformations and lysis. Membrane integrity following ALA-PDT was kept intact and undamaged in ALAD-silenced cells as examined by Annexin V-FITC/PI staining and LDH-L leakage. We conclude that ALAD, although it is present in the cell at abundant levels, has a major and limiting role in regulating PpIX synthesis and ALA-PDT outcome.