Summary
Serial assessments of measurable (or minimal) residual disease (MRD) by qPCR may identify nascent relapse in children with acute myeloid leukaemia (AML) and enable pre‐emptive therapy. We ...investigated the kinetics and prognostic impact of recurrent fusion transcripts (RUNX1‐RUNX1T1, CBFB‐MYH11, KMT2A‐MLLT3 or KMT2A‐ELL) in 774 post‐induction samples from bone marrow (BM, 347) and peripheral blood (PB, 427) from 75 children with AML. BM MRD persistence during consolidation did not increase the risk of relapse, and MRD at therapy completion did not correlate to outcome (HR = 0·64/MRD log reduction (CI: 0·32–1·26), P = 0·19). In contrast, 8/8 patients with detectable MRD in PB after first consolidation relapsed. Persistence (n = 4) and shifting from negative to positive (n = 10) in PB during follow‐up predicted relapse in 14/14 patients. All 253 PB samples collected during follow‐up from 36 patients in continuous complete remission were MRD negative. In core‐binding factor AML, persistent low‐level MRD positivity in BM during follow‐up was frequent but an increment to above 5 × 10−4 heralded subsequent haematological relapse in 12/12 patients. We demonstrate that MRD monitoring in PB after induction therapy is highly informative and propose an MRD increment above 5 × 10−4 in PB and BM as a definition of molecular relapse since it always leads to haematological relapse.
Pediatric AML is characterized by numerous genetic aberrations (chromosomal translocations, deletions, insertions) impacting its classification for risk of treatment failure. Aberrations are ...described by classical cytogenetic procedures (karyotyping, FISH), which harbor limitations (low resolution, need for cell cultivation, cost-intensiveness, experienced staff required). Optical Genome Mapping (OGM) is an emerging chip-based DNA technique combining high resolution (~500 bp) with a relatively short turnaround time. Twenty-four pediatric patients with AML, bi-lineage leukemia, and mixed-phenotype acute leukemia were analyzed by OGM, and the results were compared with cytogenetics. Results were discrepant in 17/24 (70%) cases, including 32 previously unknown alterations called by OGM only. One newly detected deletion and two translocations were validated by primer walking, breakpoint-spanning PCR, and DNA sequencing. As an added benefit, in two cases, OGM identified a new minimal residual disease (MRD) marker. Comparing impact on risk stratification in de novo AML, 19/20 (95%) cases had concordant results while only OGM unraveled another high-risk aberration. Thus, OGM considerably expands the methodological spectrum to optimize the diagnosis of pediatric AML via the identification of new aberrations. Results will contribute to a better understanding of leukemogenesis in pediatric AML. In addition, aberrations identified by OGM may provide markers for MRD monitoring.
ABSTRACTOxidative stress and inflammation are important pathogenetic mechanisms in amyotrophic lateral sclerosis (ALS). Nuclear erythroid 2-related factor 2 (Nrf2) is a basic region leucine-zipper ...transcription factor that binds to the antioxidant response element, thereby regulating the expression of many genes that are involved in cellular antioxidant and anti-inflammatory defense. Under normal conditions, Nrf2 activation is inhibited by Kelch-like ECH-associated protein 1 (Keap1). We investigated the potential involvement of the Nrf2/antioxidant response element signaling pathway in the selective degeneration of motor neurons in ALS. Nrf2 and Keap1 expression was analyzed in primary motor cortex and spinal cord postmortem tissue samples from ALS patients and controls by in situ hybridization histochemistry, quantitative real-time polymerase chain reaction, immunohistochemistry, and Western blot analysis. In ALS samples, there was a reduction of Nrf2 mRNA and protein expression in neurons, whereas Keap1 mRNA expression was increased in the motor cortex. These results suggest that alterations in this signaling cascade occur in motor neurons in ALS and that they may contribute to chronic motor neuron degeneration.
Acute myeloid leukemia (AML) is a heterogeneous malignant disease characterized by a collection of genetic and epigenetic changes. As a consequence, AML can evolve towards more aggressive subtypes ...during treatment, which require additional therapies to prevent future relapse. As we have previously detected double-stranded DNA (dsDNA) in tumor-derived extracellular vesicles (EVs), in this current study we attempted to evaluate the potential diagnostic applications of AML EV-dsDNA derived from primary bone marrow and peripheral blood plasma samples. EVs from plasma of 29 pediatric AML patients (at initial diagnosis or during treatment) were isolated by ultracentrifugation, after which dsDNA was extracted from obtained EVs and analyzed for leukemia-specific mutations using next generation sequencing (NGS) and GeneScan-based fragment-length analysis. In 18 out of 20 patients, dsDNA harvested from EVs mirrored the (leukemia-specific) mutations found in the genomic DNA obtained from primary leukemia cells. In the nanoparticle tracking analysis (NTA), a decrease in EV numbers was observed in patients after treatment compared with initial diagnosis. Following treatment, in 75 samples out of the 79, these mutations were no longer detectable in EV-dsDNA. In light of our results, we propose the use of leukemia-derived EV-dsDNA as an additional measure for mutational status and, potentially, treatment response in pediatric AML.
With an incidence of ~50%, the absence or reduced protein level of p53 is much more common than TP53 mutations in acute myeloid leukemia (AML). AML with FLT3-ITD (internal tandem duplication) ...mutations has an unfavorable prognosis and is highly associated with wt-p53 dysfunction. While TP53 mutation in the presence of FLT3-ITD does not induce AML in mice, it is not clear whether p53 haploinsufficiency or loss cooperates with FLT3-ITD in the induction of AML. Here, we generated FLT3-ITD knock-in; p53 knockout (heterozygous and homozygous) double-transgenic mice and found that both alterations strongly cooperated in the induction of cytogenetically normal AML without increasing the self-renewal potential. At the molecular level, we found the strong upregulation of Htra3 and the downregulation of Lin28a, leading to enhanced proliferation and the inhibition of apoptosis and differentiation. The co-occurrence of Htra3 overexpression and Lin28a knockdown, in the presence of FLT3-ITD, induced AML with similar morphology as leukemic cells from double-transgenic mice. These leukemic cells were highly sensitive to the proteasome inhibitor carfilzomib. Carfilzomib strongly enhanced the activity of targeting AXL (upstream of FLT3) against murine and human leukemic cells. Our results unravel a unique role of p53 haploinsufficiency or loss in the development of FLT3-ITD + AML.
Recently, studies in adults with acute promyelocytic leukemia (APL) showed high cure rates in low‐risk patients treated with all‐trans retinoid acid (ATRA) and arsenic trioxide (ATO), while ...toxicities were significantly reduced compared to the standard treatment with ATRA and chemotherapy. Here we report about first experience with 11 pediatric patients with low‐risk APL treated with ATRA and ATO. All patients stayed in molecular remission. All suffered from hyperleukocytosis. Two patients experienced reversible severe side effects. One suffered from osteonecroses at both femurs, seizures, as well as posterior reversible encephalopathy syndrome, the other patient had an abducens paresis.
Background
The small portion of leukemic stem cells (LSCs) in acute myeloid leukemia (AML) present in children and adolescents is often masked by the high background of AML blasts and normal ...hematopoietic cells. The aim of the current study was to establish a simple workflow for reliable genetic analysis of single LSC‐enriched blasts from pediatric patients.
Procedure
For three AMLs with mutations in nucleophosmin 1 and/or fms‐like tyrosine kinase 3, we performed whole genome amplification on sorted single‐cell DNA followed by whole exome sequencing (WES). The corresponding bulk bone marrow DNAs were also analyzed by WES and by targeted sequencing (TS) that included 54 genes associated with myeloid malignancies.
Results
Analysis revealed that read coverage statistics were comparable between single‐cell and bulk WES data, indicating high‐quality whole genome amplification. From 102 single‐cell variants, 72 single nucleotide variants and insertions or deletions (70%) were consistently found in the two bulk DNA analyses. Variants reliably detected in single cells were also present in TS. However, initial screening by WES with read counts between 50–72× failed to detect rare AML subclones in the bulk DNAs.
Conclusions
In summary, our study demonstrated that single‐cell WES combined with bulk DNA TS is a promising tool set for detecting AML subclones and possibly LSCs.
Fanconi anemia (FA) due to biallelic mutations in the BRCA2 gene is very rare and associated with an extremely high risk of early-onset of aggressive childhood malignancies, predominantly brain ...tumors, leukemia, and nephroblastoma. Here, we present a consanguineous family with three affected children of the D1 subtype of FA and describe the clinical consequences of the earliest known biallelic nonsense/stop-gain germ-line mutation in BRCA2, exon 5 c.469A>T, that leads to a premature stop of translation, p.Lys157*. The three patients were born with severe intrauterine growth restrictions and different degrees of congenital malformations. Altogether, they developed eight distinct malignancies and died within their first three years of life. Treatment with a reduced chemotherapy regimen was only performed in patient 2 for his first tumor, a nephroblastoma, which the patient tolerated well for eight months, until he developed myelodysplastic syndrome (MDS) and then acute myeloid leukemia (AML). Finally, the third patient experienced a hepatoblastoma, an unclassified brain tumor and MDS in parallel and died in her second year of life. Our report re-emphasizes the aggressiveness and fatality of the FA-D1 children with biallelic BRCA2 nonsense mutations, that are both located before exon 11, which contains binding domains for the RAD51 recombinase.
•A biallelic nonsense mutation in BRCA2 exon 5 is associated with severe FA type D1.•The biallelic BRCA2 mutation was associated with hematological and solid cancers.•Three FANCD1 children experienced seven malignancies within three years of life.•Malignancies in FANCD1 patients can be treated with non-DNA alkylating chemotherapy.
Acute myeloid leukemia is a life-threatening malignancy in children and adolescents treated predominantly by risk-adapted intensive chemotherapy that is partly supported by allogeneic stem cell ...transplantation. Mutations in the WT1 gene and NUP98-NSD1 fusion are predictors of poor survival outcome/prognosis that frequently occur in combination with internal tandem duplications of the juxta-membrane domain of FLT3 (FLT3-ITD). To re-evaluate the effect of these factors in contemporary protocols, 353 patients (<18 years) treated in Germany with AML-BFM treatment protocols between 2004 and 2017 were included. Presence of mutated WT1 and FLT3-ITD in blasts (n=19) resulted in low 3-year event-free survival of 29% and overall survival of 33% compared to rates of 45-63% and 67-87% in patients with only one (only FLT3-ITD; n=33, only WT1 mutation; n=29) or none of these mutations (n=272). Including NUP98-NSD1 and high allelic ratio (AR) of FLT3-ITD (AR ≥0.4) in the analysis revealed very poor outcomes for patients with co-occurrence of all three factors or any of double combinations. All these patients (n=15) experienced events and the probability of overall survival was low (27%). We conclude that co-occurrence of WT1 mutation, NUP98-NSD1, and FLT3-ITD with an AR ≥0.4 as triple or double mutations still predicts dismal response to contemporary first- and second-line treatment for pediatric acute myeloid leukemia.
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