Panel-based RNA fusion sequencing improves diagnostics of pediatric acute myeloid leukemia.

New methods like panel-based RNA fusion sequencing (RNA-FS) promise improved diagnostics in various malignancies. We here analyzed the impact of RNA-FS on the initial diagnostics of 241 cases with pediatric acute myeloid leukemia (AML). We show that, compared to classical cytogenetics (CCG), RNA-FS reliably detected risk-relevant fusion genes in pediatric AML. In addition, RNA-FS strongly improved the detection of cryptic fusion genes like NUP98::NSD1, KMT2A::MLLT10 and CBFA2T3::GLIS2 and thereby resulted in an improved risk stratification in 25 patients (10.4%). Validation of additionally detected non-risk-relevant high confidence fusion calls identified PIM3::BRD1, C22orf34::BRD1, PSPC1::ZMYM2 and ARHGAP26::NR3C1 as common genetic variants and MYB::GATA1 as recurrent aberration, which we here describe in AML subtypes M0 and M7 for the first time. However, it failed to detect rare cytogenetically confirmed fusion events like MNX1::ETV6 and other chromosome 12p-abnormalities. As add-on benefit, the proportion of patients for whom measurable residual disease (MRD) monitoring became possible was increased by RNA-FS from 44.4 to 75.5% as the information on the fusion transcripts' sequence allowed the design of new MRD assays.

Toward Cytogenomics: Technical Assessment of Long-Read Nanopore Whole-Genome Sequencing for Detecting Large Chromosomal Alterations in Mantle Cell Lymphoma.

The current advances and success of next-generation sequencing hold the potential for the transition of cancer cytogenetics toward comprehensive cytogenomics. However, the conventional use of short reads impedes the resolution of chromosomal aberrations with current next-generation sequencing modalities. Thus, this study evaluated the detection and reproducibility of extensive copy number alterations and chromosomal translocations using long-read Oxford Nanopore Technologies whole-genome sequencing compared with short-read Illumina sequencing. On the basis of the mantle cell lymphoma cell line Granta-519, almost 99% copy number reproducibility at the 100-kilobase resolution between replicates was demonstrated, with 98% concordance to Illumina. Collectively, the performance of copy number calling from 1.5 million to 7.5 million long reads was comparable to 1 billion Illumina-based reads (50× coverage). Expectedly, the long-read resolution of canonical translocation t(11; 14) (q13; q32) was superior, with a sequence similarity of 89% to the already published CCND1/IGH junction (9× coverage), spanning up to 69 kilobases. The cytogenetic profile of Granta-519 was in general agreement with the literature and karyotype, although several differences remained unresolved. In conclusion, contemporary long-read sequencing is primed for future cytogenomics or sequencing-guided cytogenetics. The combined strength of long- and short-read sequencing is apparent, where the high-precision junctional mapping complements and splits paired-end reads. The potential is emphasized by the flexible single-sample genomic data acquisition of Oxford Nanopore Technologies with the high resolution of allelic imbalances using Illumina short-read sequencing.

RUNX1 mutation has no prognostic significance in paediatric AML: a retrospective study of the AML-BFM study group.

In acute myeloid leukaemia (AML) RUNX1 mutation is characterised by certain clinicopathological features with poor prognosis and adverse risk by the European LeukemiaNet recommendation. Though initially considered as provisional category, the recent World Health Organisation (WHO) classification of 2022 removed RUNX1-mutated AML from the unique entity. However, the significance of RUNX1 mutation in paediatric AML remains unclear. We retrospectively analysed a German cohort of 488 paediatric patients with de novo AML, enroled in the AMLR12 or AMLR17 registry of the AML-BFM Study Group (Essen, Germany). A total of 23 paediatric AML patients (4.7%) harboured RUNX1 mutations, 18 of which (78%) had RUNX1 mutation at initial diagnosis. RUNX1 mutations were associated with older age, male gender, number of coexisting alterations and presence of FLT3-ITD but mutually exclusive of KRAS, KIT and NPM1 mutation. RUNX1 mutations did not prognostically impact overall or event-free survival. Response rates did not differ between patients with and without RUNX1 mutations. This comprehensive study, comprising the largest analysis of RUNX1 mutation in a paediatric cohort to date, reveals distinct but not unique clinicopathologic features, with no prognostic significance of RUNX1-mutated paediatric AML. These results broaden the perspective on the relevance of RUNX1 alterations in leukaemogenesis in AML.

Circulating cell-free messenger RNA enables non-invasive pan-tumour monitoring of melanoma therapy independent of the mutational genotype.

BACKGROUND: Plasma-derived tumour-specific cell-free nucleic acids are increasingly utilized as a minimally invasive, real-time biomarker approach in many solid tumours. Circulating tumour DNA of melanoma-specific mutations is currently the best studied liquid biopsy biomarker for melanoma. However, the combination of hotspot genetic alterations covers only around 80% of all melanoma patients. Therefore, alternative approaches are needed to enable the follow-up of all genotypes, including wild-type. METHODS: We identified KPNA2, DTL, BACE2 and DTYMK messenger RNA (mRNA) upregulated in melanoma versus nevi tissues by unsupervised data mining (N = 175 melanoma, N = 20 normal skin, N = 6 benign nevi) and experimentally confirmed differential mRNA expression in vitro (N = 18 melanoma, N = 8 benign nevi). Circulating cell-free RNA (cfRNA) was analysed in 361 plasma samples (collected before and during therapy) from 100 melanoma patients and 18 healthy donors. Absolute cfRNA copies were quantified on droplet digital PCR. RESULTS: KPNA2, DTL, BACE2 and DTYMK cfRNA demonstrated high diagnostic accuracy between melanoma patients' and healthy donors' plasma (AUC > 86%, p < .0001). cfRNA copies increased proportionally with increasing tumour burden independently of demographic variables and even remained elevated in individuals with radiological absence of disease. Re-analysis of single-cell transcriptomes revealed a pan-tumour origin of cfRNA, including endothelial, cancer-associated fibroblasts, macrophages and B cells beyond melanoma cells as cellular sources. Low baseline cfRNA levels were associated with significantly longer progression-free survival (PFS) (KPNA2 HR = .54, p = .0362; DTL HR = .60, p = .0349) and overall survival (KPNA2 HR = .52, p = .0237; BACE2 HR = .55, p = .0419; DTYMK HR = .43, p = .0393). Lastly, we found that cfRNA copies significantly increased during therapy in non-responders compared to responders regardless of therapy and mutational subtypes and that the increase of KPNA2 (HR = 1.73, p = .0441) and DTYMK (HR = 1.82, p = .018) cfRNA during therapy was predictive of shorter PFS. CONCLUSIONS: In sum, we identified a new panel of cfRNAs for a pan-tumour liquid biopsy approach and demonstrated its utility as a prognostic, therapy-monitoring tool independent of the melanoma mutational genotype.

Molecular Measurable Residual Disease Assessment before Hematopoietic Stem Cell Transplantation in Pediatric Acute Myeloid Leukemia Patients: A Retrospective Study by the I-BFM Study Group.

Hematopoietic stem cell transplantation (HSCT) is a curative post-remission treatment in patients with acute myeloid leukemia (AML), but relapse after transplant is still a challenging event. In recent year, several studies have investigated the molecular minimal residual disease (qPCR-MRD) as a predictor of relapse, but the lack of standardized protocols, cut-offs, and timepoints, especially in the pediatric setting, has prevented its use in several settings, including before HSCT. Here, we propose the first collaborative retrospective I-BFM-AML study assessing qPCR-MRD values in pretransplant bone marrow samples of 112 patients with a diagnosis of AML harboring t(8;21)(q22; q22)RUNX1::RUNX1T1, or inv(16)(p13q22)CBFB::MYH11, or t(9;11)(p21;q23)KMT2A::MLLT3, or FLT3-ITD genetic markers. We calculated an ROC cut-off of 2.1 × 10-4 that revealed significantly increased OS (83.7% versus 57.1%) and EFS (80.2% versus 52.9%) for those patients with lower qPCR-MRD values. Then, we partitioned patients into three qPCR-MRD groups by combining two different thresholds, 2.1 × 10-4 and one lower cut-off of 1 × 10-2, and stratified patients into low-, intermediate-, and high-risk groups. We found that the 5-year OS (83.7%, 68.6%, and 39.2%, respectively) and relapse-free survival (89.2%, 73.9%, and 67.9%, respectively) were significantly different independent of the genetic lesion, conditioning regimen, donor, and stem cell source. These data support the PCR-based approach playing a clinical relevance in AML transplant management.

Efficient Small Extracellular Vesicles (EV) Isolation Method and Evaluation of EV-Associated DNA Role in Cell-Cell Communication in Cancer.

Small extracellular vesicles (sEVs) play essential roles in intercellular signaling both in normal and pathophysiological conditions. Comprehensive studies of dsDNA associated with sEVs are hampered by a lack of methods, allowing efficient separation of sEVs from free-circulating DNA and apoptotic bodies. In this work, using controlled culture conditions, we enriched the reproducible separation of sEVs from free-circulated components by combining tangential flow filtration, size-exclusion chromatography, and ultrafiltration (TSU). EV-enriched fractions (F2 and F3) obtained using TSU also contained more dsDNA derived from the host genome and mitochondria, predominantly localized inside the vesicles. Three-dimensional reconstruction of high-resolution imaging showed that the recipient cell membrane barrier restricts a portion of EV-DNA. Simultaneously, the remaining EV-DNA overcomes it and enters the cytoplasm and nucleus. In the cytoplasm, EV-DNA associates with dsDNA-inflammatory sensors (cGAS/STING) and endosomal proteins (Rab5/Rab7). Relevant to cancer, we found that EV-DNA isolated from leukemia cell lines communicates with mesenchymal stromal cells (MSCs), a critical component in the BM microenvironment. Furthermore, we illustrated the arrangement of sEVs and EV-DNA at a single vesicle level using super-resolution microscopy. Altogether, employing TSU isolation, we demonstrated EV-DNA distribution and a tool to evaluate the exact EV-DNA role of cell-cell communication in cancer.

Pediatric T-ALL type-1 and type-2 relapses develop along distinct pathways of clonal evolution.

The mechanisms underlying T-ALL relapse remain essentially unknown. Multilevel-omics in 38 matched pairs of initial and relapsed T-ALL revealed 18 (47%) type-1 (defined by being derived from the major ancestral clone) and 20 (53%) type-2 relapses (derived from a minor ancestral clone). In both types of relapse, we observed known and novel drivers of multidrug resistance including MDR1 and MVP, NT5C2 and JAK-STAT activators. Patients with type-1 relapses were specifically characterized by IL7R upregulation. In remarkable contrast, type-2 relapses demonstrated (1) enrichment of constitutional cancer predisposition gene mutations, (2) divergent genetic and epigenetic remodeling, and (3) enrichment of somatic hypermutator phenotypes, related to BLM, BUB1B/PMS2 and TP53 mutations. T-ALLs that later progressed to type-2 relapses exhibited a complex subclonal architecture, unexpectedly, already at the time of initial diagnosis. Deconvolution analysis of ATAC-Seq profiles showed that T-ALLs later developing into type-1 relapses resembled a predominant immature thymic T-cell population, whereas T-ALLs developing into type-2 relapses resembled a mixture of normal T-cell precursors. In sum, our analyses revealed fundamentally different mechanisms driving either type-1 or type-2 T-ALL relapse and indicate that differential capacities of disease evolution are already inherent to the molecular setup of the initial leukemia.

A unique role of p53 haploinsufficiency or loss in the development of acute myeloid leukemia with FLT3-ITD mutation.

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.

Impact of KMT2A Rearrangement and CSPG4 Expression in Pediatric Acute Myeloid Leukemia.

KMT2A rearrangements (KMT2A-r) are among the most common structural aberrations in pediatric acute myeloid leukemia (AML) and are very important for the risk group stratification of patients. Here, we report the outcome of 967 pediatric AML patients with a known KMT2A-r status. The large cohort was characterized by morphology, multicolor flow cytometry, classical cytogenetics and mutation analysis via panel sequencing. In total, the blasts of 241 patients (24.9%) showed KMT2A-r. KMT2A-r is associated with FAB M5, a high white blood cell count and younger age at diagnosis. When subgroups were combined, KMT2A-r had no impact on event-free survival (EFS) and overall survival (OS); however, various subgroups showed a different prognosis, ranging from a <50% OS for KMT2A/AFDN (n = 11) to a 100% chance of survival for patients harboring the rare translocation KMT2A/SEPTIN9 (n = 3, follow up of 3.7 to 9.6 years). A positive correlation of KMT2A-r with KRAS mutations (p < 0.001) existed, albeit without any prognostic impact. In addition, FLT3-ITDs were detected less frequently in AML with KMT2A-r (p < 0.001). Furthermore, KMT2A-r were mutually exclusive, with mutations in NPM1 (p = 0.002), KIT (p = 0.036), WT1 (p < 0.001) and CEBPA (p = 0.006), and translocations NUP98/NSD1 (p = 0.009), RUNX1/RUNX1T1 (p = 0.003) and CBFB/MYH11 (p = 0.006). In the 346 patients tested for CSPG4 expression, a correlation between CSPG4 expression and KMT2A-r was confirmed. However, CSPG4 expression also occurred in patients without KMT2A-r and had no significant prognostic impact on EFS and OS.

Advisory opinion of the AWMF Ad hoc Commission In-vitro Diagnostic Medical Devices regarding in-vitro diagnostic medical devices manufactured and used only within health institutions established in the Union according to Regulation (EU) 2017/746 (IVDR).

In view of the approaching application date of Regulation (EU) 2017/746 ("IVDR") and the resulting EU-wide, harmonized requirements for in-vitro diagnostic medical devices (IVD) manufactured and used within European health institutions, the Ad hoc Commission IVD of the German Association of the Scientific Medical Societies (AWMF) takes a national position on the details of the requirements and conditions related to the use of these IVD products. The Ad hoc Commission IVD emphasizes the relevance of examination procedures developed in medical laboratories, especially in the field of orphan diseases and new diagnostic markers. The IVDR sets an adequate regulatory framework for IVD manufactured and used within health institutions as long as these requirements are fulfilled with reliability and in accordance with the current state of the art in medical laboratory sciences. At the same time, the IVDR requirements have to be regarded under a pragmatic view and in accordance with the quality management systems approved within the different EU Member States. On the one hand, the mandatory requirements of the RiLiBÄK play an essential role in Germany. On the other hand, elements of voluntarily applicable international standards may support the fulfilment of product requirements for safety and performance according to Annex I of the IVDR. Both the complexity and possible solutions for the implementation of the IVDR requirements are discussed on the basis of examples such as the required documentation, performance evaluation and software validation. The Ad hoc Commission IVD recommends that, while aiming at a preferably EU-wide harmonized interpretation of the IVDR requirements, the flexibility in medical laboratory diagnostics necessary for patient care, including the use of IVD from in-house production, should be emphasized.

A homozygous nonsense mutation early in exon 5 of BRCA2 is associated with very severe Fanconi anemia.

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.

Second Relapse of Pediatric Patients with Acute Myeloid Leukemia: A Report on Current Treatment Strategies and Outcome of the AML-BFM Study Group.

Successful management of relapse is critical to improve outcomes of children with acute myeloid leukemia (AML). We evaluated response, survival and prognostic factors after a second relapse of AML. Among 1222 pediatric patients of the population-based AML-Berlin-Frankfurt-Munster (BFM) study group (2004 until 2017), 73 patients met the quality parameters for inclusion in this study. Central review of source documentation warranted the accuracy of reported data. Treatment approaches included palliation in 17 patients (23%), intensive therapy with curative intent (n = 46, 63%) and other regimens (n = 10). Twenty-five patients (35%) received hematopoietic stem cell transplantation (HSCT), 21 of whom (88%) had a prior HSCT. Survival was poor, with a five-year probability of overall survival (pOS) of 15 ± 4% and 31 ± 9% following HSCT (n = 25). Early second relapse (within one year after first relapse) was associated with dismal outcome (pOS 2 ± 2%, n = 44 vs. 33 ± 9%, n = 29; p < 0.0001). A third complete remission (CR) is required for survival: 31% (n = 14) of patients with intensive treatment achieved a third CR with a pOS of 36 ± 13%, while 28 patients (62%) were non-responders (pOS 7 ± 5%). In conclusion, survival is poor but possible, particularly after a late second relapse and an intensive chemotherapy followed by HSCT. This analysis provides a baseline for future treatment planning.

Application of Circulating Cell-Free Tumor DNA Profiles for Therapeutic Monitoring and Outcome Prediction in Genetically Heterogeneous Metastatic Melanoma.

PURPOSE: Circulating cell-free tumor DNA (ctDNA) reflects the heterogeneous spectrum of tumor-specific mutations, especially in systemic disease. We validated plasma-based assays that allow the dynamic quantitative detection of ctDNA as a prognostic biomarker for tumor load and prediction of therapy response in melanoma. MATERIALS AND METHODS: We analyzed plasma-derived ctDNA from a large training cohort (n = 96) of patients with advanced-stage melanoma, with assays for the BRAF V600E and NRAS Q61 driver mutations as well as TERT C250T and TERT C228T promoter mutations. An independent patient cohort (n = 35) was used to validate the utility of ctDNA monitoring under mitogen-activated protein kinase-targeted or immune checkpoint therapies. RESULTS: Elevated plasma ctDNA level at baseline was an independent prognostic factor of disease progression when compared with serum S100 and lactate dehydrogenase levels in multivariable analyses (hazard ratio [HR], 7.43; 95% CI, 1.01 to 55.19; P = .05). The change in ctDNA levels during therapy correlated with treatment response, where increasing ctDNA was predictive for shorter progression-free survival (eg, for BRAF V600E ctDNA, HR, 3.70; 95% CI, 1.86 to 7.34; P < .001). Increasing ctDNA levels predicted disease progression significantly earlier than did routine radiologic scans (P < .05), with a mean lead time of 3.5 months. NRAS-mutant ctDNA was detected in a significant proportion of patients with BRAF-mutant tumors under therapy, but unexpectedly also at baseline. In vitro sensitivity studies suggested that this represents higher-than-expected intratumoral heterogeneity. The detection of NRAS Q61 ctDNA in baseline samples of patients with BRAF V600E mutation who were treated with mitogen-activated protein kinase inhibitors significantly correlated with shorter progression-free survival (HR, 3.18; 95% CI, 1.31 to 7.68; P = .03) and shorter overall survival (HR, 4.08; 95% CI, 1.57 to 10.58; P = .01). CONCLUSION: Our results show the potential role of ctDNA measurement as a sensitive monitoring and prediction tool for the early assessment of disease progression and therapeutic response in patients with metastatic melanoma.

Exposure of Patient-Derived Mesenchymal Stromal Cells to TGFB1 Supports Fibrosis Induction in a Pediatric Acute Megakaryoblastic Leukemia Model.

Bone marrow fibrosis (BMF) is a rare complication in acute leukemia. In pediatrics, it predominantly occurs in acute megakaryoblastic leukemia (AMKL) and especially in patients with trisomy 21, called myeloid leukemia in Down syndrome (ML-DS). Defects in mesenchymal stromal cells (MSC) and cytokines specifically released by the myeloid blasts are thought to be the main drivers of fibrosis in the bone marrow niche (BMN). To model the BMN of pediatric patients with AMKL in mice, we first established MSCs from pediatric patients with AMKL (n = 5) and ML-DS (n = 9). Healthy donor control MSCs (n = 6) were generated from unaffected children and adolescents ≤18 years of age. Steady-state analyses of the MSCs revealed that patient-derived MSCs exhibited decreased adipogenic differentiation potential and enrichment of proliferation-associated genes. Importantly, TGFB1 exposure in vitro promoted early profibrotic changes in all three MSC entities. To study BMF induction for longer periods of time, we created an in vivo humanized artificial BMN subcutaneously in immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice, using a mixture of MSCs, human umbilical vein endothelial cell, and Matrigel. Injection of AMKL blasts as producers of TGFB1 into this BMN after 8 weeks induced fibrosis grade I/II in a dose-dependent fashion over a time period of 4 weeks. Thus, our study developed a humanized mouse model that will be instrumental to specifically examine leukemogenesis and therapeutic targets for AMKL blasts in future. IMPLICATIONS: TGFB1 supports fibrosis induction in a pediatric AMKL model generated with patient-derived MSCs. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/18/10/1603/F1.large.jpg.

Measurable residual disease assessment by qPCR in peripheral blood is an informative tool for disease surveillance in childhood acute myeloid leukaemia.

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.

Phospho-Profiling Linking Biology and Clinics in Pediatric Acute Myeloid Leukemia.

Aberrant activation of key signaling-molecules is a hallmark of acute myeloid leukemia (AML) and may have prognostic and therapeutic implications. AML summarizes several disease entities with a variety of genetic subtypes. A comprehensive model spanning from signal activation patterns in major genetic subtypes of pediatric AML (pedAML) to outcome prediction and pre-clinical response to signaling inhibitors has not yet been provided. We established a high-throughput flow-cytometry based method to assess activation of hallmark phospho-proteins (phospho-flow) in 166 bone-marrow derived pedAML samples under basal and cytokine stimulated conditions. We correlated levels of activated phospho-proteins at diagnosis with relapse incidence in intermediate (IR) and high risk (HR) subtypes. In parallel, we screened a set of signaling inhibitors for their efficacy against primary AML blasts in a flow-cytometry based ex vivo cytotoxicity assay and validated the results in a murine xenograft model. Certain phospho-signal patterns differ between genetic subtypes of pedAML. Some are consistently seen through all AML subtypes such as pSTAT5. In IR/HR subtypes high levels of GM-CSF stimulated pSTAT5 and low levels of unstimulated pJNK correlated with increased relapse risk overall. Combination of GM-CSF/pSTAT5high and basal/pJNKlow separated three risk groups among IR/HR subtypes. Out of 10 tested signaling inhibitors, midostaurin most effectively affected AML blasts and simultaneously blocked phosphorylation of multiple proteins, including STAT5. In a mouse xenograft model of KMT2A-rearranged pedAML, midostaurin significantly prolonged disease latency. Our study demonstrates the applicability of phospho-flow for relapse-risk assessment in pedAML, whereas functional phenotype-driven ex vivo testing of signaling inhibitors may allow individualized therapy.

Evaluation of dsDNA from extracellular vesicles (EVs) in pediatric AML diagnostics.

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.

MTF1 binds to metal-responsive element e within the ATP7B promoter and is a strong candidate in regulating the ATP7B expression.

Wilson's disease is an autosomal recessive disorder resulting from copper excess. Some patients with clinical Wilson's disease symptoms exhibit no or only heterozygous pathogenic variants in the coding region of the disease-causing ATP7B gene. Therefore, the ATP7B promoter region is of special interest. Metal-responsive elements (MREs) located in the ATP7B promoter are promising motifs in modulating the ATP7B expression. We studied protein interaction of MREe, MREc, and MREd by electrophoretic mobility shift assays and revealed specific interactions for all MREs. We further narrowed down the specific binding site. Proteins potentially binding to the three MREs were identified by MatInspector analyses. Metal regulatory transcription factor 1 (MTF1) could be validated to bind to MREe by electrophoretic mobility shift assays. ATP7B promoter-driven reporter gene expression was significantly increased because of this interaction. MTF1 is a strong candidate in regulating the ATP7B expression through MREe binding.

Mutated WT1, FLT3-ITD, and NUP98-NSD1 Fusion in Various Combinations Define a Poor Prognostic Group in Pediatric Acute Myeloid Leukemia.

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.