Generation of human induced pluripotent stem cell line MHHi029-A from a male Fabry disease patient carrying c.959A > T mutation.

Fabry disease (FD) is a rare and inherited monogenetic disease caused by mutations in the X-chromosomal alpha-galactosidase A gene GLA concomitant with accumulation of its substrate globotriaosylceramide (Gb3) and multi-organ symptoms. We derived an induced pluripotent stem cell line, MHHi029-A, from a male FD patient carrying a c.959A > T missense mutation in the GLA gene. The hiPSCs show a normal karyotype, expression of pluripotency markers and trilineage differentiation capacity. Importantly, they present the patient-specific mutation in the GLA gene and are therefore a valuable resource for investigating the FD mechanism and identifying novel therapies.

Matrix-free human pluripotent stem cell manufacturing by seed train approach and intermediate cryopreservation.

BACKGROUND: Human pluripotent stem cells (hPSCs) have an enormous therapeutic potential, but large quantities of cells will need to be supplied by reliable, economically viable production processes. The suspension culture (three-dimensional; 3D) of hPSCs in stirred tank bioreactors (STBRs) has enormous potential for fuelling these cell demands. In this study, the efficient long-term matrix-free suspension culture of hPSC aggregates is shown. METHODS AND RESULTS: STBR-controlled, chemical aggregate dissociation and optimized passage duration of 3 or 4 days promotes exponential hPSC proliferation, process efficiency and upscaling by a seed train approach. Intermediate high-density cryopreservation of suspension-derived hPSCs followed by direct STBR inoculation enabled complete omission of matrix-dependent 2D (two-dimensional) culture. Optimized 3D cultivation over 8 passages (32 days) cumulatively yielded ≈4.7 × 1015 cells, while maintaining hPSCs' pluripotency, differentiation potential and karyotype stability. Gene expression profiling reveals novel insights into the adaption of hPSCs to continuous 3D culture compared to conventional 2D controls. CONCLUSIONS: Together, an entirely matrix-free, highly efficient, flexible and automation-friendly hPSC expansion strategy is demonstrated, facilitating the development of good manufacturing practice-compliant closed-system manufacturing in large scale.

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.

Fludarabine, cytarabine, and idarubicin with or without venetoclax in patients with relapsed/refractory acute myeloid leukemia.

Treatment options for relapsed and refractory acute myeloid leukemia patients (R/R AML) are limited. This retrospective cohort study compares safety and efficacy of fludarabine, cytarabine, and idarubicin (FLA-IDA) without or with venetoclax (FLAVIDA) in patients with R/R AML. Thirty-seven and 81 patients received one course FLA-IDA with or without a 7-day course of venetoclax, respectively. The overall response rate (ORR) was significantly higher in FLAVIDA compared to FLAIDA- treated patients (78% vs. 47%; P=0.001), while measurable residual disease was negative at a similar proportion in responding patients (50% vs. 57%), respectively. Eighty-one percent and 79% of patients proceeded to allogeneic hematopoietic cell transplantation or donor lymphocyte infusion after FLAVIDA and FLA-IDA, respectively. Event-free and overall survival were similar in FLAVIDA- and FLA-IDA-treated patients. Refractory patients could be salvaged more successfully after FLA-IDA compared to FLAVIDA pretreatment. Neutrophil and platelet recovery times were similar in the venetoclax and the control group. In conclusion, short-term venetoclax in combination with FLA-IDA represents an effective treatment regimen in R/R AML identifying chemosensitive patients rapidly and inducing measurable residual disease-negative remission in a high proportion of R/R AML patients.

Spontaneous remission and loss of monosomy 7: a window of opportunity for young children with SAMD9L syndrome.

Monosomy 7 is the most common cytogenetic abnormality in pediatric myelodysplastic syndrome (MDS) and associated with a high risk of disease progression. However, in young children, spontaneous loss of monosomy 7 with concomitant hematologic recovery has been described, especially in the presence of germline mutations in SAMD9 and SAMD9L genes. Here, we report on our experience of close surveillance instead of upfront hematopoietic stem cell transplantation (HSCT) in seven patients diagnosed with SAMD9L syndrome and monosomy 7 at a median age of 0.6 years (range, 0.4-2.9). Within 14 months from diagnosis, three children experienced spontaneous hematological remission accompanied by a decrease in monosomy 7 clone size. Subclones with somatic SAMD9L mutations in cis were identified in five patients, three of whom attained hematological remission. Two patients acquired RUNX1 and EZH2 mutations during the observation period, of whom one progressed to myelodysplastic syndrome with excess of blasts (MDS-EB). Four patients underwent allogeneic HSCT at a median time of 26 months (range, 14-40) from diagnosis for MDSEB, necrotizing granulomatous lymphadenitis, persistent monosomy 7, and severe neutropenia. At last follow-up, six patients were alive, while one passed away due to transplant-related causes. These data confirm previous observations that monosomy 7 can be transient in young children with SAMD9L syndrome. However, they also indicate that delaying HSCT poses a substantial risk of severe infection and disease progression. Finally, surveillance of patients with SAMD9L syndrome and monosomy 7 is critical to define the evolving genetic landscape and to determine the appropriate timing of HSCT (clinicaltrials gov. Identifier: NCT00662090).

Germ line variant GFI1-36N affects DNA repair and sensitizes AML cells to DNA damage and repair therapy.

ABSTRACT: Growth factor independence 1 (GFI1) is a DNA-binding transcription factor and a key regulator of hematopoiesis. GFI1-36N is a germ line variant, causing a change of serine (S) to asparagine (N) at position 36. We previously reported that the GFI1-36N allele has a prevalence of 10% to 15% among patients with acute myeloid leukemia (AML) and 5% to 7% among healthy Caucasians and promotes the development of this disease. Using a multiomics approach, we show here that GFI1-36N expression is associated with increased frequencies of chromosomal aberrations, mutational burden, and mutational signatures in both murine and human AML and impedes homologous recombination (HR)-directed DNA repair in leukemic cells. GFI1-36N exhibits impaired binding to N-Myc downstream-regulated gene 1 (Ndrg1) regulatory elements, causing decreased NDRG1 levels, which leads to a reduction of O6-methylguanine-DNA-methyltransferase (MGMT) expression levels, as illustrated by both transcriptome and proteome analyses. Targeting MGMT via temozolomide, a DNA alkylating drug, and HR via olaparib, a poly-ADP ribose polymerase 1 inhibitor, caused synthetic lethality in human and murine AML samples expressing GFI1-36N, whereas the effects were insignificant in nonmalignant GFI1-36S or GFI1-36N cells. In addition, mice that received transplantation with GFI1-36N leukemic cells treated with a combination of temozolomide and olaparib had significantly longer AML-free survival than mice that received transplantation with GFI1-36S leukemic cells. This suggests that reduced MGMT expression leaves GFI1-36N leukemic cells particularly vulnerable to DNA damage initiating chemotherapeutics. Our data provide critical insights into novel options to treat patients with AML carrying the GFI1-36N variant.

Generation of two human NRF2 knockout iPSC clones using CRISPR/Cas9 editing.

The nuclear factor erythroid 2-related factor 2 (NFE2L2, known as NRF2) regulates the expression of antioxidative and anti-inflammatory proteins. In order to investigate its impact during viral infections and testing of antiviral compounds, we applied CRISPR/Cas9 editing to eliminate NRF2 in the human iPS cell line MHHi001-A and generated two NRF2 knockout iPSC clones MHHi001-A-6 and MHHi001-A-7. After differentiation into epithelia or endothelial cells, these cells are useful tools to examine the antiviral effects of activators of the NRF2 signaling pathway.

European standard clinical practice - Key issues for the medical care of individuals with familial leukemia.

Although hematologic malignancies (HM) are no longer considered exclusively sporadic, additional awareness of familial cases has yet to be created. Individuals carrying a (likely) pathogenic germline variant (e.g., in ETV6, GATA2, SAMD9, SAMD9L, or RUNX1) are at an increased risk for developing HM. Given the clinical and psychological impact associated with the diagnosis of a genetic predisposition to HM, it is of utmost importance to provide high-quality, standardized patient care. To address these issues and harmonize care across Europe, the Familial Leukemia Subnetwork within the ERN PaedCan has been assigned to draft an European Standard Clinical Practice (ESCP) document reflecting current best practices for pediatric patients and (healthy) relatives with (suspected) familial leukemia. The group was supported by members of the German network for rare diseases MyPred, of the Host Genome Working Group of SIOPE, and of the COST action LEGEND. The ESCP on familial leukemia is proposed by an interdisciplinary team of experts including hematologists, oncologists, and human geneticists. It is intended to provide general recommendations in areas where disease-specific recommendations do not yet exist. Here, we describe key issues for the medical care of familial leukemia that shall pave the way for a future consensus guideline: (i) identification of individuals with or suggestive of familial leukemia, (ii) genetic analysis and variant interpretation, (iii) genetic counseling and patient education, and (iv) surveillance and (psychological) support. To address the question on how to proceed with individuals suggestive of or at risk of familial leukemia, we developed an algorithm covering four different, partially linked clinical scenarios, and additionally a decision tree to guide clinicians in their considerations regarding familial leukemia in minors with HM. Our recommendations cover, not only patients but also relatives that both should have access to adequate medical care. We illustrate the importance of natural history studies and the need for respective registries for future evidence-based recommendations that shall be updated as new evidence-based standards are established.

iPSC-Derived Striatal Medium Spiny Neurons from Patients with Multiple System Atrophy Show Hypoexcitability and Elevated α-Synuclein Release.

Multiple system atrophy of the parkinsonian type (MSA-P) is a rare, fatal neurodegenerative disease with sporadic onset. It is still unknown if MSA-P is a primary oligodendropathy or caused by neuronal pathophysiology leading to severe, α-synuclein-associated neurodegeneration, mainly in the striatum. In this study, we generated and differentiated induced pluripotent stem cells (iPSCs) from patients with the clinical diagnosis of probable MSA-P (n = 3) and from three matched healthy controls into GABAergic striatal medium spiny neurons (MSNs). We found a significantly elevated release and neuronal distribution for α-synuclein, as well as hypoexcitability in the MSNs derived from the MSA-P patients compared to the healthy controls. These data suggest that the striatal hypoexcitable neurons of MSA-P patients contribute to a pathological α-synuclein burden which is likely to spread to neighboring cells and projection targets, facilitating disease progression.

Rare and potentially fatal - Cytogenetically cryptic TNIP1::PDGFRB and PCM1::FGFR1 fusion leading to myeloid/lymphoid neoplasms with eosinophilia in children.

Myeloid/lymphoid neoplasms with eosinophilia (MLN-eos) are rare haematological neoplasms primarily affecting adults. The heterogeneous clinical picture and the rarity of the disease, especially in children, may delay an early diagnosis. MLN-eos are characterized by constitutive tyrosine kinase (TK) activity due to gene fusions. It is thus of importance to obtain a prompt genetic diagnosis to start a specific therapy. Here, we outline the clinical, genetic, and biochemical background of TK driven MLN-eos and report two extremely rare paediatric cases of MLN-eo, the used diagnostic methods, therapy and clinical outcomes. Our results demonstrate that, standard cytogenetic and molecular methods may not be sufficient to diagnose MLN-eo due to cytogenetically cryptic aberrations. We therefore recommend performing additional evaluation with fluorescence in-situ hybridization and molecular genetic methods (array-based comparative genomic hybridization and RNA sequencing) which will lead to the correct diagnosis. Following this diagnostic route we detected a TNIP1::PDGFRB and a PCM1::FGFR1 fusion in our patients. Thus, genetic diagnosis must be precise and quick in order to initiate adequate therapies with tyrosine kinase inhibitors or HSCT.

Generation of human induced pluripotent stem cell line encoding for a genetically encoded voltage indicator Arclight A242.

Genetically encoded voltage indicators (GEVIs) allow for monitoring membrane potential changes in neurons and cardiomyocytes (CMs) as an alternative to patch-clamp techniques. GEVIs facilitate non-invasive, high throughput screening of electrophysiological properties of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). A dual transgenic hiPSC line with Arclight A242 (GEVI) and an antibiotic resistance cardiac selection cassette was successfully generated from an earlier established hiPSC line MHHi001-A. After cardiac differentiation and selection, purified populations of CMs with constitutive GEVI expression can be utilized for studying cardiac development, disease modeling, and drug testing.

Molecular characterization and clinical outcome of B-cell precursor acute lymphoblastic leukemia with IG-MYC rearrangement.

Rarely, immunophenotypically immature B-cell precursor acute lymphoblastic leukemia (BCP-ALL) carries an immunoglobulin- MYC rearrangement (IG-MYC-r). This can result in diagnostic confusion with Burkitt lymphoma/leukemia and use of individualized treatment schedules of unproven efficacy. Here we compare the molecular characteristics of these conditions and investigate historic clinical outcome data. We identified 90 cases registered in a national BCP-ALL clinical trial/registry. When present, diagnostic material underwent cytogenetic, exome, methylome and transcriptome analyses. The outcomes analyzed were 3-year event-free survival and overall survival. IG-MYC-r was identified in diverse cytogenetic backgrounds, co-existing with either established BCP-ALL-specific abnormalities (high hyperdiploidy, n=3; KMT2A-rearrangement, n=6; iAMP21, n=1; BCR-ABL1, n=1); BCL2/BCL6-rearrangements (n=15); or, most commonly, as the only defining feature (n=64). Within this final group, precursor-like V(D)J breakpoints predominated (8/9) and KRAS mutations were common (5/11). DNA methylation identified a cluster of V(D)J-rearranged cases, clearly distinct from Burkitt leukemia/lymphoma. Children with IG-MYC-r within that subgroup had a 3-year event-free survival of 47% and overall survival of 60%, representing a high-risk BCP-ALL. To develop effective management strategies this group of patients must be allowed access to contemporary, minimal residual disease-adapted, prospective clinical trial protocols.

Wildtype heterogeneity contributes to clonal variability in genome edited cells.

Genome editing tools such as CRISPR/Cas9 enable the rapid and precise manipulation of genomes. CRISPR-based genome editing has greatly simplified the study of gene function in cell lines, but its widespread use has also highlighted challenges of reproducibility. Phenotypic variability among different knockout clones of the same gene is a common problem confounding the establishment of robust genotype-phenotype correlations. Optimized genome editing protocols to enhance reproducibility include measures to reduce off-target effects. However, even if current state-of-the-art protocols are applied phenotypic variability is frequently observed. Here we identify heterogeneity of wild-type cells as an important and often neglected confounding factor in genome-editing experiments. We demonstrate that isolation of individual wild-type clones from an apparently homogenous stable cell line uncovers significant phenotypic differences between clones. Strikingly, we observe hundreds of differentially regulated transcripts (477 up- and 306 downregulated) when comparing two populations of wild-type cells. Furthermore, we show a variety of cellular and biochemical alterations in different wild-type clones in a range that is commonly interpreted as biologically relevant in genome-edited cells. Heterogeneity of wild-type cells thus contributes to variability in genome-edited cells when these are generated through isolation of clones. We show that the generation of monoclonal isogenic wild-type cells prior to genomic manipulation reduces phenotypic variability. We therefore propose to generate matched isogenic control cells prior to genome editing to increase reproducibility.

Generation of two TMEM16A knockout iPSC clones each from a healthy human iPSC line, from a Cystic Fibrosis patient specific line with p.Phe508del mutation and from the gene corrected iPSC line.

The Transmembrane member 16A (TMEM16A), also known as anoctamin-1 (ANO1), is a calcium-activated chloride channel present in the airway epithelium. It is known to be involved in the apical chloride secretion indicating that TMEM16A could be addressed for the treatment of chloride secretion defects like in Cystic- Fibrosis (CF). In this paper we generated knockout cell lines using CRISPR/Cas9-mediated ablation in a healthy human iPSC line (MHHi001-A), in a CF patient iPSC line (MHHi002-A) and in its corrected counterpart (MHHi002-A-1). These lines can be used for gaining information about the role of TMEM16A for mucus secretion and/or production and evaluating its therapeutic potential.

Generation of an induced pluripotent stem cell line (DHMCi009-A) from an individual with TUBB2A tubulinopathy.

TUBB2A tubulinopathy is a rare neurodevelopmental disorder with developmental delay, epilepsy, and less frequent malformations of cortical development compared to other tubulinopathies. Peripheral blood mononuclear cells (PBMCs) from a male subject harboring the heterozygous de novo TUBB2A variant c.[743C>T] (p.[Ala248Val]) were reprogrammed to induced pluripotent stem cells (iPSCs) using the CytoTune™-iPS 2.0 Sendai Reprogramming Kit (Invitrogen). Generated iPSCs showed a normal karyotype, expression of pluripotency markers, spontaneous in vitro differentiation in all three germ layers, and are a suitable human disease model to analyze pathomechanisms underlying TUBB2A tubulinopathy and potential therapeutic targets.

Targeted biallelic integration of an inducible Caspase 9 suicide gene in iPSCs for safer therapies.

Drug-inducible suicide systems may help to minimize risks of human induced pluripotent stem cell (hiPSC) therapies. Recent research challenged the usefulness of such systems since rare drug-resistant subclones were observed. We have introduced a drug-inducible Caspase 9 suicide system (iCASP9) into the AAVS1 safe-harbor locus of hiPSCs. In these cells, apoptosis could be efficiently induced in vitro. After transplantation into mice, drug treatment generally led to rapid elimination of teratomas, but single animals subsequently formed tumor tissue from monoallelic iCASP9 hiPSCs. Very rare drug-resistant subclones of monoallelic iCASP9 hiPSCs appeared in vitro with frequencies of ∼ 3 × 10-8. Besides transgene elimination, presumably via loss of heterozygosity (LoH), silencing via aberrant promoter methylation was identified as a major underlying mechanism. In contrast to monoallelic iCASP9 hiPSCs, no escapees from biallelic iCASP9 cells were observed after treatment of up to 0.8 billion hiPSCs. The highly increased safety level provided by biallelic integration of the iCASP9 system may substantially contribute to the safety level of iPSC-based therapies.

Clonal Evolution at First Sight: A Combined Visualization of Diverse Diagnostic Methods Improves Understanding of Leukemic Progression.

Patients with myeloid neoplasia are classified by the WHO classification systems. Besides clinical and hematological criteria, cytogenetic and molecular genetic alterations highly impact treatment stratification. In routine diagnostics, a combination of methods is used to decipher different types of genetic variants. Eight patients were comprehensively analyzed using karyotyping, fluorescence in situ hybridization, array-CGH and a custom NGS panel. Clonal evolution was reconstructed manually, integrating all mutational information on single nucleotide variants (SNVs), insertions and deletions (indels), structural variants and copy number variants (CNVs). To allow a correct integration, we differentiate between three scenarios: 1) CNV occurring prior to the SNV/indel, but in the same cells. 2) SNV/indel occurring prior to the CNV, but in the same cells. 3) SNV/indel and CNV existing in parallel, independent of each other. Applying this bioinformatics approach, we reconstructed clonal evolution for all patients. This generalizable approach offers the possibility to integrate various data to analyze identification of driver and passenger mutations as well as possible targets for personalized medicine approaches. Furthermore, this model can be used to identify markers to assess the minimal residual disease.

Optical Genome Mapping as a Diagnostic Tool in Pediatric Acute Myeloid Leukemia.

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.