Genetic alterations in lymphoblastic leukaemia / lymphoma - a practical guide to WHO HAEM5.

We present a practical guide for analyzing the genetic aspects of lymphoblastic leukaemia/lymphoma according to the 5th edition of the World Health Organization (WHO) classification of haematolymphoid neoplasms (WHO-HAEM5) issued in 2024. The WHO-HAEM5 acknowledges the increasing importance of genetics in the diagnosis of lymphoid neoplasia. Classification is based on the established genetic subtypes according to cell lineage, with precursor cell neoplasms followed by mature malignancies. This guide describes those genetic abnormalities in acute precursor B- and T-cell neoplasms required for risk stratification, and for treatment, providing diagnostic algorithms under the headings of 'essential' and 'desirable' diagnostic criteria.

Current genetic diagnostics in inborn errors of immunity.

New technologies in genetic diagnostics have revolutionized the understanding and management of rare diseases. This review highlights the significant advances and latest developments in genetic diagnostics in inborn errors of immunity (IEI), which encompass a diverse group of disorders characterized by defects in the immune system, leading to increased susceptibility to infections, autoimmunity, autoinflammatory diseases, allergies, and malignancies. Various diagnostic approaches, including targeted gene sequencing panels, whole exome sequencing, whole genome sequencing, RNA sequencing, or proteomics, have enabled the identification of causative genetic variants of rare diseases. These technologies not only facilitated the accurate diagnosis of IEI but also provided valuable insights into the underlying molecular mechanisms. Emerging technologies, currently mainly used in research, such as optical genome mapping, single cell sequencing or the application of artificial intelligence will allow even more insights in the aetiology of hereditary immune defects in the near future. The integration of genetic diagnostics into clinical practice significantly impacts patient care. Genetic testing enables early diagnosis, facilitating timely interventions and personalized treatment strategies. Additionally, establishing a genetic diagnosis is necessary for genetic counselling and prognostic assessments. Identifying specific genetic variants associated with inborn errors of immunity also paved the way for the development of targeted therapies and novel therapeutic approaches. This review emphasizes the challenges related with genetic diagnosis of rare diseases and provides future directions, specifically focusing on IEI. Despite the tremendous progress achieved over the last years, several obstacles remain or have become even more important due to the increasing amount of genetic data produced for each patient. This includes, first and foremost, the interpretation of variants of unknown significance (VUS) in known IEI genes and of variants in genes of unknown significance (GUS). Although genetic diagnostics have significantly contributed to the understanding and management of IEI and other rare diseases, further research, exchange between experts from different clinical disciplines, data integration and the establishment of comprehensive guidelines are crucial to tackle the remaining challenges and maximize the potential of genetic diagnostics in the field of rare diseases, such as IEI.

Deciphering the molecular complexity of the IKZF1plus genomic profile using Optical Genome Mapping.

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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.

CAR-NK Cells Targeting HER1 (EGFR) Show Efficient Anti-Tumor Activity against Head and Neck Squamous Cell Carcinoma (HNSCC).

(1) Background: HNSCC is a highly heterogeneous and relapse-prone form of cancer. We aimed to expand the immunological tool kit against HNSCC by conducting a functional screen to generate chimeric antigen receptor (CAR)-NK-92 cells that target HER1/epidermal growth factor receptor (EGFR). (2) Methods: Selected CAR-NK-92 cell candidates were tested for enhanced reduction of target cells, CD107a expression and IFNγ secretion in different co-culture models. For representative HNSCC models, patient-derived primary HNSCC (pHNSCC) cell lines were generated by employing an EpCAM-sorting approach to eliminate the high percentage of non-malignant cells found. (3) Results: 2D and 3D spheroid co-culture experiments showed that anti-HER1 CAR-NK-92 cells effectively eliminated SCC cell lines and primary HNSCC (pHNSCC) cells. Co-culture of tumor models with anti-HER1 CAR-NK-92 cells led to enhanced degranulation and IFNγ secretion of NK-92 cells and apoptosis of target cells. Furthermore, remaining pHNSCC cells showed upregulated expression of putative cancer stem cell marker CD44v6. (4) Conclusions: These results highlight the promising potential of CAR-NK cell therapy in HNSCC and the likely necessity to target multiple tumor-associated antigens to reduce currently high relapse rates.

Parallel deletion and duplication at 7q11.23 in a silent carrier for two reciprocal syndromic disorders.

Partial deletions at chromosome 7q11.23 are causative for the autosomal-dominant Williams-Beuren syndrome (WBS), whereas the partial duplication of this region leads to the 7q11.23 duplication syndrome. Both syndromes are highly penetrant and occur with a frequency of 1:7500-10,000 (WBS) and 1:13,000-20,000 (7q11.23 duplication syndrome). They are associated with multiple organ defects, intellectual disability, and typical facial dysmorphisms showing broad phenotypic variability. The 7q11.23 region is susceptible to chromosomal rearrangements due to flanking segmental duplications and regions of long repetitive DNA segments. Here, we report on a family with two children affected by WBS and clinically unaffected parents. Interestingly, metaphase fluorescence in situ hybridization (FISH) revealed a deletion on 7q11.23 in the father. Intensive genetic testing, using interphase FISH, whole genome sequencing and optical genome mapping led to the confirmation of a 1.5 Mb deletion at one 7q11.23 allele and the identification of a reciprocal 1.8 Mb duplication at the other allele. This finding is highly important regarding genetic counseling in this family. The father is a silent carrier for two syndromic disorders, thus his risk to transmit a disease-causing allele is 100%. To the best of our knowledge we, here, report on the first case in which the phenotype of a microdeletion/microduplication syndrome was compensated by its reciprocal counterpart.

Phenotypic spectrum in recessive STING-associated vasculopathy with onset in infancy: Four novel cases and analysis of previously reported cases.

Gain-of-function variants in the stimulator of interferon response cGAMP interactor 1 (STING1) gene cause STING-Associated Vasculopathy with onset in Infancy (SAVI). Previously, only heterozygous and mostly de novo STING1 variants have been reported to cause SAVI. Interestingly, one variant that only leads to SAVI when homozygous, namely c.841C>T p.(Arg281Trp), has recently been described. However, there are no entries in public databases regarding an autosomal recessive pattern of inheritance. Here, we report four additional unrelated SAVI patients carrying c.841C>T in homozygous state. All patients had interstitial lung disease and displayed typical interferon activation patterns. Only one child displayed cutaneous vasculitis, while three other patients presented with a relatively mild SAVI phenotype. Steroid and baricitinib treatment had a mitigating effect on the disease phenotype in two cases, but failed to halt disease progression. Heterozygous c.841C>T carriers in our analysis were healthy and showed normal interferon activation. Literature review identified eight additional cases with autosomal recessive SAVI caused by c.841C>T homozygosity. In summary, we present four novel and eight historic cases of autosomal recessive SAVI. We provide comprehensive clinical data and show treatment regimens and clinical responses. To date, SAVI has been listed as an exclusively autosomal dominant inherited trait in relevant databases. With this report, we aim to raise awareness for autosomal recessive inheritance in this rare, severe disease which may aid in early diagnosis and development of optimized treatment strategies.

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.

Copy Number Analysis in a Large Cohort Suggestive of Inborn Errors of Immunity Indicates a Wide Spectrum of Relevant Chromosomal Losses and Gains.

Inborn errors of immunity (IEI) are genetically driven disorders. With the advancement of sequencing technologies, a rapidly increasing number of gene defects has been identified, thereby mirroring the high heterogeneity in immunological and clinical presentations observed in patients. However, for a large majority of patients, no causative single nucleotide variant (SNV) or small indel can be identified using next-generation sequencing. First studies have shown that also copy number variants (CNVs) can cause IEI. Unfortunately, CNVs are not well examined in many routine diagnostic settings and the aim of this study was to assess the number of clinically relevant chromosomal losses and gains in a large cohort. We identified a total of 20 CNVs using whole exome sequencing data of a cohort of 191 patients with a suspected IEI. A definite molecular diagnosis could be made in five patients (2.6%), including pathogenic deletions affecting ICOS, TNFAIP3, and 22q11.2. CNVs of uncertain significance were observed in fifteen patients (7.9%), including deletions of 11q22.1q22.3 and 16p11.2 but also duplications affecting entire or parts of genes previously associated with IEI. Importantly, five patients carrying a CNV of uncertain significance also carried pathogenic or likely pathogenic SNVs (PIK3R1, NFKB1, NLRC4, DOCK2), or SNVs of unknown significance (NFKB2). This cooccurrence of SNVs and CNVs suggests modifying effects in some patients, and functional follow-up is warranted now in order to better understand phenotypic heterogeneity. In summary, the diagnostic yield of IEI can be increased substantially by evaluating CNVs, which allows an improved therapeutic management in those patients.

Cryptic TCF3 fusions in childhood leukemia: Detection by RNA sequencing.

Acute lymphoblastic leukemia (ALL) is the most frequent malignancy in childhood and adolescence. In more than 60% of cases of this heterogeneous disease, a genetic marker is identified via cytogenetic or molecular analyses. TCF3 gene fusions occur in 5%-11% of ALL patients. In < 1%, the TCF3 alteration in ALL leads to a TCF3-HLF fusion gene. Even though this is a very rare event, the detection of a TCF3-HLF fusion gene is associated with a very poor prognosis with incurable relapses in almost all patients. The frequent TCF3-PBX1 fusion gene, which is detectable in 5%-10% of childhood B-cell precursor ALLs and ~3.8% of adult B-cell precursor ALLs, is associated with a rather good prognosis, that is, an observed event-free 5-year survival of approximately 85%. Thus, the distinction of the different partner genes fused to TCF3 is essential for risk assessment. To verify RNA sequencing as a tool for detection of known and unknown fusion genes, we screened 200 cases of pediatric B-cell precursor ALL with "targeted" RNA sequencing in a pilot project in comparison to classical cytogenetic analyses (chromosome R-banding analysis), fluorescence in situ hybridization, and PCR. We observed a TCF3 fusion gene in 6.5% (13/200) of the patients. Ten (5%) patients displayed a TCF3-PBX1 fusion gene, two (1%) patients a TCF3-FLI1 fusion gene, and one (0.5%) patient a TCF3-HLF fusion gene. For the TCF3 fusions, we obtained discrepant results with the different methods, which are described in the article. Taken together, translocations leading to TCF3 fusion genes might appear cryptic and may remain undetected by a single method.

A selectable all-in-one CRISPR prime editing piggyBac transposon allows for highly efficient gene editing in human cell lines.

CRISPR prime-editors are emergent tools for genome editing and offer a versatile alternative approach to HDR-based genome engineering or DNA base-editors. However, sufficient prime-editor expression levels and availability of optimized transfection protocols may affect editing efficiencies, especially in hard-to-transfect cells like hiPSC. Here, we show that piggyBac prime-editing (PB-PE) allows for sustained expression of prime-editors. We demonstrate proof-of-concept for PB-PE in a newly designed lentiviral traffic light reporter, which allows for estimation of gene correction and defective editing resulting in indels, based on expression of two different fluorophores. PB-PE can prime-edit more than 50% of hiPSC cells after antibiotic selection. We also show that improper design of pegRNA cannot simply be overcome by extended expression, but PB-PE allows for estimation of effectiveness of selected pegRNAs after few days of cultivation time. Finally, we implemented PB-PE for efficient editing of an amyotrophic lateral sclerosis-associated mutation in the SOD1-gene of patient-derived hiPSC. Progress of genome editing can be monitored by Sanger-sequencing, whereas PB-PE vectors can be removed after editing and excised cells can be enriched by fialuridine selection. Together, we present an efficient prime-editing toolbox, which can be robustly used in a variety of cell lines even when non-optimized transfection-protocols are applied.

The Clinical Utility of Optical Genome Mapping for the Assessment of Genomic Aberrations in Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is the most prevalent type of cancer occurring in children. ALL is characterized by structural and numeric genomic aberrations that strongly correlate with prognosis and clinical outcome. Usually, a combination of cyto- and molecular genetic methods (karyotyping, array-CGH, FISH, RT-PCR, RNA-Seq) is needed to identify all aberrations relevant for risk stratification. We investigated the feasibility of optical genome mapping (OGM), a DNA-based method, to detect these aberrations in an all-in-one approach. As proof of principle, twelve pediatric ALL samples were analyzed by OGM, and results were validated by comparing OGM data to results obtained from routine diagnostics. All genomic aberrations including translocations (e.g., dic(9;12)), aneuploidies (e.g., high hyperdiploidy) and copy number variations (e.g., IKZF1, PAX5) known from other techniques were also detected by OGM. Moreover, OGM was superior to well-established techniques for resolution of the more complex structure of a translocation t(12;21) and had a higher sensitivity for detection of copy number alterations. Importantly, a new and unknown gene fusion of JAK2 and NPAT due to a translocation t(9;11) was detected. We demonstrate the feasibility of OGM to detect well-established as well as new putative prognostic markers in an all-in-one approach in ALL. We hope that these limited results will be confirmed with testing of more samples in the future.

Genetic Correction of IL-10RB Deficiency Reconstitutes Anti-Inflammatory Regulation in iPSC-Derived Macrophages.

Patient material from rare diseases such as very early-onset inflammatory bowel disease (VEO-IBD) is often limited. The use of patient-derived induced pluripotent stem cells (iPSCs) for disease modeling is a promising approach to investigate disease pathomechanisms and therapeutic strategies. We successfully developed VEO-IBD patient-derived iPSC lines harboring a mutation in the IL-10 receptor ß-chain (IL-10RB) associated with defective IL-10 signaling. To characterize the disease phenotype, healthy control and VEO-IBD iPSCs were differentiated into macrophages. IL-10 stimulation induced characteristic signal transducer and activator of transcription 3 (STAT3) and suppressor of cytokine signaling 3 (SOCS3) downstream signaling and anti-inflammatory regulation of lipopolysaccharide (LPS)-mediated cytokine secretion in healthy control iPSC-derived macrophages. In contrast, IL-10 stimulation of macrophages derived from patient iPSCs did not result in STAT3 phosphorylation and subsequent SOCS3 expression, recapitulating the phenotype of cells from patients with IL-10RB deficiency. In line with this, LPS-induced cytokine secretion (e.g., IL-6 and tumor necrosis factor-α (TNF-α)) could not be downregulated by exogenous IL-10 stimulation in VEO-IBD iPSC-derived macrophages. Correction of the IL-10RB defect via lentiviral gene therapy or genome editing in the adeno-associated virus integration site 1 (AAVS1) safe harbor locus led to reconstitution of the anti-inflammatory response. Corrected cells showed IL-10RB expression, IL-10-inducible phosphorylation of STAT3, and subsequent SOCS3 expression. Furthermore, LPS-mediated TNF-α secretion could be modulated by IL-10 stimulation in gene-edited VEO-IBD iPSC-derived macrophages. Our established disease models provide the opportunity to identify and validate new curative molecular therapies and to investigate phenotypes and consequences of additional individual IL-10 signaling pathway-dependent VEO-IBD mutations.

iPSC modeling of stage-specific leukemogenesis reveals BAALC as a key oncogene in severe congenital neutropenia.

Severe congenital neutropenia (CN) is a pre-leukemic bone marrow failure syndrome that can evolve to acute myeloid leukemia (AML). Mutations in CSF3R and RUNX1 are frequently observed in CN patients, although how they drive the transition from CN to AML (CN/AML) is unclear. Here we establish a model of stepwise leukemogenesis in CN/AML using CRISPR-Cas9 gene editing of CN patient-derived iPSCs. We identified BAALC upregulation and resultant phosphorylation of MK2a as a key leukemogenic event. BAALC deletion or treatment with CMPD1, a selective inhibitor of MK2a phosphorylation, blocked proliferation and induced differentiation of primary CN/AML blasts and CN/AML iPSC-derived hematopoietic stem and progenitor cells (HSPCs) without affecting healthy donor or CN iPSC-derived HSPCs. Beyond detailing a useful method for future investigation of stepwise leukemogenesis, this study suggests that targeting BAALC and/or MK2a phosphorylation may prevent leukemogenic transformation or eliminate AML blasts in CN/AML and RUNX1 mutant BAALC(hi) de novo AML.