The E592K variant of SF3B1 creates unique RNA missplicing and associates with high-risk MDS without ring sideroblasts.

Among the most common genetic alterations in the myelodysplastic syndromes (MDS) are mutations in the spliceosome gene SF3B1. Such mutations induce specific RNA missplicing events, directly promote ring sideroblast (RS) formation, and generally associate with more favorable prognosis. However, not all SF3B1 mutations are the same, and little is known about how distinct hotspots influence disease. Here we report that the E592K variant of SF3B1 associates with high-risk disease features in MDS, including a lack of RS, increased myeloblasts, a distinct co-mutation pattern, and a lack of the favorable survival seen with other SF3B1 mutations. Moreover, compared to other hotspot SF3B1 mutations, E592K induces a unique RNA missplicing pattern, retains an interaction with the splicing factor SUGP1, and preserves normal RNA splicing of the sideroblastic anemia genes TMEM14C and ABCB7. These data have implications for our understanding of the functional diversity of spliceosome mutations, as well as the pathobiology, classification, prognosis, and management of SF3B1-mutant MDS.

A lineage-specific STAT5BN642H mouse model to study NK-cell leukemia.

Patients with T- and NK-cell neoplasms frequently have somatic STAT5B gain-of-function mutations. The most frequent STAT5B mutation is STAT5BN642H, which is known to drive murine T-cell leukemia although its role in NK-cell malignancies is unclear. Introduction of the STAT5BN642H mutation into human NK-cell lines enhances their potential to induce leukemia in mice. We have generated a mouse model that enables tissue-specific expression of STAT5BN642H and have selectively expressed the mutated STAT5B in hematopoietic cells (N642Hvav/+) or exclusively in NK cells (N642HNK/NK). All N642Hvav/+ mice rapidly develop an aggressive T-/NK T-cell leukemia, whereas N642HNK/NK mice display an indolent NK-large granular lymphocytic leukemia (NK-LGLL) that progresses to an aggressive leukemia with age. Samples from NK-cell leukemia patients have a distinctive transcriptional signature driven by mutant STAT5B, which overlaps with that of murine leukemic N642HNK/NK NK cells. We have generated the first reliable STAT5BN642H-driven pre-clinical mouse model that displays an indolent NK-LGLL progressing to aggressive NK-cell leukemia. This novel in vivo tool will enable us to explore the transition from an indolent to an aggressive disease and will thus permit the study of prevention and treatment options for NK-cell malignancies.

Comparing malignant monocytosis across the updated WHO and ICC classifications of 2022.

ABSTRACT: The World Health Organization (WHO) classification of hematolymphoid tumors and the International Consensus Classification (ICC) of 2022 introduced major changes to the definition of chronic myelomonocytic leukemia (CMML). To assess its qualitative and quantitative implications for patient care, we started with 3311 established CMML cases (according to WHO 2017 criteria) and included 2130 oligomonocytosis cases fulfilling the new CMML diagnostic criteria. Applying both 2022 classification systems, 356 and 241 of oligomonocytosis cases were newly classified as myelodysplastic (MD)-CMML (WHO and ICC 2022, respectively), most of which were diagnosed as myelodysplastic syndrome (MDS) according to the WHO 2017 classification. Importantly, 1.5 times more oligomonocytosis cases were classified as CMML according to WHO 2022 than based on ICC, because of different diagnostic criteria. Genetic analyses of the newly classified CMML cases showed a distinct mutational profile with strong enrichment of MDS-typical alterations, resulting in a transcriptional subgroup separated from established MD and myeloproliferative CMML. Despite a different cytogenetic, molecular, immunophenotypic, and transcriptional landscape, no differences in overall survival were found between newly classified and established MD-CMML cases. To the best of our knowledge, this study represents the most comprehensive analysis of routine CMML cases to date, both in terms of clinical characterization and transcriptomic analysis, placing newly classified CMML cases on a disease continuum between MDS and previously established CMML.

Developmental trajectories and cooperating genomic events define molecular subtypes of BCR::ABL1-positive ALL.

ABSTRACT: Distinct diagnostic entities within BCR::ABL1-positive acute lymphoblastic leukemia (ALL) are currently defined by the International Consensus Classification of myeloid neoplasms and acute leukemias (ICC): "lymphoid only", with BCR::ABL1 observed exclusively in lymphatic precursors, vs "multilineage", where BCR::ABL1 is also present in other hematopoietic lineages. Here, we analyzed transcriptomes of 327 BCR::ABL1-positive patients with ALL (age, 2-84 years; median, 46 years) and identified 2 main gene expression clusters reproducible across 4 independent patient cohorts. Fluorescence in situ hybridization analysis of fluorescence-activated cell-sorted hematopoietic compartments showed distinct BCR::ABL1 involvement in myeloid cells for these clusters (n = 18/18 vs n = 3/16 patients; P < .001), indicating that a multilineage or lymphoid BCR::ABL1 subtype can be inferred from gene expression. Further subclusters grouped samples according to cooperating genomic events (multilineage: HBS1L deletion or monosomy 7; lymphoid: IKZF1-/- or CDKN2A/PAX5 deletions/hyperdiploidy). A novel HSB1L transcript was highly specific for BCR::ABL1 multilineage cases independent of HBS1L genomic aberrations. Treatment on current German Multicenter Study Group for Adult ALL (GMALL) protocols resulted in comparable disease-free survival (DFS) for multilineage vs lymphoid cluster patients (3-year DFS: 70% vs 61%; P = .530; n = 91). However, the IKZF1-/- enriched lymphoid subcluster was associated with inferior DFS, whereas hyperdiploid cases showed a superior outcome. Thus, gene expression clusters define underlying developmental trajectories and distinct patterns of cooperating events in BCR::ABL1-positive ALL with prognostic relevance.

Collaborative effect of Csnk1a1 haploinsufficiency and mutant p53 in Myc induction can promote leukemic transformation.

ABSTRACT: It is still not fully understood how genetic haploinsufficiency in del(5q) myelodysplastic syndrome (MDS) contributes to malignant transformation of hematopoietic stem cells. We asked how compound haploinsufficiency for Csnk1a1 and Egr1 in the common deleted region on chromosome 5 affects hematopoietic stem cells. Additionally, Trp53 was disrupted as the most frequently comutated gene in del(5q) MDS using CRISPR/Cas9 editing in hematopoietic progenitors of wild-type (WT), Csnk1a1-/+, Egr1-/+, Csnk1a1/Egr1-/+ mice. A transplantable acute leukemia only developed in the Csnk1a1-/+Trp53-edited recipient. Isolated blasts were indefinitely cultured ex vivo and gave rise to leukemia after transplantation, providing a tool to study disease mechanisms or perform drug screenings. In a small-scale drug screening, the collaborative effect of Csnk1a1 haploinsufficiency and Trp53 sensitized blasts to the CSNK1 inhibitor A51 relative to WT or Csnk1a1 haploinsufficient cells. In vivo, A51 treatment significantly reduced blast counts in Csnk1a1 haploinsufficient/Trp53 acute leukemias and restored hematopoiesis in the bone marrow. Transcriptomics on blasts and their normal counterparts showed that the derived leukemia was driven by MAPK and Myc upregulation downstream of Csnk1a1 haploinsufficiency cooperating with a downregulated p53 axis. A collaborative effect of Csnk1a1 haploinsufficiency and p53 loss on MAPK and Myc upregulation was confirmed on the protein level. Downregulation of Myc protein expression correlated with efficient elimination of blasts in A51 treatment. The "Myc signature" closely resembled the transcriptional profile of patients with del(5q) MDS with TP53 mutation.

Tumor necrosis factor α promotes clonal dominance of KIT D816V+ cells in mastocytosis: role of survivin and impact on prognosis.

ABSTRACT: Systemic mastocytosis (SM) is defined by the expansion and accumulation of neoplastic mast cells (MCs) in the bone marrow (BM) and extracutaneous organs. Most patients harbor a somatic KIT D816V mutation, which leads to growth factor-independent KIT activation and accumulation of MC. Tumor necrosis factor α (TNF) is a proapoptotic and inflammatory cytokine that has been implicated in the clonal selection of neoplastic cells. We found that KIT D816V increases the expression and secretion of TNF. TNF expression in neoplastic MCs is reduced by KIT-targeting drugs. Similarly, knockdown of KIT or targeting the downstream signaling cascade of MAPK and NF-κB signaling reduced TNF expression levels. TNF reduces colony formation in human BM cells, whereas KIT D816V+ cells are less susceptible to the cytokine, potentially contributing to clonal selection. In line, knockout of TNF in neoplastic MC prolonged survival and reduced myelosuppression in a murine xenotransplantation model. Mechanistic studies revealed that the relative resistance of KIT D816V+ cells to TNF is mediated by the apoptosis-regulator BIRC5 (survivin). Expression of BIRC5 in neoplastic MC was confirmed by immunohistochemistry of samples from patients with SM. TNF serum levels are significantly elevated in patients with SM and high TNF levels were identified as a biomarker associated with inferior survival. We here characterized TNF as a KIT D816V-dependent cytokine that promotes clonal dominance. We propose TNF and apoptosis-associated proteins as potential therapeutic targets in SM.

Massive parallel sequencing unveils homologous recombination deficiency in follicular dendritic cell sarcoma.

Standardized treatment options are lacking for patients with unresectable or multifocal follicular dendritic cell sarcoma (FDCS) and disease-related mortality is as high as 20%. Applying whole genome sequencing (WGS) in one case and whole exome sequencing (WES) in additional twelve, this study adds information on the molecular landscape of FDCS, expanding knowledge on pathobiological mechanisms and identifying novel markers of potential theragnostic significance. Massive parallel sequencing showed high frequency of mutations on oncosuppressor genes, particularly in RB1, CARS and BRCA2 and unveiled alterations on homologous recombination DNA damage repair related genes in 70% (9/13) of cases. This indicates that patients with high stage FDCS may be eligible for poly ADP ribose polymerase inhibition protocols. Low tumor mutational burden was confirmed in this study despite common PDL1 expression in FDCS arguing on the efficacy of immune checkpoint inhibitors. CDKN2A deletion, detected by WGS and confirmed by FISH in 41% of cases (9/22) indicates that impairment of cell cycle regulation may sustain oncogenesis in FDCS. Absence of mutations in the RAS/RAF/MAPK pathway and lack of clonal hematopoiesis related mutations in FDCS sanction its differences from dendritic cell-derived neoplasms of haematopoietic derivation. WGS and WES in FDCS provides additional information on the molecular landscape of this rare tumor, proposing novel candidate genes for innovative therapeutical approaches to improve survival of patients with multifocal disease.

The Gene Expression Classifier ALLCatchR Identifies B-cell Precursor ALL Subtypes and Underlying Developmental Trajectories Across Age.

Current classifications (World Health Organization-HAEM5/ICC) define up to 26 molecular B-cell precursor acute lymphoblastic leukemia (BCP-ALL) disease subtypes by genomic driver aberrations and corresponding gene expression signatures. Identification of driver aberrations by transcriptome sequencing (RNA-Seq) is well established, while systematic approaches for gene expression analysis are less advanced. Therefore, we developed ALLCatchR, a machine learning-based classifier using RNA-Seq gene expression data to allocate BCP-ALL samples to all 21 gene expression-defined molecular subtypes. Trained on n = 1869 transcriptome profiles with established subtype definitions (4 cohorts; 55% pediatric / 45% adult), ALLCatchR allowed subtype allocation in 3 independent hold-out cohorts (n = 1018; 75% pediatric / 25% adult) with 95.7% accuracy (averaged sensitivity across subtypes: 91.1% / specificity: 99.8%). High-confidence predictions were achieved in 83.7% of samples with 98.9% accuracy. Only 1.2% of samples remained unclassified. ALLCatchR outperformed existing tools and identified novel driver candidates in previously unassigned samples. Additional modules provided predictions of samples blast counts, patient's sex, and immunophenotype, allowing the imputation in cases where these information are missing. We established a novel RNA-Seq reference of human B-lymphopoiesis using 7 FACS-sorted progenitor stages from healthy bone marrow donors. Implementation in ALLCatchR enabled projection of BCP-ALL samples to this trajectory. This identified shared proximity patterns of BCP-ALL subtypes to normal lymphopoiesis stages, extending immunophenotypic classifications with a novel framework for developmental comparisons of BCP-ALL. ALLCatchR enables RNA-Seq routine application for BCP-ALL diagnostics with systematic gene expression analysis for accurate subtype allocation and novel insights into underlying developmental trajectories.

Mechanisms of antigen escape from BCMA- or GPRC5D-targeted immunotherapies in multiple myeloma.

B cell maturation antigen (BCMA) target loss is considered to be a rare event that mediates multiple myeloma (MM) resistance to anti-BCMA chimeric antigen receptor T cell (CAR T) or bispecific T cell engager (TCE) therapies. Emerging data report that downregulation of G-protein-coupled receptor family C group 5 member D (GPRC5D) protein often occurs at relapse after anti-GPRC5D CAR T therapy. To examine the tumor-intrinsic factors that promote MM antigen escape, we performed combined bulk and single-cell whole-genome sequencing and copy number variation analysis of 30 patients treated with anti-BCMA and/or anti-GPRC5D CAR T/TCE therapy. In two cases, MM relapse post-TCE/CAR T therapy was driven by BCMA-negative clones harboring focal biallelic deletions at the TNFRSF17 locus at relapse or by selective expansion of pre-existing subclones with biallelic TNFRSF17 loss. In another five cases of relapse, newly detected, nontruncating, missense mutations or in-frame deletions in the extracellular domain of BCMA negated the efficacies of anti-BCMA TCE therapies, despite detectable surface BCMA protein expression. In the present study, we also report four cases of MM relapse with biallelic mutations of GPRC5D after anti-GPRC5D TCE therapy, including two cases with convergent evolution where multiple subclones lost GPRC5D through somatic events. Immunoselection of BCMA- or GPRC5D-negative or mutant clones is an important tumor-intrinsic driver of relapse post-targeted therapies. Mutational events on BCMA confer distinct sensitivities toward different anti-BCMA therapies, underscoring the importance of considering the tumor antigen landscape for optimal design and selection of targeted immunotherapies in MM.

Interplay of TP53 allelic state, blast count, and complex karyotype on survival of patients with AML and MDS.

Several clinical and genetic factors impact overall survival (OS) in myelodysplastic neoplasms (MDS) and acute myeloid leukemia (AML), including complex karyotype (CK), TP53 allelic state, and blast count. We analyzed the interplay of these factors by performing Cox regression analysis and by determining the frequency of TP53 single-hit (sh) and double-hit (dh) events and OS in MDS (n = 747) with <5% blasts, with ≥5% but <10% blasts, and ≥10% but <20% blasts and AML (n = 772). MDS with <5% blasts showed the best outcome, followed by with ≥5% but <10% blasts, and ≥10% but <20% blasts, and AML (median OS: 75, 54, 27, and 18 months, respectively). The same hierarchy was observed when each subgroup was divided into TP53sh, TP53dh, and without TP53 alterations (alt), revealing a dismal outcome of TP53dh in all subgroups (17, 10, 8, and 1 month[s], respectively). MDS with <5% blasts differed from the other subgroups by showing predominantly TP53sh (76% of TP53alt cases), and by an independent adverse impact of CK on OS (hazard ratio, 5.2; P < .001). The remaining subgroups displayed many similarities, with TP53dh found at high frequencies (67%, 91%, and 71%, respectively) and only TP53alt but not CK independently influencing OS, and TP53dh showing the strongest influence. When the total cohort was split based on TP53 state, only the blast count and not CK had an independent adverse impact on OS in all subgroups. Thus, TP53dh is the strongest prognostic factor, further supporting its integration into risk stratification guidelines and classification as a separate entity. However, the blast count also influences OS independent of TP53 state, whereas CK plays a minor prognostic role.

The E592K variant of SF3B1 creates unique RNA missplicing and associates with high-risk MDS without ring sideroblasts.

Among the most common genetic alterations in the myelodysplastic syndromes (MDS) are mutations in the spliceosome gene SF3B1. Such mutations induce specific RNA missplicing events, directly promote ring sideroblast (RS) formation, generally associate with more favorable prognosis, and serve as a predictive biomarker of response to luspatercept. However, not all SF3B1 mutations are the same, and here we report that the E592K variant of SF3B1 associates with high-risk disease features in MDS, including a lack of RS, increased myeloblasts, a distinct co-mutation pattern, and decreased survival. Moreover, in contrast to canonical SF3B1 mutations, E592K induces a unique RNA missplicing pattern, retains an interaction with the splicing factor SUGP1, and preserves normal RNA splicing of the sideroblastic anemia genes TMEM14C and ABCB7. These data expand our knowledge of the functional diversity of spliceosome mutations, and they suggest that patients with E592K should be approached differently from low-risk, luspatercept-responsive MDS patients with ring sideroblasts and canonical SF3B1 mutations.

Challenging gold standard hematology diagnostics through the introduction of whole genome sequencing and artificial intelligence.

The diagnosis of hematological malignancies is rather complex and requires the application of a plethora of different assays, techniques and methodologies. Some of the methods, like cytomorphology, have been in use for decades, while other methods, such as next-generation sequencing or even whole genome sequencing (WGS), are relatively new. The application of the methods and the evaluation of the results require distinct skills and knowledge and place different demands on the practitioner. However, even with experienced hematologists, diagnostic ambiguity remains a regular occurrence and the comprehensive analysis of high-dimensional WGS data soon exceeds any human's capacity. Hence, in order to reduce inter-observer variability and to improve the timeliness and accuracy of diagnoses, machine learning based approaches have been developed to assist in the decision making process. Moreover, to achieve the goal of precision oncology, comprehensive genomic profiling is increasingly being incorporated into routine standard of care.

Novel causative variants of VEXAS in UBA1 detected through whole genome transcriptome sequencing in a large cohort of hematological malignancies.

UBA1 is an X-linked gene and encodes an ubiquitin-activating enzyme. Three somatic mutations altering the alternative start codon (M41) in UBA1 in hematopoietic precursor cells have recently been described, resulting in a syndrome of severe inflammation, cytopenias, and the presence of intracellular vacuoles in hematopoietic precursors - termed VEXAS syndrome, a predominantly male disease. Here we present a patient with clinical features of VEXAS who harbored two novel somatic variants in UBA1 (I894S and N606I). To better understand the clinical relevance and biological consequences of non-M41 (UBA1non-M41) variants, we analyzed the whole genome and transcriptome data of 4168 patients with hematological malignancies and detected an additional 16 UBA1non-M41 putative somatic variants with a clear sex-bias in patients with myeloid malignancies. Patients diagnosed with myeloid malignancies carrying UBA1non-M41 putative somatic variants either had vacuoles or immunodysregulatory symptoms. Analysis of the transcriptome confirmed neutrophil activation in VEXAS patients compared to healthy controls but did not result in a specific transcriptomic signature of UBA1M41 patients in comparison with MDS patients. In summary, we have described multiple putative novel UBA1non-M41 variants in patients with various hematological malignancies expanding the genomic spectrum of VEXAS syndrome.

Identification of a specific immunophenotype associated with a consistent pattern of genetic mutations including SRFS2 and gene expression profile in MDS.

BACKGROUND: Myelodysplastic syndromes (MDS) comprise a heterogeneous group of diseases classified by comprehensive diagnostics. Identification of homogeneous subgroups is desirable to understand differences in clinical course and to develop targeted treatment approaches. We identified a specific CD11b/CD16 expression pattern in granulocytes associated with reduced CD45 expression in myeloid progenitor cells (MPC) in MDS cases and assessed its genetic background by whole genome (WGS) and whole transcriptome sequencing (WTS). METHODS: The cohort consisted of 32 MDS cases with the specific aberrant immunophenotype. Since all these 32 cases were found to be SRSF2 mutated additional 51 SRSF2 mutated MDS cases without this specific immunophenotype were selected as controls. For all cases WGS and WTS were performed. RESULTS: The immunophenotype newly identified in SRSF2 mutated MDS patients is characterized (1) by a specific maturation pattern, i.e. an increase of CD11b expression without CD16 expression followed by an increase in CD16 expression without further CD11b expression and (2) by only dim CD45 expression of MPC. STAG2 mutations were exclusively found in MDS cases with the specific immunophenotype (17/32, 53% vs. 0%, p < 0.001). Hence, >50% of cases with the specific immunophenotype were characterized by co-mutations in SRSF2 and STAG2. In addition, cluster analysis revealed a specific gene expression profile of such cases. CONCLUSION: We here for the first time describe a specific immunophenotype which defines MDS cases with SRSF2 mutations and a consistent and specific mutational and gene expression profile. This comprehensive data warrants analysis of further such cases to assess the feasibility of defining a new sub-entity of MDS.